A bioactive peptide PCT-2 and its preparation method and application

Abstract

The present invention discloses a bioactive peptide PCT-2 and its preparation method and application, belonging to the field of biotechnology. The amino acid sequence of the bioactive peptide PCT-2 is MTCEDR, as shown in the sequence table SEQ ID NO: 6, and has antioxidant and intestinal flora regulating functions. The bioactive peptide PCT-2 is extracted from spirulina protein peptide, which can significantly increase the activity of SOD and GSH-Px in serum, significantly reduce the MDA content in serum, and its in vivo antioxidant activity is equivalent to that of the positive control Trolox. At the same time, compared with the blank control group, the bioactive peptide PCT-2 can also reduce the α diversity index of the intestinal flora, change the overall structure of the intestinal flora, regulate the abundance of each classification level of the intestinal flora, and significantly increase the content of acetic acid and butyric acid, which can be applied to antioxidant and intestinal flora regulating functional foods.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide and a preparation method and application thereof, and in particular to a small molecule bioactive peptide PCT-2 with antioxidant and intestinal flora regulating functions and a preparation method thereof and application thereof in antioxidant and intestinal flora regulating functional foods, belonging to the field of biotechnology. Background Art

[0002] Human intestinal microorganisms mainly include bacteria, yeast and parasites, which carry more than 100 times the number of human genes and are closely related to obesity, hypertension, intestinal inflammation and cardiovascular diseases. They are helpful in regulating intestinal flora and are written into the "Catalogue of Health Functions Allowed for Health Food Claims". The common way to regulate intestinal flora is to supplement probiotics; in addition, bioactive peptides have also been found to have intestinal flora regulation. Chen Ge described the regulatory effect of sturgeon peptide LLLE on the intestinal flora of obese mice in the paper "Study on the mechanism of sturgeon peptide LLLE improving chlorpyrifos-induced obesity in high-fat diet mice"; Hu Xuejia described the regulatory effect of red lady fish meat antihypertensive peptide on the intestinal flora of hypertensive mice in the paper "Preparation of red lady fish meat antihypertensive peptide and study of its antihypertensive mechanism"; Siqing Fan et al. described the intestinal flora regulation effect of hexapeptide GPAGPR and hexapeptide GPSGRP in the article "Novel anti-hyperuricemic hexapeptides derived from Apostichopus japonicus hydrolysateand their modulation effects on the gut microbiota and host microRNA profile". The bioactive peptides with intestinal flora regulating function disclosed in the above-mentioned documents have diverse structures and no obvious sequence structure regularity.

[0003] Free radicals and the body's oxidative balance are also closely related to human health. Once imbalanced, it can lead to aging, cancer, cardiovascular disease, Alzheimer's disease, etc. The human body has a good mechanism for maintaining the dynamic balance of free radicals, which can play a role through antioxidant enzymes, chelating metal ions and scavenging free radicals. If the imbalance of free radicals in the human body exceeds the self-regulation level, exogenous antioxidants need to be supplemented. Traditional antioxidants are generally derived from chemical synthesis, and have disadvantages such as poor stability and strong toxic side effects. Antioxidant peptides derived from protein hydrolysis have the advantages of safety, non-toxicity, strong stability, etc., which are more in line with consumer demand. Wu Yuanyuan et al. reviewed the discovery and optimization of the preparation process of hairtail antioxidant peptides in "Progress in the Enzymatic Preparation and Functional Properties of Hairtail Bioactive Peptides"; Meng Le et al. described the preparation of antioxidant peptides using thick-shelled mussels as raw materials and antioxidant activity as screening indicators, using enzymatic hydrolysis technology and modern separation and purification technology; Zuo Yijin described in detail the antioxidant activity of tilapia skin enzymatic hydrolysate in the paper "The Effect of the Separation and Purification Process of Tilapia Skin Antioxidant Peptides on Their Structure and Activity", as well as the core functional antioxidant peptide components of the decapeptide PGIIGLPGPA, octapeptide AVGPVGPS and octapeptide ERGPPGPP. The bioactive peptides with antioxidant functions disclosed in the above literature have diverse structures and no obvious sequence structure rules.

[0004] In the article "Research Progress on Nutritional and Health Properties of Spirulina Bioactive Peptides", Liu Jing et al. reviewed the activities of Spirulina in anti-oxidation, anti-inflammation, regulating blood lipid and blood sugar metabolism, lowering blood pressure and anti-cancer. In the article "Process Optimization of Preparation of Antioxidant Active Peptides from Spirulina by Alkaline Protease Hydrolysis", Ma Yanfang et al. described the process of optimizing the preparation of antioxidant peptides using Spirulina protein as raw material, but did not report on the specific antioxidant peptide components and the regulatory effects on intestinal flora. Summary of the invention

[0005] The object of the present invention is to provide a small molecule active peptide screened from spirulina protein peptides, having antioxidant and intestinal flora regulating functions, and can be applied to antioxidant and intestinal flora regulating functional foods.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A bioactive peptide PCT-2, the amino acid sequence of the bioactive peptide PCT-2 is MTCEDR, as shown in the sequence table SEQ ID NO: 6, and has antioxidant and intestinal flora regulating functions.

[0008] The application of the aforementioned bioactive peptide PCT-2 in antioxidant and intestinal flora regulating functional foods.

[0009] A method for preparing the aforementioned bioactive peptide PCT-2 is a solid phase synthesis method, specifically:

[0010] The solid phase synthesis was carried out by using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier and adopting the Fmoc solid phase synthesis strategy.

[0011] Another method for preparing the aforementioned bioactive peptide PCT-2 is to use an enzymatic method, specifically:

[0012] (1) taking spirulina protein, adding water of 9 times the mass of the spirulina protein, adding a composite protease at an enzyme-substrate ratio of 0.5% for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 55° C., the pH value is 8.0, the enzymatic hydrolysis time is 6 h, and an enzymatic hydrolysis product is obtained, wherein the composite protease is a mixture of alkaline protease and neutral protease at a mass ratio of 1:1;

[0013] (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through eight layers of gauze to remove the residue and obtain the spirulina protein peptide enzymatic hydrolysate;

[0014] (3) freeze-drying the above-mentioned spirulina protein peptide hydrolysate to obtain spirulina protein peptide powder, wherein the spirulina protein peptide powder contains a large amount of bioactive peptide PCT-2.

[0015] The benefits of the present invention are as follows: the bioactive peptide PCT-2 screened from spirulina protein peptides can significantly increase the activities of SOD and GSH-Px in serum, significantly reduce the MDA content in serum, and its in vivo antioxidant activity is equivalent to that of the positive control Trolox; at the same time, compared with the blank control group, PCT-2 treatment can reduce the α diversity index of the intestinal flora, change the overall structure of the intestinal flora (β diversity index), regulate the abundance of each classification level of the intestinal flora, and significantly increase the content of acetic acid and butyric acid; the bioactive peptide PCT-2 can be applied to antioxidant and intestinal flora regulating functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the binding mode of the bioactive peptide PCT-2 and Keap1;

[0017] Figure 2 yes Figure 1 A partial enlarged schematic diagram of

[0018] Figure 3 The figure is the effect of bioactive peptide PCT-2 treatment on the physiological indicators of mice, among which A is the effect on the weight gain of mice, B is the effect on the spleen coefficient of mice, and C is the effect on the liver coefficient of mice. * indicates P<0.05;

[0019] Figure 4: is the effect of bioactive peptide PCT-2 treatment on mouse serum antioxidant indexes, where A is the effect on mouse serum MDA content, B is the effect on mouse serum SOD activity, C is the effect on mouse serum CAT activity, and D is the effect on mouse serum GSH-Px activity. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001;

[0020] Figure 5 The bioactive peptide PCT-2 treatment in mice keap1 Effects of gene transcription levels on the mouse brain keap1 Figure 2. Effects on gene transcription levels. (B) on mouse liver keap1 Effect diagram of gene transcription level, * indicates P < 0.05, ** indicates P < 0.01;

[0021] Figure 6 This is a graph showing the effect of bioactive peptide PCT-2 treatment on the β-diversity of intestinal flora in mice;

[0022] Figure 7 The following are the effects of bioactive peptide PCT-2 treatment on the content of short-chain fatty acids in the intestine of mice, where A is the effect on the content of acetic acid in the intestine of mice, B is the effect on the content of propionic acid in the intestine of mice, and C is the effect on the content of butyric acid in the intestine of mice. ** indicates P<0.01, and *** indicates P<0.001. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0024] 1. Preparation of Spirulina Protein Peptide Samples

[0025] The spirulina was washed, dried, crushed and sieved in sequence to obtain spirulina powder. Ice was made with distilled water, and the ice was crushed into 20-40 meshes to obtain ice powder. Spirulina powder and ice powder were mixed in a mass ratio of 1:10, and the mixture was placed in a stirring tank and subjected to a wall-breaking treatment for 100 minutes at 1000rpm and 0°C. After the ice powder melted naturally, a wall-breaking liquid (containing spirulina protein) was obtained. The wall-breaking liquid was centrifuged to remove the solid residue, and the supernatant was filtered with a filter cloth with a pore size of 25μm and a filter membrane with a pore size of 0.45μm in turn to obtain a crude extract of spirulina protein. The crude extract of spirulina protein was ultrafiltered and concentrated using ultrafiltration membranes with molecular weight cutoffs of 5kDa and 10kDa, respectively, and the ultrafiltration fractions with a molecular weight range of 5kD to 10kD were collected and spray-dried to obtain spirulina protein.

[0026] Take spirulina protein, add water 9 times the mass of spirulina protein (the mass concentration of spirulina protein is 10%), add composite protease (mixed by alkaline protease and neutral protease in a mass ratio of 1:1) at an enzyme-substrate ratio of 0.5% for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 55°C, the pH value is 8.0, and the enzymatic hydrolysis time is 6 hours.

[0027] After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered with eight layers of gauze to remove the residue and obtain the spirulina protein peptide enzymatic hydrolysate.

[0028] The spirulina protein peptide enzymatic hydrolyzate is freeze-dried to obtain spirulina protein peptide powder.

[0029] 2. Obtaining the polypeptide sequence of Spirulina protein peptide

[0030] The Spirulina protein peptides obtained above were determined by LC-MS / MS, and the determination results were analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0031] The LC-MS / MS assay conditions were:

[0032] (1) In the liquid phase method: the chromatographic column is C18, 3μm, 250mm×75μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300nL / min, the injection volume is 4μL, and the chromatographic gradient is 60min. The specific elution gradient is: 0-48min, phase A is uniformly reduced from 95% to 60%; 48-55min, phase A is uniformly reduced from 60% to 30%; 55-56min, phase A is uniformly reduced from 30% to 0; 56-60min, maintain 0% phase A;

[0033] (2) Mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120,000, AGC setting of 300, scanning range of 200-1600 m / z. MIPS mode of peptide, valence states of 1-5, secondary resolution of 15,000, separation window of 1.6 m / z.

[0034] 3. Screening peak area ≥ 2×10 7 Active peptides with amino acid number ≤ 6

[0035] From the several peptide sequences obtained above, 17 peptide sequences with peak areas ≥2×10 7 The screening results of active peptides with amino acid number ≤ 6 are shown in Table 1.

[0036] Table 1 Peak area ≥2×10 in Spirulina protein peptides 7 Active peptide sequence with amino acid number ≤ 6

[0037]

[0038] 4. Screening of active peptides with strong binding ability to Keap1

[0039] The 17 peptide sequences in Table 1 were docked with Keap1 using Discovery Studio software. Before docking, the 2D structure of the peptide was converted into a 3D structure by energy minimization, and peptide sequences with strong binding ability with Keap1 were screened. The 3D structure of protein Keap1 can be downloaded from the RCSB protein database (PDB ID: 4IFJ). The docking results are expressed as docking scores (-CiE). The larger the -CiE value, the stronger the interaction between the peptide and Keap1.

[0040] The molecular docking results of 17 peptide sequences are shown in Table 2.

[0041] Table 2 Prediction results of the interaction between Spirulina protein peptide components and Keap1

[0042]

[0043] 5. Molecular docking analysis

[0044] In all peaks with an area ≥ 5 × 10 7 Among the active peptides, the hexapeptide MTCEDR (denoted as PCT-2, SEQ ID NO: 6) had the largest -CiE (88.7351 kcal / mol) when docked with Keap1. Therefore, the hexapeptide MTCEDR was selected for further prediction analysis.

[0045] After analysis, the binding mode of the hexapeptide MTCEDR and Keap1 is as follows Figure 1 and Figure 2 The molecular docking situation is as follows:

[0046] There are 9 HH bond interactions, 2 CH bond interactions and 3 electrostatic interactions between the hexapeptide MTCEDR and Keap1, and 9 amino acid residues are involved in the interaction between the hexapeptide MTCEDR and Keap1.

[0047] VI. Evaluation of the Antioxidant Function of Hexapeptide MTCEDR

[0048] 1. Solid phase synthesis of hexapeptide MTCEDR

[0049] The hexapeptide MTCEDR, namely PCT-2, was synthesized in the solid phase by adopting the Fmoc solid phase synthesis strategy, using Fmoc protected amino acids as raw materials and polystyrene resin as a solid phase carrier.

[0050] 2. Animal grouping and active peptide treatment

[0051] Positive control: 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox).

[0052] Eight-week-old male ICR mice were selected, and the temperature was 20℃-26℃, the air humidity was 50%-60%, the light and dark were alternating for 12 hours, and food and water were freely available. After 7 days of adaptive feeding, they were randomly divided into groups (control group, Trolox group, PCT-2 group), with 12 mice in each group. Among them, the control group was gavaged with normal saline once a day, the Trolox group was gavaged with Trolox (dissolved in normal saline, the concentration was 1 mg / mL) once a day, and the PCT-2 group was gavaged with solid-phase synthesized PCT-2 (dissolved in normal saline, the concentration was 1 mg / mL) once a day, with a gavage volume of 100μL / 10g, for 30 consecutive days. During the experiment, each group had free access to food and water.

[0053] 3. Detection of the effects of PCT-2 treatment on physiological indicators and serum antioxidant indicators of mice

[0054] After the experimental period, all mice were anesthetized with enflurane, and blood was collected from the orbital plexus, incubated in a 37°C water bath for 10 min, and centrifuged at 4°C and 3000 rpm for 15 min to separate the upper serum, which was then stored in a −80°C refrigerator for later use.

[0055] The mice were killed by cervical dislocation and their spleen, liver and brain were quickly separated, weighed and stored in a -80°C refrigerator for later use.

[0056] The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) in serum were detected using corresponding kits.

[0057] Effects of PCT-2 treatment on physiological parameters of mice (weight gain, spleen coefficient, liver coefficient) are shown in Figure 3 The effects on serum antioxidant indexes (MDA content, SOD activity, CAT activity, GSH-Px activity) of mice are shown in Figure 4 .

[0058] Depend on Figure 3 It can be seen that: compared with the blank control group, Trolox positive treatment only significantly increased the spleen coefficient of mice (p<0.05) but had no significant effect on the weight gain and liver coefficient of mice (p>0.05), PCT-2 treatment had no significant effect on the weight gain, spleen coefficient and liver coefficient of mice (p>0.05); compared with Trolox positive treatment, PCT-2 treatment had no significant effect on the weight gain, spleen coefficient and liver coefficient of mice (p>0.05).

[0059] Depend on Figure 4 It can be seen that compared with the control group, Trolox positive treatment significantly increased the activities of SOD (p<0.01), CAT (p<0.05) and GSH-Px (p<0.05) in mouse serum and significantly reduced the content of MDA (p<0.05) in serum. PCT-2 treatment significantly increased the activities of SOD (p<0.001) and GSH-Px (p<0.01) in mouse serum and significantly reduced the content of MDA (p<0.01) in serum, but had no effect on the activity of CAT in serum (p>0.05). Compared with Trolox positive treatment, PCT-2 treatment had no significant effect on the content of MDA, SOD activity, CAT activity and GSH-Px activity in mouse serum (p>0.05).

[0060] 4. Real-time fluorescence quantitative PCR detection

[0061] (1) Real-time fluorescence quantitative PCR primer design

[0062] Keap1 protein was selected, and real-time fluorescence quantitative PCR (qRT-PCR) primers were designed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) and Primer 5 software from Premier Company of Canada. The primer information is shown in Table 3.

[0063] Table 3 Primer sequences for real-time fluorescence quantitative PCR

[0064]

[0065] (2) Total RNA extraction from tissues

[0066] 100 mg of mouse brain tissue and liver tissue were taken in a sterile mortar precooled with liquid nitrogen, and liquid nitrogen was added for rapid and thorough grinding. Then 1 mL of TrasZol Up reagent was added, and the mixture was transferred to a 1.5 mL centrifuge tube without DNase, and 0.2 mL of chloroform was added. The mixture was fully vortexed for 30 seconds, allowed to stand for 10 minutes, and then centrifuged at 4°C and 12000×g for 15 minutes. The supernatant was taken to another 1.5 mL centrifuge tube without RNase, and 0.5 mL of isopropanol was added. The liquid in the tube was gently mixed, allowed to stand at room temperature for 10 minutes, and then centrifuged again at 4°C and 12000×g for 10 minutes. The supernatant was discarded, and 1 mL of 75% ethanol prepared with RNase-free water was added to the precipitate. The precipitate was gently washed, and centrifuged at 4°C and 7500×g for 5 minutes. The supernatant was discarded, and the centrifuge tube cap was opened to evaporate the ethanol. Then 100 μL of RNase-free water was added, and the total RNA concentration and quality of the sample were quickly determined using Nano Drop 2000c. The RNA was aliquoted into RNase-free centrifuge tubes and stored at -80°C.

[0067] (3) cDNA template synthesis

[0068] Take 1 µg of total RNA and synthesize cDNA using the TransScript All-in-One First-Strand cDNA SynthesisSuperMix for qPCR (One-Step gDNA Removal) Kit.

[0069] (4) Gene expression detection

[0070] Preparation of qRT-PCR system: After appropriately diluting the cDNA template, take 5 μL and add it to the total system. Take 0.4 μL of forward primer and reverse primer respectively and add them to the total system. Then take 10 μL of 2×TransStartTM Green qPCR SuperMix (containing SYBR Green dye) and 4.2 μL of enzyme-free water and add them to the total system. The total system is 20 μL.

[0071] The qRT-PCR three-step method was as follows: denaturation at 94°C for 10 min; amplification reaction was performed for 40 cycles, including denaturation at 94°C for 15 s, annealing at 50°C for 15 s, and extension at 72°C for 45 s; and extension at 72°C for 10 min.

[0072] Effects of PCT-2 treatment on the transcriptional level of keap1 gene in mouse brain and liver are shown in Figure 5 .

[0073] Depend on Figure 5 It can be seen that compared with the blank control group, Trolox positive treatment significantly downregulated the transcription level of keap1 gene in the mouse brain and liver, while PCT-2 treatment only significantly downregulated the transcription level of keap1 gene in the mouse brain, and the transcription level of keap1 gene in the liver had no significant difference from the blank control group (p>0.05).

[0074] VII. Evaluation of the intestinal flora regulatory function of hexapeptide MTCEDR

[0075] 1. Animal grouping and active peptide treatment

[0076] Eight-week-old male ICR mice were selected, and the temperature was 20℃-26℃, the air humidity was 50%-60%, the light and dark were alternating for 12 hours, and food and water were freely available. After 7 days of adaptive feeding, they were randomly divided into two groups (control group and PCT-2 group), with 12 mice in each group. Among them, the control group was gavaged with normal saline once a day, and the PCT-2 group was gavaged with the previously solid-phase synthesized PCT-2 (dissolved in normal saline, concentration of 1 mg / mL) once a day, with a gavage volume of 100μL / 10g, for 30 consecutive days. During the experiment, each group had free access to food and water.

[0077] 2. Detection of the effect of PCT-2 treatment on the intestinal flora of mice

[0078] After the experimental period, the mouse feces were collected and stored in a -80°C refrigerator for future use.

[0079] (1) Sequencing of bacterial flora in mouse feces

[0080] The total DNA from mouse feces was extracted using the QIAamp DNA Stool Mini extraction kit and stored in a -20°C refrigerator until use.

[0081] DNA concentration was determined using a Thermo NanoDrop 2000 UV-Vis.

[0082] PCR primers were designed based on the highly variable region of the 16S rRNA gene, wherein the nucleotide sequence of the forward primer was: 5′-ACTCCTACGGGAGGCAGCAG-3′, and the nucleotide sequence of the reverse primer was: 5′-GGACTACHVGGGTWTCTAAT-3′.

[0083] PCR amplification was performed, and the PCR amplification reaction system was 25µL, including: 2µL of genomic DNA with a concentration of 10 ng / ul, 2µL of forward primer with a concentration of 0.1 μM, 2µL of reverse primer with a concentration of 0.1 μM, 12.5μL of premix, and 25µL of ddH2O. The specific PCR amplification program for this round was: denaturation at 95℃ for 5min; denaturation at 95℃ for 30s, annealing at 54℃ for 30s, extension at 72℃ for 40s, 20 cycles; extension at 72℃ for 5min. After the amplification, the PCR products obtained by PCR amplification were detected by agarose gel electrophoresis and recovered by gel excision. After accurate quantification, they were sequenced on the Illumina Miseq platform.

[0084] The original FASTQ files were processed using QIIME 1.8.0 as follows:

[0085] (i) FLASH 1.2.11 was used to splice paired reads into one sequence based on the overlap relationship between PE reads, and the sequences that could not be merged were deleted;

[0086] (ii) remove the barcode after identifying the sample source of the data;

[0087] (iii) using Prinseq software, we removed fragments shorter than 50 bp and bases with a quality value of less than 20 at the tail of reads, and finally filtered low-complexity sequences to remove non-amplified regions;

[0088] (iv) The cluster algorithm (Mothur 1.36.0) was used to correct sequencing errors and remove chimeras in the sequences (Uchime 4.2.40).

[0089] The alpha diversity of intestinal flora was analyzed by Mothur 1.36.0, and principal coordinate analysis was performed by Muscle 3.3.31.

[0090] RDP classifier software was used to perform species annotation and community change analysis on the processed sequences.

[0091] The effects of PCT-2 treatment on the α-diversity of intestinal flora are shown in Table 4.

[0092] Table 4 Effects of PCT-2 treatment on α-diversity of intestinal flora

[0093]

[0094] As shown in Table 4, compared with the control group, PCT-2 treatment reduced the ACE index, Chao index, Shannon index and Simpson index of the intestinal flora in mice.

[0095] The effect of PCT-2 treatment on the overall composition of the intestinal flora of mice was analyzed by weighted principal coordinates. Figure 6 .

[0096] Depend on Figure 6 It can be seen that PCT-2 treatment will change the overall structure (β diversity) of the intestinal flora in mice to a certain extent.

[0097] At the phylum level, the effects of PCT-2 treatment on the composition of the intestinal flora in mice are shown in Table 5 .

[0098] Table 5 Effects of PCT-2 treatment on the composition of intestinal flora in mice at the phylum level

[0099]

[0100] As shown in Table 5, compared with the blank control group, PCT-2 treatment increased the abundance of Bacteroidota, Firmicutes and Patescibacteria in the intestine of mice, and reduced the abundance of Campilobacterota, Actinobacteriota and Desulfobacterota It can be seen that PCT-2 treatment has an effect on the composition of the mouse intestinal flora at the phylum level.

[0101] At the family level, the effects of PCT-2 treatment on the composition of the intestinal flora in mice are shown in Table 6 .

[0102] Table 6 Effects of PCT-2 treatment on the composition of intestinal flora in mice at the family level

[0103]

[0104] As shown in Table 6, compared with the control group, PCT-2 treatment increased the abundance of Muribaculaceae, Lactobacillaceae, Lachnospiraceae, Bacteroidaceae and Erysipelotrichaceae in the intestine of mice, and reduced the abundance of Bacillaceae, norank_o__Clostridia_UCG-014, Helicobacteraceae, Prevotellaceae and Bifidobacteriaceae. It can be seen that PCT-2 treatment has an effect on the composition of the intestinal flora of mice at the family level.

[0105] The differentially expressed ASVs in the intestinal flora after PCT-2 treatment were analyzed (p<0.05). The analysis results are shown in Table 7.

[0106] Table 7 Analysis results of differentially expressed ASVs after PCT-2 treatment

[0107]

[0108]

[0109] As shown in Table 7, compared with the control group, PCT-2 treatment upregulated the abundance of 23 ASVs and downregulated the abundance of 26 ASVs. It can be seen that PCT-2 treatment has an effect on the expression of ASVs.

[0110] (2) Analysis of short-chain fatty acids in mouse feces

[0111] Weigh 30 mg of mouse dry feces, add 0.8 mL of water, shake and mix, then add 0.2 mL of 50% sulfuric acid, mix and add 1 mL of ether, mix thoroughly for 30 min, centrifuge at 10000 rpm for 10 min, add calcium chloride to the supernatant to absorb the water, filter with 0.22 μm filter membrane, and take 1 mL of filtrate for sample. Using the external standard method, standard curves were made for acetic acid, propionic acid and butyric acid, respectively. GC-MS (Agilent 7890 gas phase combined with general analysis M7-80E mass spectrometer) was used, the detector was a hydrogen flame detector (FID), the chromatographic column was DB-WAX (30m×0.25mm×0.25μm), the carrier gas was helium, the split ratio was 20:1, the flow rate was 1mL / min, the injection volume was 1μL, the initial temperature of the column oven was 90℃, increased to 150℃ at a rate of 12℃ / min, and then increased to 220℃ at a rate of 20℃ / min, and maintained at 220℃ for 4.5min. The temperatures of the transmission line and ion source were set to 220℃ and 230℃, respectively, and the solvent delay time was 3min.

[0112] After PCT-2 treatment, the content of acetic acid, propionic acid and butyric acid in the intestine of mice was detected. Figure 7 .

[0113] Depend on Figure 7 It can be seen that compared with the control group, PCT-2 treatment can significantly increase the acetic acid content (p<0.01) and butyric acid content (p<0.001) in mouse feces, but has no effect on the propionic acid content.

[0114] In summary, the hexapeptide MTCEDR (PCT-2) screened from spirulina protein peptides in the present invention has antioxidant and intestinal flora regulating functions and can be applied to antioxidant and intestinal flora regulating functional foods.

[0115] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A bioactive peptide PCT-2, characterized in that: The amino acid sequence of the bioactive peptide PCT-2 is MTCEDR, as shown in SEQ ID NO: 6 in the sequence table, and has antioxidant and intestinal flora regulating functions.

2. Use of the bioactive peptide PCT-2 according to claim 1 in the preparation of antioxidant and intestinal flora regulating functional foods.

3. The method for preparing the bioactive peptide PCT-2 according to claim 1, characterized in that: The solid phase synthesis method is adopted, specifically: The solid phase synthesis was carried out by using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier and adopting the Fmoc solid phase synthesis strategy.

4. The method for preparing the bioactive peptide PCT-2 according to claim 1, characterized in that: Enzymatic hydrolysis is used, specifically: (1) taking spirulina protein, adding water of 9 times the mass of the spirulina protein, adding a composite protease at an enzyme-substrate ratio of 0.5% for enzymatic hydrolysis, the enzymatic hydrolysis temperature is 55° C., the pH value is 8.0, the enzymatic hydrolysis time is 6 h, and an enzymatic hydrolysis product is obtained, wherein the composite protease is a mixture of alkaline protease and neutral protease at a mass ratio of 1:1; (2) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is filtered through eight layers of gauze to remove the residue and obtain the spirulina protein peptide enzymatic hydrolysate; (3) freeze-drying the above-mentioned spirulina protein peptide hydrolysate to obtain spirulina protein peptide powder, which contains a large amount of bioactive peptide PCT-2.

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