Bioactive peptide PCT-1, preparation method therefor, and use thereof

Abstract

A bioactive peptide PCT-1, a preparation method therefor, and a use thereof, relating to the technical field of biology. The amino acid sequence of the bioactive peptide PCT-1 is LNRTFE, as shown in SEQ ID NO: 1 in a sequence listing, and the bioactive peptide PCT-1 has antioxidant and intestinal flora regulation functions. The bioactive peptide PCT-1 is extracted from a spirulina protein peptide and can significantly improve the activity of SOD and GSH-Px in serum and significantly reduce the content of MDA in the serum. The in vivo antioxidant activity of the bioactive peptide is equivalent to that of a positive control Trolox. Moreover, compared with a blank control group, the bioactive peptide PCT-1 can also reduce the α diversity index of the intestinal flora, change the overall structure of the intestinal flora, regulate the abundance of intestinal flora at various taxonomic levels, and significantly increase the content of acetic acid and butyric acid, and can be applied to foods having antioxidant and intestinal flora regulation functions.

Description

A bioactive peptide PCT-1, its preparation method and application Technical Field

[0001] This invention relates to a small molecule peptide, its preparation method, and its application, specifically to a small molecule bioactive peptide PCT-1 with antioxidant and gut microbiota regulation functions, its preparation method, and its application in foods with antioxidant and gut microbiota regulation functions, belonging to the field of biotechnology. Background Technology

[0002] Obesity, aging, and various diseases such as cancer, cardiovascular disease, and Alzheimer's disease have been proven to be closely related to the excessive production of free radicals. The normal body can regulate oxidative balance through various antioxidant mechanisms, including scavenging free radicals to block their chain reactions, chelating metal ions to inhibit free radical production, and regulating endogenous glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) to eliminate free radicals. When endogenous oxidative balance is disrupted, exogenous antioxidants are needed. Traditional antioxidants are generally chemically synthesized, which has disadvantages such as poor stability and strong toxicity. In contrast, antioxidant peptides derived from protein hydrolysis have advantages such as safety, non-toxicity, and high stability, making them more suitable for consumer demands.

[0003] The human gut contains approximately 1,000 species and 100 trillion microorganisms, mainly including bacteria, yeast, and parasites. These gut microbes carry more than 100 times the number of genes in the human body, earning them the title of "invisible organ." Gut microbiota imbalance is closely related to obesity, hypertension, intestinal inflammation, and cardiovascular disease, and regulating gut microbiota is one of the functions permitted for health supplement claims in the "List of Permitted Health Functions for Health Foods (Non-Nutritional Supplements, 2023 Edition)." Besides directly supplementing with probiotics, bioactive peptides have also been reported to have gut microbiota-regulating functions. Technical issues

[0004] In her graduation thesis, "The Influence of the Isolation and Purification Process of Antioxidant Peptides from Tilapia Skin on Their Structure and Activity," Zuo Yijin described in detail the antioxidant activity of tilapia skin enzymatic hydrolysate, as well as its core functional antioxidant peptide components, including the decapeptide PGIIGLPGPA, octapeptide AVGPVGPS, and octapeptide ERGPPGPP. Wang Yiju, in her graduation thesis, "Isolation, Identification, and Functional Verification of Active Peptides from Antarctic Krill," described the process of preparing antioxidant peptides from Antarctic krill, demonstrating that the strong antioxidant components are mainly enriched in low molecular weight peptides, and identifying the heptapeptide APGELPY, hexapeptide DIFDPL, and hexapeptide LDVAPL as having good antioxidant effects. Yu Hui, in "Isolation and Identification of Antioxidant Peptides from Enzymatic Hydrolysate of Meaty Crab By-products," described the evaluation of the antioxidant activity of trypsin hydrolysate of meaty crab by-products, and isolated two antioxidant peptides with strong ABTS+ scavenging ability: the tripeptide YEG and the dipeptide YE. Furthermore, Feng Ziqi et al., in their article "The Effects of Thick-Shelled Mussel Active Peptides on Alcohol-Induced Liver Injury and Gut Microbiota in Mice," described the regulatory effect of thick-shelled mussel active peptides on the gut microbiota of mice with alcohol-induced liver injury. Chen Xin'ai, in her graduation thesis "Study on the Effects of Giant Salamander Active Peptides on Gut Microbiota," specifically studied the regulatory effects of giant salamander active peptides on the gut microbiota structure and metabolism in high-fat diet-induced obese mice. Han Mengyao, in her graduation thesis "Preparation and Activity Study of Largemouth Bass Skin Collagen Peptides," mentioned the regulatory effect of largemouth bass skin collagen peptides on the gut microbiota of immunocompromised mice. Further, Jiaojiao Han et al., in their article "The novel peptides ICRD and LCGEC screened from tuna roe show antioxidative activity via Keap1 / Nrf2-ARE pathway regulation and gut microbiota modulation," described the gut microbiota regulatory effects of the antioxidant peptides tetrapeptide ICRD and pentapeptide LCGEC. From the structures of bioactive peptides disclosed in the above literature, we can find that bioactive peptides with antioxidant and gut microbiota regulation functions have diverse structures and no obvious sequence structure regularity. Technical solutions

[0005] The purpose of this invention is to provide a small molecule active peptide screened from spirulina protein peptides, which has antioxidant and gut microbiota regulation functions and can be applied to foods with antioxidant and gut microbiota regulation functions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A bioactive peptide PCT-1, the amino acid sequence of which is LNRTFE, as shown in SEQ ID NO: 1 of the sequence listing, has antioxidant and gut microbiota regulation functions.

[0008] The aforementioned bioactive peptide PCT-1 is used in functional foods that have antioxidant and gut microbiota regulation properties.

[0009] One method for preparing the aforementioned bioactive peptide PCT-1 employs a solid-phase synthesis method, specifically:

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

[0011] Another method for preparing the aforementioned bioactive peptide PCT-1 involves enzymatic hydrolysis, specifically:

[0012] (1) Take spirulina protein, add water at a mass ratio of 9 times that of spirulina protein, add compound protease at a ratio of 0.5% for enzymatic hydrolysis, the hydrolysis temperature is 55℃, the pH value is 8.0, the hydrolysis time is 6h, and the hydrolysis product is obtained. The compound protease is composed of alkaline protease and neutral protease mixed in 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 hydrolysate.

[0014] (3) Freeze-dry the above-mentioned Spirulina protein peptide hydrolysate to obtain Spirulina protein peptide powder, which contains a large amount of bioactive peptide PCT-1. Beneficial effects

[0015] The advantages of this invention are as follows: The bioactive peptide PCT-1 obtained by screening spirulina protein peptides can significantly increase the activity of SOD and GSH-Px in serum, significantly reduce the content of MDA in serum, and its in vivo antioxidant activity is comparable to that of the positive control Trolox. At the same time, compared with the blank control group, PCT-1 treatment can reduce the α diversity index of intestinal flora, change the overall structure of intestinal flora (β diversity index), regulate the abundance of various taxonomic levels of intestinal flora, and significantly increase the content of acetic acid and butyric acid. This bioactive peptide PCT-1 can be applied to antioxidant and intestinal flora regulation functional foods. Attached Figure Description

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

[0017] Figure 2 is a partially enlarged schematic diagram of Figure 1;

[0018] Figure 3 shows the effects of treatment with the bioactive peptide PCT-1 on physiological indicators in mice. In the figure, A shows the effect on weight gain in mice, B shows the effect on spleen coefficient in mice, and C shows the effect on liver coefficient in mice. * indicates P<0.05, and ** indicates P<0.01.

[0019] Figure 4 shows the effect of treatment with the bioactive peptide PCT-1 on the antioxidant indicators of mouse serum. In the figure, 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, and ** indicates P<0.01.

[0020] Figure 5 shows the effect of treatment with the bioactive peptide PCT-1 on the transcriptional level of the keap1 gene in mice. In this figure, A shows the effect on the transcriptional level of the keap1 gene in the mouse brain, and B shows the effect on the transcriptional level of the keap1 gene in the mouse liver. * indicates P<0.05, and ** indicates P<0.01.

[0021] Figure 6 shows the effect of treatment with the bioactive peptide PCT-1 on the β diversity of the intestinal flora in mice.

[0022] Figure 7 shows the effect of treatment with the bioactive peptide PCT-1 on the content of short-chain fatty acids in the mouse intestine. In the figure, A shows the effect on the content of acetic acid in the mouse intestine, B shows the effect on the content of propionic acid in the mouse intestine, and C shows the effect on the content of butyric acid in the mouse intestine. ** indicates P<0.01, and *** indicates P<0.001. Embodiments of the present invention

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0024] I. Preparation of Spirulina Protein Peptide Samples

[0025] Spirulina was washed, dried, pulverized, and sieved sequentially to obtain spirulina powder. The spirulina powder was added to a 0.01M phosphate buffer solution (pH 6.8) at a ratio of 1 kg:10 L and thoroughly mixed in a granulator. Then, it was circulated and ground using a colloid mill. The resulting mixture was centrifuged to remove solid residue. The supernatant was filtered sequentially through a 25 μm filter cloth and a 0.45 μm filter membrane to obtain a crude spirulina protein extract. This crude extract was concentrated by ultrafiltration using ultrafiltration membranes with molecular weight cutoffs of 5 kDa and 10 kDa. The ultrafiltration fractions with molecular weights in the range of 5 kDa to 10 kDa were collected and spray-dried to obtain spirulina protein.

[0026] Take spirulina protein and add 9 times the mass of water (the mass concentration of spirulina protein is 10%). Add a complex protease (made of alkaline protease and neutral protease mixed in a 1:1 mass ratio) at an enzyme base ratio of 0.5% for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 55℃, the pH value is 8.0, and the enzymatic hydrolysis time is 6h.

[0027] After enzymatic hydrolysis, the hydrolysate was filtered through eight layers of gauze to remove residue, yielding spirulina protein peptide hydrolysate.

[0028] The above-mentioned spirulina protein peptide hydrolysate was freeze-dried to obtain spirulina protein peptide powder.

[0029] II. Obtaining the polypeptide sequence from spirulina protein peptides

[0030] The previously obtained spirulina protein peptides were analyzed using LC-MS / MS, and the results were analyzed using mass spectrometry software to obtain several polypeptide sequences.

[0031] The LC-MS / MS determination conditions are as follows:

[0032] (1) In the liquid chromatography 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 as follows: 0-48min, phase A decreases uniformly from 95% to 60%; 48-55min, phase A decreases uniformly from 60% to 30%; 55-56min, phase A decreases uniformly from 30% to 0%; 56-60min, phase A is maintained at 0%;

[0033] (2) In the mass spectrometry method: Orbitrap Exploris 480 (Thermofisher), positive ion detection mode, primary resolution of 120000, AGC set to 300, scan range of 200-1600 m / z. MIPS mode is peptide, valence state 1-5 is selected, secondary resolution is 15000, separation window is 1.6 m / z.

[0034] III. Screening bioactive peptides with peak area ≥ 2 × 10⁷ and amino acid count ≤ 6

[0035] From the previously obtained polypeptide sequences, 17 peak areas ≥ 2 × 10⁻⁶ were finally selected. 7 The screening results for active peptides with ≤6 amino acids are shown in Table 1.

[0036] Table 1. Peak areas ≥2×10⁻⁶ in Spirulina protein peptides 7 Active peptide sequences with ≤6 amino acids

[0037]

[0038] IV. Screening for bioactive peptides with strong binding affinity to Keap1

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

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

[0041] Table 2. Predicted Interactions Between Spirulina Protein Peptide Components and Keap1

[0042]

[0043] V. Molecular docking analysis

[0044] Among bioactive peptides with a peak area ≥ 10 × 10⁷, the hexapeptide LNRTFE (denoted as PCT-1, SEQ ID NO:1) showed the largest -CiE (82.7197 kcal / mol) when docked with Keap1, and also had the largest peak area (44.32 × 10⁷). Therefore, the hexapeptide LNRTFE was selected for further predictive analysis.

[0045] Analysis revealed the binding modes of the hexapeptide LNRTFE to Keap1 as shown in Figures 1 and 2, with the specific molecular docking details as follows:

[0046] The hexapeptide LNRTFE forms five HH bond interactions, three CH bond interactions, one salt bridge interaction, and two electrostatic interactions with Keap1. Five amino acid residues are involved in the interaction between the hexapeptide LNRTFE and Keap1.

[0047] VI. Evaluation of the antioxidant function of hexapeptide LNRTFE

[0048] 1. Solid-phase synthesis of hexapeptide LNRTFE

[0049] Using the Fmoc solid-phase synthesis strategy, Fmoc-protected amino acids were used as raw materials, and polystyrene resin was selected as the solid-phase support to synthesize the hexapeptide LNRTFE, i.e., PCT-1.

[0050] 2. Animal grouping and active peptide treatment

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

[0052] Eight-week-old male ICR mice were selected and housed at a temperature of 20℃-26℃ and an air humidity of 50%-60%, with 12-hour light-dark cycles and free access to food and water. After 7 days of acclimatization, the mice were randomly divided into three groups (control group, Trolox group, and PCT-1 group), with 12 mice in each group. The control group was administered physiological saline by gavage once daily, the Trolox group was administered Trolox (dissolved in physiological saline at a concentration of 1 mg / mL) by gavage once daily, and the PCT-1 group was administered solid-phase synthesized PCT-1 (dissolved in physiological saline at a concentration of 1 mg / mL) by gavage once daily. The gavage volume was 100 μL / 10g, and the administration continued for 30 days. During the experiment, all groups had free access to food and water.

[0053] 3. Effects of PCT-1 treatment on physiological parameters and serum antioxidant parameters in mice.

[0054] After the experimental period ended, all mice were anesthetized with enflurane, and blood was collected from the orbital plexus. The blood was then incubated in a 37°C water bath for 10 min, centrifuged at 4°C and 3000 rpm for 15 min, and the supernatant serum was separated, aliquoted and stored at -80°C for later use.

[0055] After euthanizing the mice by dislocation, the spleen, liver, and brain were quickly separated, weighed, and stored in a -80°C freezer for later use.

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

[0057] The effects of PCT-1 treatment on physiological indicators (weight gain, spleen coefficient, liver coefficient) in mice are shown in Figure 3, and the effects on serum antioxidant indicators (MDA content, SOD activity, CAT activity, GSH-Px activity) in mice are shown in Figure 4.

[0058] As shown in Figure 3, compared with the 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-1 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-1 treatment significantly reduced the weight gain of mice (p<0.01) but had no significant effect on the spleen coefficient and liver coefficient of mice (p>0.05).

[0059] As shown in Figure 4, 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 serum and significantly decreased the content of MDA (p<0.05) in serum. PCT-1 treatment significantly increased the activities of SOD (p<0.05) and GSH-Px (p<0.05) in serum and significantly decreased 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-1 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 quantitative PCR detection

[0061] (1) Primer design for real-time quantitative PCR

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

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

[0064]

[0065] (2) Extraction of total RNA from tissues

[0066] Take 100 mg each of mouse brain and liver tissue and place them in a sterile mortar pre-cooled with liquid nitrogen. Grind rapidly and thoroughly with liquid nitrogen. Then add 1 mL of TrasZol Up reagent and transfer the mixture to a DNase-free 1.5 mL centrifuge tube. Add 0.2 mL of chloroform, vortex thoroughly for 30 seconds, let stand for 10 minutes, and then centrifuge at 4 °C and 12000 × g for 15 minutes. Take the supernatant and transfer it to another RNase-free 1.5 mL centrifuge tube. Add 0.5 mL of isopropanol, gently mix the liquid in the tube, let stand at room temperature for 10 minutes, and then centrifuge again at 4 °C and 12000 × g for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol prepared with RNase-free water to the precipitate, gently wash the precipitate, centrifuge at 4 °C and 7500 × g for 5 minutes, discard the supernatant, open the centrifuge tube cap to allow the ethanol to evaporate completely, and then add 100 μL of RNase-free water. Use a Nano Drop 2000c to quickly determine the concentration and mass of total RNA in the sample. The RNA was aliquoted into RNase-free centrifuge tubes and stored at -80°C.

[0067] (3) cDNA template synthesis

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

[0069] (4) Gene expression detection

[0070] Preparation of the qRT-PCR system: After appropriately diluting the cDNA template, add 5 μL to the total system. Add 0.4 μL of the forward primer and reverse primer to the total system, and then add 10 μL of 2×TransStart™ Green qPCR SuperMix (containing SYBR Green dye) and 4.2 μL of enzyme-free water to the total system. The total system volume is 20 μL.

[0071] qRT-PCR three-step method: denaturation at 94℃ for 10 min; amplification reaction for 40 cycles: denaturation at 94℃ for 15 s, annealing at 50℃ for 15 s, extension at 72℃ for 45 s; extension at 72℃ for 10 min.

[0072] The effect of PCT-1 treatment on the transcriptional level of the keap1 gene in the mouse brain and liver is shown in Figure 5.

[0073] As shown in Figure 5, compared with the control group, both Trolox-positive treatment and PCT-1 treatment significantly downregulated the transcription level of the keap1 gene in the brain and liver, while there was no significant difference between Trolox-positive treatment and PCT-1 treatment.

[0074] VII. Evaluation of the gut microbiota regulation function of hexapeptide LNRTFE

[0075] 1. Animal grouping and active peptide treatment

[0076] Eight-week-old male ICR mice were selected and housed at a temperature of 20℃-26℃ and an air humidity of 50%-60%, with 12 hours of light-dark alternation, and free access to food and water. After 7 days of acclimatization, they were randomly divided into two groups (control group and PCT-1 group), with 12 mice in each group. The control group was administered physiological saline by gavage once a day, while the PCT-1 group was administered PCT-1 (dissolved in physiological saline at a concentration of 1 mg / mL) by gavage once a day, at a volume of 100 μL / 10g, for 30 consecutive days. During the experiment, all groups had free access to food and water.

[0077] 2. Detection of the effect of PCT-1 treatment on the gut microbiota of mice

[0078] After the experiment was completed, mouse feces were collected and stored in a -80°C refrigerator for later use.

[0079] (1) Sequencing of microbial community in mouse feces

[0080] Total DNA was extracted from mouse feces using the QIAamp DNA Stool Mini extraction kit and stored at -20°C for later use.

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

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

[0083] PCR amplification was performed in a 25 µL reaction volume, specifically comprising: 2 µL of genomic DNA at a concentration of 10 ng / μL, 2 µL of 0.1 μM forward primer, 2 µL of 0.1 μM reverse primer, 12.5 µL of premixed buffer, and ddH2O to bring the volume to 25 µL. The PCR amplification program was as follows: 95 °C denaturation for 5 min; 95 °C denaturation for 30 s, 54 °C annealing for 30 s, 72 °C extension for 40 s, 20 cycles; 72 °C extension for 5 min.

[0084] After amplification, the PCR products obtained from PCR amplification were detected by agarose gel electrophoresis and recovered by gel excision. After accurate quantification, the products were sequenced on the Illumina miseq platform.

[0085] The original FASTQ file was processed using QiIME 1.8.0 as follows:

[0086] (i) Based on the overlap relationship between PE reads, use FLASH 1.2.11 to concatenate pairs of reads into a sequence and delete sequences that cannot be merged;

[0087] (ii) Remove the barcode after identifying the sample source of the data;

[0088] (iii) Use Prinseq software to remove fragments shorter than 50bp and bases with a quality value below 20 at the end of reads. Finally, filter low-complexity sequences and remove non-amplified regions.

[0089] (iv) Sequencing error correction was performed using the cluster algorithm (Mothur 1.36.0) while removing chimeras from the sequences (Uchime 4.2.40).

[0090] Gut microbiota α diversity was analyzed using Mothur 1.36.0, and principal coordinate analysis was performed using Muscle 3.3.31.

[0091] RDP classifier software was used to perform species annotation and community change analysis on the processed sequences.

[0092] The effects of PCT-1 treatment on gut microbiota α diversity are shown in Table 4.

[0093] Table 4. Effects of PCT-1 treatment on gut microbiota α-diversity

[0094]

[0095] As shown in Table 4, compared with the control group, PCT-1 treatment reduced the ACE index, Chao index, Shannon index and Simpson index of the intestinal flora in mice.

[0096] The effect of PCT-1 treatment on the overall composition of the mouse gut microbiota was analyzed using weighted principal coordinates. The results are shown in Figure 6.

[0097] As shown in Figure 6, PCT-1 treatment can alter the overall structure (β diversity) of the gut microbiota in mice to some extent.

[0098] At the phylum level, the effects of PCT-1 treatment on the composition of the intestinal flora in mice are shown in Table 5.

[0099] Table 5. Effects of PCT-1 treatment on the composition of mouse gut microbiota at the phylum level.

[0100]

[0101] Table 5 shows that, compared with the control group, PCT-1 treatment increased the abundance of Actinobacteriota and Patescibacteria in the mouse gut, while decreasing the abundance of Bacteroidota, Firmicutes, Campilobacterota, and Desulfobacterota. This indicates that PCT-1 treatment affects the composition of the mouse gut microbiota at the phylum level.

[0102] At the scientific level, the effects of PCT-1 treatment on the composition of the intestinal flora in mice are shown in Table 6.

[0103] Table 6. Effects of PCT-1 treatment on the composition of gut microbiota in mice at the scientific level.

[0104]

[0105] Table 6 shows that, compared with the control group, PCT-1 treatment increased the abundance of Muribauculaceae, Lactobacillaceae, Prevotellaceae, Erysipelotrichaceae, and Bifidobacteriaceae in the mouse gut, while decreasing the abundance of Lachnospiraceae, Bacillaceae, norank_o__Clostridia_UCG-014, Bacteroidaceae, and Helicobacteraceae. This indicates that PCT-1 treatment also affects the composition of the mouse gut microbiota at the family level.

[0106] Differentially expressed ASVs in the gut microbiota after PCT-1 treatment were analyzed (p<0.05), and the results are shown in Table 7.

[0107] Table 7 Results of differentially expressed ASVs after PCT-1 treatment

[0108]

[0109]

[0110] Table 7 shows that, compared with the control group, PCT-1 treatment upregulated the abundance of 25 ASVs and downregulated the abundance of 24 ASVs. This indicates that PCT-1 treatment also affects ASV expression.

[0111] (2) Analysis of short-chain fatty acid content in mouse feces

[0112] Weigh 30 mg of dried mouse feces, add 0.8 mL of water, shake to mix, then add 0.2 mL of 50% sulfuric acid, mix well, then 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 through a 0.22 μm filter membrane, and take 1 mL of the filtrate for loading. Standard curves for acetic acid, propionic acid, and butyric acid were established using the external standard method. GC-MS (Agilent 7890 gas chromatography-purposive chromatography-mass spectrometry M7-80E) was employed with a flame ionization (FID) detector, a DB-WAX column (30m × 0.25mm × 0.25μm), helium as the carrier gas, a split ratio of 20:1, a flow rate of 1 mL / min, and an injection volume of 1 μL. The initial column temperature was 90℃, increased to 150℃ at a rate of 12℃ / min, then increased to 220℃ at a rate of 20℃ / min and held at 220℃ for 4.5 min. The transfer line and ion source temperatures were set to 220℃ and 230℃, respectively, with a solvent delay time of 3 min.

[0113] The results of detecting the content of acetic acid, propionic acid and butyric acid in the intestine of mice after PCT-1 treatment are shown in Figure 7.

[0114] As shown in Figure 7, compared with the control group, PCT-1 treatment significantly increased the content of acetic acid (p<0.001) and butyric acid (p<0.01) in mouse feces, but had no effect on the content of propionic acid.

[0115] In summary, the hexapeptide LNRTFE (PCT-1) obtained from spirulina protein peptides in this invention has antioxidant and gut microbiota regulation functions, and can be applied to foods with antioxidant and gut microbiota regulation functions. Industrial applicability

[0116] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the protection scope of this invention.

Claims

1. A bioactive peptide PCT-1, characterized in that, The amino acid sequence of the bioactive peptide PCT-1 is LNRTFE, as shown in SEQ ID NO: 1 in the sequence listing, and it has antioxidant and gut microbiota regulation functions.

2. The application of the bioactive peptide PCT-1 according to claim 1 in antioxidant and gut microbiota regulation functional foods.

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

4. The method for preparing the bioactive peptide PCT-1 according to claim 1, characterized in that, Enzymatic hydrolysis is used, specifically: (1) Take spirulina protein, add water at a mass ratio of 9 times that of spirulina protein, add compound protease at a ratio of 0.5% for enzymatic hydrolysis, the hydrolysis temperature is 55℃, the pH value is 8.0, the hydrolysis time is 6h, and the hydrolysis product is obtained. The compound protease is composed of alkaline protease and neutral protease mixed in 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 hydrolysate. (3) Freeze-dry the above-mentioned Spirulina protein peptide hydrolysate to obtain Spirulina protein peptide powder, which contains a large amount of bioactive peptide PCT-1.

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