Pea peptide, preparation method of modified pea peptide and use thereof for improving gastrointestinal function

Abstract

The present invention relates to the technical field of pea peptides, and specifically to pea peptides, a preparation method of modified pea peptides, and uses for improving gastrointestinal function. The amino acid sequence of the pea peptide is shown in SEQ ID NO.1. The preparation method of the modified pea peptide is prepared by reacting the pea peptide shown in SEQ ID NO.1 with monomethoxypolyethylene glycol-butyraldehyde. The uses include preparing intestinal antibacterial or antibacterial products, preparing products for improving intestinal immune function, preparing products for treating intestinal inflammation, preparing products for improving inflammatory intestinal morphology, preparing products for improving gastrointestinal hormone disorders, and preparing products for increasing intestinal short-chain fatty acid content.

Description

Technical Field

[0001] The present invention relates to the technical field of pea peptides, and in particular to a preparation method of pea peptides, modified pea peptides and uses thereof for improving gastrointestinal function. Background Art

[0002] Pea, scientific name Pisum sativum Linn., is rich in nutrition, with a starch content of 55-68%, a protein content of 20-30%, a crude fiber content of 8-10%, and a fat content of less than 2%. Because peas are rich in starch, in addition to being eaten, they are mainly used to produce pea starch and related products. Pea protein is an important component of peas, with a biological value of 48-64%, an efficacy ratio of 0.6-1.2, and is not prone to allergies, with high nutritional value. The amino acid ratio of pea protein is relatively balanced, easy to digest and absorb, and a high lysine content, making it a good source of protein. Therefore, this paper reviews the extraction methods of pea protein and its antioxidant peptides in recent years, analyzes the physicochemical properties of pea protein and the biological activity of its antioxidant peptides, and provides a reference for the production and comprehensive development and utilization of pea protein. Summary of the invention

[0003] The inventors have studied the amino acid sequence and preliminary functions of the pea hypothetical protein KIW84_055578 and found that a short peptide in the sequence has a biological function unique to the hypothetical protein, namely, the pea peptide has the functions of antibacterial, antibacterial, improving intestinal immune function, treating intestinal inflammation, improving inflammatory intestinal morphology, improving gastrointestinal hormone disorders and increasing the content of intestinal short-chain fatty acids.

[0004] Based on this, the present invention provides a pea peptide, whose amino acid sequence is shown in SEQ ID NO.1.

[0005] The present invention provides a method for preparing a modified pea peptide, comprising reacting a pea peptide as shown in SEQ ID NO.1 with monomethoxy polyethylene glycol-butyraldehyde.

[0006] Furthermore, the reaction addition ratio of the pea peptide and the monomethoxypolyethylene glycol-butyraldehyde is 1:2.

[0007] Furthermore, the reaction system is a 100 mM acetic acid-sodium acetate buffer at pH 5.0.

[0008] Furthermore, the reaction time is 40 to 60 minutes.

[0009] Furthermore, the reaction is terminated with ethanolamine.

[0010] The invention provides a modified pea peptide prepared by the preparation method.

[0011] The present invention provides a use in preparing a product for improving intestinal health.

[0012] The present invention provides at least one of the following uses of the prepared modified pea peptide:

[0013] 1) Preparation of intestinal antibacterial or antibacterial products;

[0014] 2) Preparation of products for improving intestinal immune function;

[0015] 3) Preparation of products for treating intestinal inflammation;

[0016] 4) Preparation of products for improving intestinal morphology of inflammation;

[0017] 5) Preparation of products for improving gastrointestinal hormone disorders;

[0018] 6) Prepare products for increasing the content of short-chain fatty acids in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the ZORBAX SB-C18 reverse phase chromatogram of the pea peptide modification product, A is the modification reaction product of the experimental group, and B is the modification reaction product of the control group.

[0020] Figure 2 Time-kill curves of pea peptide (Pep) and modified pea peptide (Mod-pep1).

[0021] Figure 3 The bactericidal efficiency of pea peptide (Pep), modified pea peptide 1 (Mod-pep1) and modified pea peptide 2 (Mod-pep2) at different pH values, “*” indicates statistical difference, p<0.05.

[0022] Figure 4 The bactericidal efficiency of pea peptide (Pep), modified pea peptide (Mod-pep1) and modified pea peptide (Mod-pep2) after being treated with pepsin (3000-3500NFU), trypsin (250NFU) and α-chymotrypsin (1200U), respectively. "*" indicates statistical difference, p<0.05.

[0023] Figure 5 The expression levels of sIgA in the duodenum, jejunum and ileum of mice in the model group (MOD), experimental group I (EXP1) and experimental group II (EXP2). "*" indicates statistical difference, p<0.05.

[0024] Figure 6The acetic acid content in the intestine of mice in the normal group (NOM), model group (MOD), experimental group I (EXP1) and experimental group II (EXP2), "*" indicates statistical difference, p < 0.05.

[0025] Figure 7 The propionic acid content in the intestine of mice in the normal group (NOM), model group (MOD), experimental group I (EXP1) and experimental group II (EXP2), "*" indicates statistical difference, p < 0.05.

[0026] Figure 8 Butyrate content in the intestine of mice in the normal group (NOM), model group (MOD), experimental group I (EXP1) and experimental group II (EXP2), "*" indicates statistical difference, p < 0.05. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The reagents not described separately in detail in the present invention are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0028] Example 1. Synthesis of pea peptide

[0029] Based on the amino acid sequence of pea (Pisumsativum L.) protein KAI5410143.1hypothetical protein KIW84_055578 disclosed by NCBI and previous experimental studies, the present invention mainly focuses on studying the biological activity of pea peptide ISQIQRPVKELAFP. To this end, the pea peptide was synthesized by a chemical solid phase synthesis method. The obtained pea peptide has good water solubility, a molecular weight of 1625.91 g / mol, an isoelectric point of about 10.12, and can be stored in a -20°C refrigerator for a long time.

[0030] Example 2: Modification of pea peptide

[0031] Even though the pea peptide prepared by the present invention has the characteristic of high water solubility, it is easily destroyed by hydrolases and easily cleared by glomerular filtration in the body due to its high water solubility, resulting in problems such as difficulty in absorption in the body. Therefore, this embodiment is conducive to the modification of pea peptide with polyethylene glycol to obtain a polyethylene glycol-modified pea peptide. The details are as follows:

[0032] 1. Modification reaction

[0033] Experimental group: 100 mM acetic acid-sodium acetate at pH 5.0 was used as the buffer system, and the reaction was carried out at 20°C. The initial concentration of pea peptide in the reaction system was 1.5 mg / mL. mPEG-ButyrALD 5000 (monomethoxypolyethylene glycol-butyraldehyde, molecular weight MW5000, Guangdong Wengjiang Chemical Reagent Co., Ltd.) was used as the modifier with an initial concentration of 1.5 mg / mL. The reaction was fully reacted for 50 minutes, and an excess of ethanolamine (20 mg / mL, half of the total reaction volume) was added to terminate the reaction. The product was analyzed after dilution 5 times to obtain modified pea peptide 1.

[0034] Control group: mPEG-ButyrALD 5000 (monomethoxypolyethylene glycol-butyraldehyde, molecular weight MW20000Da, Guangdong Wengjiang Chemical Reagent Co., Ltd.) was used, and other reaction conditions were the same as above to obtain modified pea peptide 2.

[0035] 2. Analysis of modified products

[0036] The ZORBAX SB-C18 reverse phase column was used for gradient elution. The mobile phase A was 100mM acetic acid-sodium acetate buffer (pH5.0) (containing 0.1 trifluoroacetic acid, w / v); phase B was methanol containing 0.1 trifluoroacetic acid, w / v); flow rate was 0.9mL / min; and the detection wavelength was 280nm. The gradient elution program was: 0→50min, 95%A→20%A. Standard curve: 50mg of pea peptide was accurately weighed and prepared into different gradients with 100mM acetic acid-sodium acetate buffer (pH5.0). 20μL was injected under the above chromatographic conditions to establish a standard curve of chromatographic peak area and corresponding pea peptide concentration. The results are shown in Figure 2. Figure 1 As shown, under the condition of 280nm, a large amount of modified pea peptides can be obtained in a reaction time of 50min, while the chromatographic peak area of ​​the modified pea peptide in the control group is smaller (indicating that its reaction yield is lower).

[0037] 3. Isolation and purification of modified pea peptides

[0038] The reaction product was loaded onto Sp Sepharose Fast Flow (column volume 10 ml) for chromatography purification. The modified pea peptide was purified by 20 column volumes of 0%→100% system with a pH of 4 and 0.02M acetate solution containing 0.5M NaCl. The purity was above 96% (the above liquid phase purity).

[0039] Example 3. In vitro bactericidal activity and stress resistance of pea peptides and modified pea peptides

[0040] 1. Reagents and instruments

[0041] Pea peptide was provided by Olinbell Technology Co., Ltd., which was secreted through the Bacillus subtilis expression system; LB medium and MH broth medium were purchased from Qingdao Haibo Biotechnology; ampicillin was purchased from Sangon Biotechnology; McFarland turbidimeter tube was purchased from Sinotec; pepsin, trypsin and α-chymotrypsin were purchased from Beijing Solebow Technology Co., Ltd.; pH meter (Shanghai Reitz pHS-3C); microplate reader (BioTek, USA), etc.

[0042] 2. Test strains and test products

[0043] Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Staphylococcus aureus ATCC25923, Helicobacter pylori ATCC43504 and Pasteurella multocida ATCC19427 were all from the American Type Culture Collection (ATCC, USA).

[0044] The test products are the pea peptide provided in Example 1 and the modified pea peptide provided in Example 2.

[0045] 3. Determination of antibacterial effect

[0046] The bactericidal effect of pea peptides and modified pea peptides was visually evaluated by the inhibition zone-Oxford cup method, and the antibacterial effect of pea peptides and modified pea peptides was determined by the minimum inhibitory concentration (MIC). MIC was determined by liquid microdilution method. The specific steps are as follows: 1) Prepare LB solid culture medium, inoculate bacteria on the clean bench after sterilization, and culture in a 37°C constant temperature incubator for 16-24 hours; 2) Prepare MH broth culture medium, pick monoclonal clones on LB solid culture medium and transfer them to MH broth culture medium after sterilization, and culture at 37°C and 220r / min for 8 hours; 3) Take 10mL MH broth culture medium and place it in a sterilized centrifuge tube, dilute it with physiological saline to 0.5 McFarland turbidimeter tube, and the concentration is about 1.5×10 8 CFU / mL; 4) Add freshly prepared sterile MH broth medium and dilute to about 5×105 CFU / mL; 5) Add 180 μL, 5×10 5 CFU / mL of the above solution; 6) Add 20 μL of pea peptide solution and gently blow to mix. 7) Place in a 37°C constant temperature incubator for 16-24 hours, observe the growth of bacteria with the naked eye, and the concentration that does not turn turbid is the minimum inhibitory concentration.

[0047] The results show that the MICs of pea peptides against Escherichia coli ATCC25922, Salmonella typhimurium ATCC14028, Pasteurella ATCC19427, Helicobacter pylori ATCC43504 and Staphylococcus aureus ATCC25923 are 0.25, 0.5, 0.7, 0.6 and 1.4 μg / mL respectively; the MICs of the modified pea peptides obtained in the experimental group are 0.5, 0.5, 0.8, 0.8 and 1.6 μg / mL respectively. The MICs of the modified pea peptides obtained in the control group are 1.6, 0.8, 1.2, 5.0 and 5.0 μg / mL respectively.

[0048] 4. Time-killing curve of pea peptide and modified pea peptide

[0049] The time-kill curve is also one of the bactericidal effects of antimicrobial peptides. By comparing the time and dose, the kinetics of the bactericidal effect of pea peptides was preliminarily shown. The specific steps are as follows: 1) Escherichia coli was used as an indicator bacteria and inoculated on LB solid culture medium. After culturing at 37°C for 24 hours, a single clone was picked and transferred to MH broth medium for 8 hours; 2) The above bacterial solution was diluted to 1×10 6 CFU / mL, take 180μL in a 96-well plate; 3) Take 20μL of pea peptide at concentrations of 0, 0.25, 1 and 10 times the MIC value, add it to a 96-well plate, and set up three replicate wells; 4) Put the 96-well plate into a 37℃ constant temperature incubator for 16h, and at 1, 2, 4, 8 and 16h, detect the OD value of each well, calculate the statistics, and settle the results according to the formula. 5) The formula is as follows: Bactericidal efficiency = (A-A1) / (A1-A2)×100%, where A, A1 and A2 represent the absorbance values ​​of the negative control, different treatment factors and positive control tests at the A520 wavelength of the UV spectrophotometer after the reaction.

[0050] In this experiment, Escherichia coli ATCC25922 was used as the indicator bacteria, and pea peptide or modified pea peptide 1 with 1 times MIC value was selected to detect OD value at different time points (1st, 2nd, 4th, 8th, 16th hour). Figure 2 .Depend on Figure 2 It was found that with the extension of the sterilization time, the sterilization efficiency of pea peptide or modified pea peptide reached a peak at the 6th hour and then began to decline; after falling to the 10th hour, the sterilization efficiency began to level off.

[0051] 5. Determination of stress resistance of pea peptide

[0052] The stress resistance test mainly includes the effects of pH and protease on the antibacterial effect of pea peptides. The specific steps are as follows: 2) Use a pH meter to prepare 6 solutions with pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, take 100 μL of solutions with different pH values ​​in equal volumes and mix them evenly with 40 μg / mL pea peptide, incubate them in a water bath at 37°C for 30 min, and set up three replicates; 4) Prepare pepsin (3000-3500 NFU), trypsin (250 NFU), and α-chymotrypsin (1200 U) solutions, take 100 μL of equal volumes and mix them evenly with 40 μg / mL pea peptide, incubate them in a water bath at 37°C for 30 min, and set up three replicates; 5) Take 20 μL of the treated pea peptide in a 96-well plate, and add 180 μL of the reserved 5×10 5 The bacterial solution was mixed by pipetting, placed in a 37°C constant temperature incubator for 24 hours, and the OD value was detected; 6) The calculation formula is consistent with the time-killing curve calculation formula.

[0053] The results are as follows Figure 3 As shown ( Figure 3 In the figure, "*" indicates that there is a significant difference in the bactericidal efficiency of pea peptide at different pH values, p<0.05). The bactericidal efficiency of pea peptide at different pH values ​​is not as good as that of modified pea peptide 1, indicating that modified pea peptide 1 is more suitable for bactericidal or antibacterial in the acidic environment of the stomach in vivo, and the bactericidal efficiency of modified pea peptide 2 prepared in the control group at different pH values ​​is not significantly improved compared with pea peptide. Figure 4 As shown in the figure, after pea peptide and modified pea peptide 1 were treated with pepsin (Pepsin, 3000-3500NFU), trypsin (Trypsin, 250NFU) and α-chymotrypsin (α-chymotrypsin, 1200U) respectively, the antibacterial activity of pea peptide was significantly reduced, while the modified pea peptide 1 had no obvious reduction, indicating that the modified pea peptide obtained by the above modification method improves the antibacterial property of pea peptide and is more conducive to its antibacterial function in vivo.

[0054] Example 4: In vivo test

[0055] 1. Materials and methods

[0056] (1) Experimental animals and test subjects

[0057] SPF grade Kunming mice were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd., and the experimental animal production license number is SCXK Lu) 2019-0003. The ambient temperature was 22°C ± 2°C, the relative humidity was 50% ± 10%, and the photoperiod was set to 12h automatic light and dark control. The test products were pea peptides and modified pea peptides (modified pea peptides prepared in the experimental group) provided in the above examples, which were diluted with sterile water for gavage of mice.

[0058] (2) Acute toxicity test

[0059] After continuous gavage at a dose of 20 g / kg·bw for 14 days, both sexes of mice were in good condition, behaved normally, and no obvious poisoning symptoms or death were observed. After the test animals were killed and anatomical examinations were performed, no obvious abnormal changes were found in the main organs such as liver, spleen, kidney, heart, lung, stomach and intestine. It is believed that the maximum tolerated dose (MTD) of the test samples for SPF grade Kunming mice is greater than 20 g / kg·bw (LD 50 >20g / kg bw), which is classified as practically non-toxic according to the oral acute toxicity classification standard.

[0060] (3) Establishment of chronic gastritis mouse model and grouping experiment

[0061] The method for establishing a mouse model of chronic gastritis is as follows: 1×10 9 CFU / mL Hp bacterial suspension (Helicobacter pylori, Hp standard strain SSI, China Pharmaceutical and Biological Products Identification Institute) was gavaged into SPF-grade Kunming mice at a dose of 0.2 mL, once every 2 days for 5 consecutive times, and the mice were fasted and deprived of water for 6 hours before and 0.5 hours after gavage. Four weeks after the last gavage of Hp bacterial suspension, the mice were anesthetized and killed, and the gastric antrum tissue was taken for rapid urease test, pathological detection and Hp culture experiment. Gram staining results were red, rapid urease results were positive (phenol red color turned red), and Hp bacterial culture results were positive, which was judged as Hp infection, indicating that the Hp infection model was successfully prepared.

[0062] The group experiment was divided into normal group, model group, experimental group I and experimental group II. The normal group and model group were not gavaged. Experimental group I was gavaged with pea peptide chronic gastritis model mice at a dose of 20g / kg·bw for 14 days. Experimental group II was gavaged with modified pea peptide chronic gastritis model mice at a dose of 20g / kg·bw for 14 days.

[0063] (3) Tissue sample preparation

[0064] After fasting for 8 hours, 15-day-old mice were randomly selected from each replicate in each treatment, and a normal mouse with a weight close to the average weight of each replicate was randomly selected. The duodenum, jejunum, and ileum were separated, and the two ends of each segment were cut off. The middle segment with better quality was divided into two segments: one segment was gently washed with saline to remove the contents, and the middle part of about 3 cm was fixed in 4% paraformaldehyde, transferred to 4°C for paraffin section preparation; the other segment was gently washed with saline to remove the contents and cut longitudinally, and the mucosal tissue was gently scraped and placed in a cryopreservation tube, which was temporarily stored in liquid nitrogen and then transferred to -80°C for determination of the expression of small intestinal nutrient transport carriers. The cecum was separated, and the cecal chyme was gently squeezed out and placed in a sterile cryopreservation tube, which was temporarily stored in liquid nitrogen and then transferred to -80°C for storage.

[0065] (4) Intestinal tissue morphology

[0066] The intestines were separated from the visceral masses of mice in each group, and the contents of the midgut and hindgut, i.e., intestinal chyme, were collected and placed in 1.5 mL cryovials for analysis of intestinal microorganisms and enzyme activity (when scraping intestinal chyme and other contents, care was taken to prevent damage to the intestinal mucosa). Then, the midgut and hindgut intestinal tissues were collected and placed in 1.5 mL centrifuge tubes for intestinal gene fluorescence quantitative expression analysis, quick-frozen in liquid nitrogen, and stored at -80°C before use. A Nikon Ni-E / Ni-U upright fluorescence microscope was used to observe and calculate the intestinal villus height IVH / μm, intestinal villus width IVW / μm, and muscular layer thickness IWT / μm.

[0067] (5) Gastrointestinal hormone level detection

[0068] Before killing the mice, 0.2 mL of eyeball blood was collected under anesthesia and placed in an anticoagulant tube, centrifuged, and the supernatant was collected and stored at -80 ° C. Subsequently, 6 mice were randomly killed, gastric tissue was separated, 0.2 g of gastric tissue was taken, ground into tissue homogenate, centrifuged, and the supernatant was stored at -80 ° C. Enzyme-linked immunosorbent assay was used to detect the levels of motilin (MTL), 5-hydroxylamine (5-HT), gastrin (Gas), somatostatin (SS), and ghrelin in plasma and gastric tissue supernatant. The MTL kit (Shanghai Junshi Biotechnology Co., Ltd.) and other kits (Shanghai Yaji Biotechnology Co., Ltd.) were used, and the operation was strictly carried out according to the instructions of the kit.

[0069] (6) Expression of small intestinal secretory immunoglobulin A (sIgA)

[0070] Small intestinal secretory immunoglobulin A (sIgA) was detected by immunohistochemistry, with goat anti-mouse sIgA monoclonal antibody (PMA04C020258, Perfemiker) as the primary antibody. Optical microscopy was used to qualitatively analyze the small intestinal sIgA positive signals, and Image-Pro Plus 6.0 software was used for semi-quantitative analysis of the mean optical density (Mean of iod).

[0071] (7) Determination of short-chain fatty acids in intestinal chyme

[0072] Weigh 100 mg of freeze-dried intestinal chyme sample, add 1 mL of water to mix, add 200 μL of 50% sulfuric acid, add 50 μL of 1000 mg / L internal standard (cyclohexanone) solution and 1 mL of ether to homogenize for 1 min, centrifuge at 4 ° C, 12000 rpm for 10 min, take the supernatant and measure it on a gas chromatography-mass spectrometer (Japan, Shimadzu, GCMS QP2010-Ultra). Use GCMSSolution software for integration, and use the standard curve to calculate the content to obtain the value of short-chain fatty acids.

[0073] 2. Results

[0074] Table 1 shows the intestinal morphology indexes of each group of mice, where "*" indicates a significant difference relative to the model group (P < 0.05), and "#" indicates a significant difference relative to the normal group (P < 0.05). As shown in Table 1, experimental group II has significant differences relative to both the normal group and the model group, indicating that the modified pea peptide provided by the present invention can improve the intestinal morphology of inflammatory mice.

[0075] Table 1 Intestinal morphology of mice in each group (mean ± standard error)

[0076]

[0077] Table 2 Gastrointestinal hormone levels of mice in each group (mean ± standard error)

[0078] 5-HT (ng / mL) Gas (pg / mL) MTL(pg / mL) SS (pg / mL) Grelin (pg / mL) Normal group 19.03±1.97 33.42±4.16 304.23±13.26 249.37±12.36 149.03±6.32 Model Group 32.25±2.45 53.19±7.05 378.23±26.02 187.03±10.78 98.72±7.84 Experimental Group I 21.03±2.05* 37.25±4.78* 312.18±17.03* 234.25±14.78* 139.36±10.36* Experimental Group II 14.63±1.92*# 25.84±6.09*# 268.52±20.24*# 256.92±16.33*# 152.84±9.09*#

[0079] Table 2 shows the gastrointestinal hormone levels of mice in each group, where "*" indicates a significant difference relative to the model group (P < 0.05), and "#" indicates a significant difference relative to the normal group (P < 0.05). As shown in Table 2, experimental group II has significant differences relative to both the normal group and the model group. Hp infection can affect the host's gastrointestinal motility and gastrointestinal hormone secretion. This study found that the levels of 5-HT, Gas, and MTL in the peripheral blood and gastric tissue of Hp-infected mice were increased, while the levels of SS and Grelin were decreased. 5-HT can participate in the regulation of the digestive system, and most of the 5-HT synthesis and secretion comes from the digestive tract. Gas is a gastrointestinal hormone secreted by gastric mucosal G cells that stimulates gastric acid secretion and pepsin secretion, which can promote cell growth function. Hp virulence protein can activate the Gas promoter and promote Gas secretion. SS can inhibit the secretion of gastric acid and pepsin in a paracrine manner, and also has an inhibitory effect on the release of Gas, affecting the function of the gastrointestinal tract and playing a protective role in the gastric mucosa. MTL is involved in gastrointestinal motility, gastrointestinal water and electrolyte transport, Grelin plays an important role in appetite regulation, gastric emptying, and gastric acid secretion. The expression level of Grelin in the gastric mucosa of Hp infected people is reduced, and the degree of inhibition is related to the severity of gastric mucosal damage. The continuous reduction of Grelin levels can cause cellular immune response and chronic active gastritis. The results of this study show that the pea peptide and modified pea peptide provided by the present invention can regulate gastrointestinal hormone disorders caused by Hp infection.

[0080] like Figure 5 As shown, "*" indicates that the experimental group II has a significant difference compared with the model group (P < 0.05), which shows that oral administration of modified pea peptides to Hp model mice can significantly increase the expression of sIgA in the duodenum, ileum and jejunum, and has a remarkable effect on the intestinal health of the jejunum.

[0081] like Figure 6As shown, "*" indicates that the experimental group II has significant differences compared with the model group and the normal group (P < 0.05). This shows that oral administration of modified pea peptides to Hp model mice can reduce the content of short-chain fatty acids in the intestines of mice. Short-chain fatty acids regulate the physiological state of the host by lowering the pH value of the stomach and intestines of fish and inhibiting the growth of Gram-negative bacteria through acid dissociation and the production of anions in bacterial cells. Short-chain fatty acids are produced by anaerobic microbial fermentation of complex carbohydrates that are not digested and absorbed in the small intestine and enter the large intestine, of which acetic acid, propionic acid and butyric acid account for up to 85%. Short-chain fatty acids SCFAs have important physiological functions such as regulating intestinal immune function and reducing intestinal permeability, and are one of the important sources of energy for intestinal epithelial cells and liver. Butyric acid has inhibitory activity on many mutant substances and is a strong anti-cancer substance in the intestine. Butyric acid is the preferred energy substance for colon cells and is related to the regulation of cell apoptosis and cell differentiation. It can be seen that the modified pea peptide provided by the present invention can not only increase the content of short-chain fatty acids in the intestine of mice, but also improve intestinal immune function, reduce intestinal permeability, regulate intestinal cell apoptosis and differentiation, and thus regulate intestinal health.

[0082] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a modified pea peptide, characterized in that: The method comprises reacting a pea peptide as shown in SEQ ID NO.1 and monomethoxy polyethylene glycol-butyraldehyde in a 100 mM pH 5.0 acetic acid-sodium acetate buffer at a mass ratio of 1:1 for 40 to 60 minutes, and terminating the reaction with ethanolamine; wherein the pea peptide is derived from the amino acid sequence of the pea (Pisum sativum Linn.) hypothetical protein KIW84_055578, and the molecular weight of the monomethoxy polyethylene glycol-butyraldehyde is MW5000.

2. The modified pea peptide obtained by the preparation method according to claim 1.

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