Chlorella heptapeptide tGPLGSR with multi-target lipid-lowering activity, and preparation method and application thereof

Abstract

This invention discloses a Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, its preparation method, and its applications. The amino acid sequence of this heptapeptide is Thr-Gly-Pro-Leu-Gly-Ser-Arg. The preparation method includes: performing ultraviolet and electroporation combined mutagenesis on wild-type Chlorella proteoglycans to obtain a high-protein-content mutant algal strain; obtaining algal protein through fermentation and synergistic extraction and purification with alcohols; directionally digesting the protein with alkaline protease, followed by enriching the active peptide components through ultrafiltration and gel chromatography; and finally, screening the heptapeptide using liquid chromatography-mass spectrometry (LC-MS), computer-aided prediction, and molecular docking techniques. This heptapeptide exhibits potent inhibitory activity against pancreatic lipase and can dose-dependently reduce the accumulation of triglycerides and cholesterol in 3T3-L1 cells. Its mechanism involves multiple target pathways, including inhibition of MMP-9, activation of AMPK phosphorylation, and downregulation of PNLIP, and it can be used to develop lipid-lowering functional foods or health products.

Description

Technical Field

[0001] This invention belongs to the biopharmaceutical industry and involves the fields of biopharmaceutical manufacturing and functional foods. Specifically, it relates to a Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, its preparation method, and its application. Background Technology

[0002] With continued global economic growth and improved living standards, obesity has become a global public health challenge, with its prevalence steadily rising. It not only significantly increases the risk of various chronic diseases such as diabetes, cardiovascular disease, and metabolic syndrome, but is also closely related to psychological problems such as anxiety and depression, further leading to limited social functioning, decreased quality of life, and increased healthcare burden. Studies have shown that the development of obesity is closely related to the lipid digestion and absorption process. Pancreatic lipase, a key enzyme in hydrolyzing dietary fat (capable of hydrolyzing 50-70%), can be effectively slowed down by inhibiting its activity, making it an important strategy for weight control.

[0003] Currently, commonly used pancreatic lipase inhibitors in clinical practice, such as orlistat, enteric-coated pancreatic lipase capsules, and ezetimibe, while having some weight-loss effects, are often accompanied by adverse reactions such as gastrointestinal discomfort, allergies, and malabsorption of fat-soluble vitamins, limiting patient compliance and the safety of long-term use. Therefore, exploring safe, effective, and sustainably sourced pancreatic lipase inhibitors has become an urgent research need.

[0004] Naturally derived bioactive peptides are considered ideal alternatives due to their high safety, good biocompatibility, and ease of degradation. Existing research has yielded peptides with pancreatic lipase inhibitory activity through enzymatic hydrolysis of traditional plant and animal proteins such as soybeans, whey, and amaranth seeds. These peptides specifically bind to the active site of pancreatic lipase, inhibiting the binding of the enzyme to its substrate, thereby effectively blocking the breakdown of dietary fat, reducing fat absorption, and exerting a lipid-lowering effect. However, these raw materials largely rely on grain crops or livestock products, posing challenges such as competition for land and water resources with human staple foods, high production costs, unstable raw material supply, and a fragile industrial chain, which are detrimental to sustainable development.

[0005] Microalgae (such as Chlorella and Spirulina) are novel protein resources with significant advantages, including rapid growth, high protein content (up to 60-70%), no need for arable land, and the ability to be cultivated using wastewater or seawater, making them an ideal choice for sustainable protein supply. However, research on the development of bioactive peptides from microalgal proteins remains relatively scarce, especially reports on microalgal peptides with well-defined sequences and multi-target lipid-lowering functions. The main shortcomings are:

[0006] (1) Microalgae have a strong cell wall structure, resulting in low efficiency in protein extraction and enzymatic hydrolysis;

[0007] (2) The peptide fragments of the enzymatic hydrolysis products are relatively complex, making efficient screening and identification difficult;

[0008] (3) Most studies remain at the crude extract stage, lacking the discovery of bioactive peptides with clear sequences, clear mechanisms, and diverse targets.

[0009] Based on this, this invention uses a mutagenic, high-protein Chlorella strain as raw material. Through multi-step synergistic extraction, targeted enzymatic hydrolysis, graded purification, and multi-dimensional screening, a heptapeptide, TGPLGSR, with significant pancreatic lipase inhibitory activity and multi-target cellular lipid metabolism regulation function was identified for the first time from Chlorella protein hydrolysates. This peptide not only effectively inhibits pancreatic lipase activity but also significantly reduces intracellular triglyceride and cholesterol accumulation by regulating multiple targets such as MMP-9, AMPK, and PNLIP, providing a new technical pathway and material basis for developing safe, efficient, and sustainable lipid-lowering functional factors. Summary of the Invention

[0010] In view of the technical problems of existing lipid-lowering active peptide raw materials being unsustainable and having unclear sequences and mechanisms, the present invention provides a Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, its preparation method and application.

[0011] The technical solution adopted in this invention is as follows:

[0012] A Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, the heptapeptide TGPLGSR is Thr-Gly-Pro-Leu-Gly-Ser-Arg, and its amino acid sequence is shown in SEQ ID NO: 1.

[0013] The preparation method of the above-mentioned Chlorella heptapeptide TGPLGSR is as follows:

[0014] S1. Wild Chlorella proteoglycans were treated with a physical composite mutagenesis technique combining ultraviolet irradiation and electric shock to screen and obtain mutagenic Chlorella strains with high protein content, providing high-quality raw materials for subsequent preparation of bioactive peptides.

[0015] S2. Ferment and culture the mutated Chlorella strain, collect the algae and dry them to obtain Chlorella powder;

[0016] S3. Chlorella powder is extracted and purified by a combination of alcohol dissolution and enzymatic hydrolysis to prepare high-purity chlorella protein.

[0017] S4. Alkaline protease was used to enzymatically hydrolyze Chlorella proteins. The resulting hydrolysate was subjected to ultrafiltration and gel filtration chromatography in sequence, and peptide components with a molecular weight <3 kDa were collected.

[0018] S5. The amino acid sequences of peptide components with a molecular weight <3 kDa were identified by liquid chromatography-mass spectrometry (RPLC-MS). Based on bioinformatics prediction, molecular docking simulation and in vitro activity verification, Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity was screened from the identified peptide sequences.

[0019] Further in vitro activity verification showed that the above-mentioned Chlorella heptapeptide TGPLGSR has a strong inhibitory activity against pancreatic lipase, and the inhibition type is competitive inhibition.

[0020] More importantly, cell experiments (treating mouse preadipocytes 3T3-L1 with Chlorella heptapeptide) revealed that the aforementioned Chlorella heptapeptide TGPLGSR significantly inhibited 3T3-L1 preadipocyte differentiation in a dose-dependent manner, reducing intracellular triglyceride (up to 52.79%) and cholesterol (up to 36.55%) accumulation. Mechanistic studies showed that its lipid-lowering effect is not through a single pathway, but rather by targeting and inhibiting the activity of matrix metalloproteinase-9 (MMP-9), thereby blocking downstream inflammatory signal transduction, activating AMP-dependent protein kinase (AMPK) phosphorylation, and downregulating the expression of pancreatic lipase-related genes (PNLIP) and key lipid synthesis genes (such as FAS and ACC), thus achieving multi-pathway synergistic inhibition of adipocyte differentiation and lipid accumulation.

[0021] Further, in step S1, the wild-type Chlorella pyrenoidosa is used, and the conditions for synergistic mutagenesis by ultraviolet irradiation and electric shock are as follows: ultraviolet wavelength 200-250 nm, irradiation distance 10-30 cm, irradiation time 15-120 seconds; voltage 1800-3000 volts, capacitance 40-50 microfarads, resistance 300-500 ohms, and electric shock time 4-6 milliseconds.

[0022] Further, in step S3, the synergistic extraction and purification by alcohol dissolution and enzymatic hydrolysis specifically involves: extracting Chlorella powder with ethanol solution, then subjecting the residue to cellulose enzymatic hydrolysis and physical cell wall disruption treatment, combining the extracts, concentrating and drying to obtain crude protein; dissolving the crude protein and then filtering under reduced pressure and freeze-drying to obtain high-purity Chlorella protein.

[0023] Furthermore, in step S4, the conditions for enzymatic hydrolysis are: pH 8-10, temperature 40-60℃, enzyme dosage of 3-5% of the mass of Chlorella protein, substrate concentration of 80-120 mg / mL, and hydrolysis time of 8-12 h.

[0024] Furthermore, in step S4, ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa; gel filtration chromatography uses Sephadex G-25 dextran gel.

[0025] Furthermore, in step S5, the filtering includes:

[0026] (a) Confidence screening of the identified peptide sequences;

[0027] (b) The screened peptides were scored using a bioactivity prediction tool;

[0028] (c) Assess the toxicity, sensitization, and solubility of the peptide;

[0029] (d) Perform molecular docking of the candidate peptide with pancreatic lipase and calculate the binding free energy;

[0030] (e) Verify the in vitro pancreatic lipase inhibitory activity of the candidate peptide with the lowest binding free energy.

[0031] Furthermore, Chlorella heptapeptide exerts its lipid-lowering effect through at least one of the following pathways: targeting and inhibiting matrix metalloproteinase-9 (MMP-9), activating AMP-dependent protein kinase (AMPK) phosphorylation, and downregulating the expression of pancreatic lipase-related genes (PNLIP) and key genes for lipid synthesis.

[0032] Further, step S5 specifically involves: desalting peptide components with a molecular weight <3 kDa using the ZipTip C18 method, then identifying the sequences of the desalted peptide components with a molecular weight <3 kDa using RPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry), and using PEAKS software to search the database to identify the peptide sequences. Among the identified peptide sequences, further screening is performed based on a confidence level (ALC) greater than 95%. The Peptide Ranker tool is used to predict bioactivity and physicochemical properties, the AllerTOP tool to assess peptide sensitization, the ToxinPred tool to assess peptide toxicity, and the Innovagen tool to predict peptide solubility and isoelectric point.

[0033] Furthermore, the aforementioned further screened peptides were molecularly docked with pancreatic lipase, and then Discovery Studio software was used to analyze the peptide with the lowest binding free energy to pancreatic lipase, thus obtaining Chlorella heptapeptide TGPLGSR.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] (1) This invention is the first to identify a heptapeptide TGPLGSR with lipid-lowering activity from a specific mutagenized Chlorella. Its raw materials have the advantages of being environmentally friendly and resource-saving, avoiding the production constraints of traditional animal and plant protein sources.

[0036] (2) This invention forms a complete process for the characteristics of microalgal proteins by combining compound mutagenesis, synergistic extraction, targeted enzymatic hydrolysis and multi-stage purification, and combined with computer-aided screening, which significantly improves the discovery efficiency and preparation specificity of active peptides.

[0037] (3) The heptapeptide of the present invention can not only effectively inhibit pancreatic lipase activity, but also inhibit fat absorption and synthesis by regulating multiple key targets such as MMP-9, AMPK, and PNLIP. The mechanism is clear and has the potential for synergistic effect.

[0038] (4) The heptapeptides of the present invention are food-derived components with diverse mechanisms of action. They have broad application prospects in functional foods, health products and other fields, and provide new natural solutions for weight and blood lipid management. Attached Figure Description

[0039] Figure 1 The diagram shows the screening of wild Chlorella proteolyticus mutant strains and the production, crude protein powder, and purified protein powder of the mutant Chlorella. (a) Mutagenized strain plate; (b) Algal powder; (c) Crude protein powder; (d) Purified protein.

[0040] Figure 2 The image shows the ultraviolet spectrum of Chlorella proteins induced by alkaline protease digestion.

[0041] Figure 3 The Sephadex G-25 gel separation and elution curves of enzymatic peptides with a molecular weight <3 kD in the mutagenic Chlorella protein hydrolysate were obtained.

[0042] Figure 4 The inhibition rates of pancreatic lipase activity of Pe1 and Pe2, the eluted components of Chlorella protease hydrolysate with molecular weight <3 kD, were measured. In the figure, a and b on the bars are statistical significance markers. a and b indicate that there is a significant difference in the inhibition rate of pancreatic lipase activity between components Pe1 and Pe2 at the p < 0.05 level (one-way ANOVA and Duncan's multiple range test were used), and the significance level represented by a is greater than that of b.

[0043] Figure 5 To identify the Pe1 peptide sequence of the enzymatically digested peptide component with a molecular weight <3 kD from Chlorella enzyme hydrolysate, the following data were obtained: (a) total ion chromatogram of the enzymatically digested peptide component; (b) distribution of peptide molecular weight composition; and (c) distribution of peptides of different lengths.

[0044] Figure 6To identify the pancreatic lipase inhibitory activity of peptides in the Pe1 fraction of the mutagenic Chlorella protein hydrolysate with a molecular weight <3 kD, the bars a, b, and c in the figure are statistical significance markers. a, b, and c indicate that there are significant differences in the pancreatic lipase inhibitory activities of TGPLGSR, AGVAGPK, and SGLDGAK peptides at the p < 0.05 level (univariate ANOVA and Duncan's multiple range test were used), and the significance of a, b, and c decreases in that order.

[0045] Figure 7 To determine the half-inhibitory concentration (IC50) of pancreatic lipase in peptide fractions with molecular weight <3 kD from mutagenic Chlorella protein hydrolysate, Pe1 was used to identify the peptide half-inhibitory activity (IC50) of the peptides. 50 (a) the half-inhibitory activity concentration of peptide AGVAGPK against pancreatic lipase; (b) the half-inhibitory activity concentration of peptide SGLDGAK against pancreatic lipase; and (c) the half-inhibitory activity concentration of peptide TGPLGSR against pancreatic lipase.

[0046] Figure 8 To investigate the mechanism of action of Chlorella heptapeptide TGPLGSR in inhibiting pancreatic lipase.

[0047] Figure 9 The spatial conformations of the secondary structures of microalgal protein peptides from different sources are shown, including (a) peptide LPLL, (b) peptide FLGF, (c) peptide YPFW, (d) peptide YYGR, (e) peptide FLQR, and (f) peptide TGPLGSR.

[0048] Figure 10 To investigate the effect of different doses of Chlorella heptapeptide on the differentiation of mouse 3T3-L1 preadipocytes, the following images were used: (A) Oil Red O staining of the effect of different doses of Chlorella heptapeptide on the differentiation of mouse 3T3-L1 preadipocytes; (B) Effect of different doses of Chlorella heptapeptide on the lipid droplet positive area ratio of differentiated mouse 3T3-L1 preadipocytes. The bars a, b, c, and d on the images are statistical significance markers. a, b, c, and d indicate that there are significant differences in the differentiation of mouse 3T3-L1 preadipocytes at the p < 0.05 level due to different doses of Chlorella heptapeptide (using univariate ANOVA and Duncan's multiple range test), and the significance of a, b, c, and d decreases sequentially.

[0049] Figure 11To investigate the effects of different doses of mutagenic Chlorella heptapeptide on the total cholesterol and triglyceride levels in mouse 3T3-L1 preadipocytes, the following was studied: (A) the effect of different doses of mutagenic Chlorella heptapeptide on the total cholesterol level in mouse 3T3-L1 preadipocytes; (B) the effect of different doses of mutagenic Chlorella heptapeptide on the triglyceride level in mouse 3T3-L1 preadipocytes. The bars a, b, c, and d in the figure represent statistical significance markers. a, b, c, and d indicate that at the p < 0.05 level, different doses of mutagenic Chlorella heptapeptide had significant differences in their effects on the total cholesterol and triglyceride levels in mouse 3T3-L1 preadipocytes (using univariate ANOVA and Duncan's multiple range test), and the significance level of a, b, c, and d decreased sequentially.

[0050] Figure 12 To investigate the effects of different doses of Chlorella heptapeptide on the relative expression levels of lipid formation-related genes in mouse 3T3-L1 preadipocytes, the study included: (A) the effect of different doses of Chlorella heptapeptide on the relative expression level of fatty acid synthase mRNA in mouse 3T3-L1 preadipocytes; and (B) the effect of different doses of Chlorella heptapeptide on the relative expression level of acetyl-CoA carboxylase mRNA in mouse 3T3-L1 preadipocytes. The bars a, b, c, and d in the figure represent statistical significance markers. a, b, c, and d indicate that at the p < 0.05 level, different doses of Chlorella heptapeptide significantly affected the expression levels of lipid formation-related genes in mouse 3T3-L1 preadipocytes (using univariate ANOVA and Duncan's multiple range test), with the significance decreasing sequentially from a to b.

[0051] Figure 13To investigate the effects of different doses of mutagenic Chlorella heptapeptide on the expression levels of MMP-9, PNLIP, AMPK, and p-AMPK in mouse 3T3-L1 preadipocytes, the following findings were presented: (A) Immunoblot images showing the effects of different doses of mutagenic Chlorella heptapeptide on the expression levels of GAPDH, MMP-9, PNLIP, AMPK, and p-AMPK in mouse 3T3-L1 preadipocytes; (B) The effect of different doses of mutagenic Chlorella heptapeptide on the relative expression level of MMP-9 protein in mouse 3T3-L1 preadipocytes; (C) The effect of different doses of mutagenic Chlorella heptapeptide on the relative expression level of PNLIP protein in mouse 3T3-L1 preadipocytes; (D) The effect of different doses of mutagenic Chlorella heptapeptide on the relative expression level of AMPK protein in mouse 3T3-L1 preadipocytes; (E) [Further details to be added]. The effects of different doses of mutagenic Chlorella heptapeptide on the relative expression level of p-AMPK protein in mouse 3T3-L1 preadipocytes were investigated. The bars a, b, c, and d in the figure represent statistical significance markers. a, b, c, and d indicate that at the p < 0.05 level, different doses of mutagenic Chlorella heptapeptide significantly affected the expression levels of MMP-9, PNLIP, AMPK, and p-AMPK in mouse 3T3-L1 preadipocytes (using univariate ANOVA and Duncan's multiple range test). The significance of a, b, c, and d decreased sequentially. Bars without labels indicate no significant differences between groups.

[0052] Figure 14 To investigate the effects of different doses of mutagenic Chlorella heptapeptide on the expression levels of TNF-α and IL-6 in mouse 3T3-L1 preadipocytes, the following experiments were conducted: (A) Effect of different doses of mutagenic Chlorella heptapeptide on the expression level of TNF-α in mouse 3T3-L1 preadipocytes; (B) Effect of different doses of mutagenic Chlorella heptapeptide on the expression level of IL-6 in mouse 3T3-L1 preadipocytes. The bars a, b, c, and d in the figure represent statistical significance markers. a, b, c, and d indicate that at the p < 0.05 level, different doses of mutagenic Chlorella heptapeptide significantly affected the expression levels of TNF-α and IL-6 in mouse 3T3-L1 preadipocytes (using univariate ANOVA and Duncan's multiple range test), with the significance decreasing sequentially from a to b.

[0053] Figure 15To illustrate the effects of different treatment strategies on the yield of crude protein and purified protein from *Chlorella vulgaris*, the following parameters were used: A) ethanol extraction, B) cellulase hydrolysis, C) physical cell wall disruption, D) ethanol extraction + cellulase hydrolysis, E) ethanol extraction + physical cell wall disruption, and F) cellulase hydrolysis + physical cell wall disruption. The bars a, b, c, d, e, and f in the figure represent statistical significance markers. a, b, c, d, e, and f indicate significant differences in the yield of crude protein and purified protein from *Chlorella vulgaris* at the p < 0.05 level (using univariate ANOVA and Duncan's multiple range test). The significance levels of a, b, c, d, e, and f decrease sequentially.

[0054] Figure 16 The figures show the effects of different proteases on the degree of hydrolysis of mutagenic Chlorella proteins (A) and the effects of different protease-digested protein products on the inhibition rate of pancreatic lipase activity (B). The bars a, b, c, d, and e in the figures are statistical significance markers. a, b, c, d, and e indicate that there are significant differences in the degree of hydrolysis of mutagenic Chlorella proteins by different proteases at the p < 0.05 level (using univariate ANOVA and Duncan's multiple range test), and the significance levels represented by a, b, c, d, and e decrease in that order. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments, but the invention is not limited thereto. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0056] Example 1: Obtaining Chlorella strains with high protein content through synergistic mutagenesis of electric shock and ultraviolet irradiation.

[0057] Wild-type Chlorella pyrenoidosa was inoculated into modified BG11 liquid medium (composition shown in the table below, pH=6.1) and cultured until the logarithmic growth phase. 1 mL of the logarithmic growth phase culture was then diluted to 10 mL (i.e., a 10-fold dilution) to achieve a cell concentration of approximately 102. 6Cells / mL, take 1 mL of diluted culture medium into a 2 mL centrifuge tube, and resuspend three times with frozen sorbitol solution. Take 400 μL of the resuspended solution into an electroporation cuvette, and perform electroporation transformation for 4 ms under the program conditions of 4℃, 3 kV, 25 uF, and 400 ohms. Take 200 μL of the electroporated solution from each cuvette into a 2 mL centrifuge tube for serial dilution, and then take 100 μL of the diluted solution for agar plate. Then, perform UV mutagenesis by irradiation at 225 nm, 20 cm distance, and 15 W power for 45 s. After 24 h in the dark, incubate in an incubator at 26℃ for 168 h. After single colonies grow on the agar plate ( Figure 1 (As shown in a), several single colonies were randomly selected and cultured in modified BG11 liquid medium at 26℃ for 168 h. After culture, the algal sludge was collected by centrifugation and freeze-dried to obtain algal powder. The protein content of the algal powder was then determined using a Kjeldahl nitrogen analyzer. The instrument parameters were set as follows: titrant concentration 0.10 mol / L, protein coefficient 6.25, 20 mL each of boric acid and distilled water, 40 mL of alkali solution, and distillation time 5 min. Specifically, 0.3 g of Chlorella powder from each group was weighed into a digestion tube, and 6.4 g of mixed catalyst (CuSO4•5H2O:K2SO4 mass ratio of 1:15) and 12 mL of concentrated sulfuric acid were added sequentially. The tube was then placed in a graphite digestion furnace for 2.5 h of digestion. The protein content was then determined, and the mutagenic Chlorella strain with a protein content of 71-76% was selected as the target algal strain.

[0058] Table 1. Composition of the modified BG11 liquid culture medium

[0059]

[0060] Example 2: Protein extraction, separation and purification from mutagenic Chlorella strains

[0061] High-protein-content mutagenic Chlorella target strains were inoculated into modified BG11 liquid medium using standard aseptic techniques. The culture was placed in a light-dark cycle (12 h light / 12 h dark) incubator at 4000 lux, 26℃, and 120 rpm for 120 h to obtain the mutagenic Chlorella seed culture. The mutagenic Chlorella seed culture was then inoculated at a 10% volume ratio into a fermenter containing modified BG11 liquid medium and fermented under light conditions of 4000 lux, 120 rpm, and 26℃ for 168 h. The results were obtained under 8000 × g conditions (where g is the acceleration due to gravity, approximately 9.8 m / s²). 2 The fermentation culture was collected by centrifugation and then spray-dried to obtain high-protein Chlorella mutagenesis powder. Figure 1 (as shown in b).

[0062] Accurately weigh 50 g of dried Chlorella vulgaris powder and dissolve it in 1 L of anhydrous ethanol at a ratio of 1:20 g / ml. Soak at room temperature for 24 h, centrifuge at 4000 rpm for 10 min, retain the supernatant, add 0.5 L of distilled water to the precipitate, adjust the pH of the mixture to 5.0, then add 1% (w / w) of cellulase and hydrolyze at 50℃ for 3 h, followed by enzyme inactivation at 100℃ for 10-15 min. Treat the above product with an ultrasonic cell disruption and shearing device, centrifuge at 4000 rpm for 10 min, and collect the supernatant. Combine all supernatants from the above steps and freeze-dry to obtain crude Chlorella vulgaris protein powder. Figure 1 As shown in c), its protein content was 86.63%. Subsequently, 50 g of Chlorella crude protein powder was accurately weighed and dissolved in 1 L of distilled water at a ratio of 1:20 g / mL. The solution was stirred thoroughly at 4℃ for 1 h, and then placed in a 4℃ refrigerator for 5 h to ensure complete protein dissolution. The solution was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Impurities were removed by vacuum filtration, and the filtrate was freeze-dried under vacuum to obtain mutagenic Chlorella protein with high water solubility and purity. Figure 1 As shown in d), its protein content reaches 94.58%.

[0063] Example 3: Mutagenic Chlorella protein enzymatic hydrolysis and separation and purification of enzymatically hydrolyzed peptides

[0064] (1) Enzymatic hydrolysis of Chlorella mutagenesis protein: Weigh 100 mg of Chlorella mutagenesis protein powder and disperse it in ultrapure water. Adjust the pH of the dispersion to 9.5, add 5% (by weight) of alkaline protease, and hydrolyze at 50℃ for 10 h. Inactivate the enzyme in a water bath at 100℃, centrifuge at 6000 rpm for 10 min, and collect the supernatant. Determine the degree of hydrolysis of Chlorella enzymatic hydrolysis according to the colorimetric method of GB 5009.235-2016. The specific operation is as follows: Take 2 mL of the enzymatic hydrolysis supernatant into a 100 mL volumetric flask and dilute to volume with distilled water to obtain the sample dilution. Take different volumes of ammonia nitrogen standard solution and place them into 10 mL colorimetric tubes. Add 4 mL of buffer solution and colorimetric reagent respectively, and add distilled water to make up the total volume to 10 mL. Mix well, incubate in a water bath at 100℃ for 15 min, cool, and measure the absorbance at a wavelength of 400 nm to plot the standard curve. Repeat the above operation by taking 2 mL of sample dilution solution, calculate the amino nitrogen concentration in the sample test solution according to the standard curve regression equation, and calculate the degree of hydrolysis according to the following formula.

[0065]

[0066] In the formula: m represents the mass of nitrogen in the sample test solution, in micrograms (μg); m1 is the mass of the sample, in grams (g); V1 is the volume of the sample solution used for the test, and V2 is the pretreatment volume, both in milliliters (mL).

[0067] In addition, a protein hydrolysis solution of 1.0 mg / mL was prepared using distilled water, and the UV absorption spectrum was scanned in the wavelength range of 250-350 nm using a UV spectrophotometer at a scan rate of 50 nm / min. The results showed that the mutagenic Chlorella protein hydrolysate exhibited a significant hyperchromic effect in the UV spectrum at 280 nm, indicating that alkaline protease hydrolysis induced significant changes in protein structure, exposing UV-absorbing groups that were originally hidden within the protein, thereby enhancing the absorption intensity. Figure 2 (As shown in the figure). These results demonstrate that alkaline protease effectively hydrolyzes Chlorella proteins, with the calculated degree of hydrolysis reaching 25.22%.

[0068] (2) Ultrafiltration of mutagenic Chlorella protein hydrolysate: The alkaline protease hydrolysate was prepared into a 10 mg / mL solution and separated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 3 kDa, 5 kDa, and 10 kDa. Based on molecular weight, four polypeptide fractions with different molecular weights were collected and named D-1 (MW < 3 kD), D-2 (MW = 3-5 kD), D-3 (MW = 5-10 kD), and D-4 (MW > 10 kD), respectively.

[0069] (3) Ultrafiltration separation and purification of protein hydrolysate D-1 (MW < 3 kD) obtained by mutagenesis of Chlorella strain: The MW < 3 kD hydrolysate obtained after ultrafiltration was further separated by Sephadex G-25 dextran gel. The specific steps were as follows: Prepare a MW < 3 kD hydrolysate sample with a concentration of 20 mg / mL, use ultrapure water as the eluent, take 1 mL of sample for loading, and set the elution flow rate to 0.3 mL / min. Collect the eluent once every 3 min, measure the absorbance value of the eluent at 220 nm, and plot the elution curve according to the corresponding absorbance value of the eluent. Figure 3 As shown in the figure, the elution curve showed two distinct peaks, located near tubes 22 and 28, respectively. The eluent near these two peaks was then collected and purified by freeze-drying to obtain two components, which were defined as Pe1 and Pe2, respectively.

[0070] (4) Determination of pancreatic lipase inhibitory activity of Pe1 and Pe2, isolated and purified components of mutagenized Chlorella strain protein hydrolysate with MW < 3 kD: The two obtained components Pe1 and Pe2 were subjected to pancreatic lipase inhibitory activity determination. The specific steps were as follows: Pancreatic lipase was mixed with Tris buffer (100 mM Tris-HCl and 5 mM CaCl2, pH=7.0) in a certain ratio to prepare a 1 mg / mL pancreatic lipase solution. 4-Nitrophenylbutyrate was diluted to 5 mM with DMSO. 20 μL of the test sample with a concentration of 10 mg / mL, 140 μL of Tris buffer and 20 μL of pancreatic lipase solution were mixed and incubated at 37℃ for 30 min. For the blank group, 20 μL of Tris buffer was used instead of pancreatic lipase solution. Then 20 μL of PNPB solution was added to start the reaction and reacted at 37℃ for 15 min. After the reaction, the absorbance was measured at a wavelength of 405 nm, and the pancreatic lipase activity inhibition rate was calculated according to formula (2). Figure 4 The results showed that the pancreatic lipase inhibition rate of component Pe1 was 67.51%, which was significantly higher than that of component Pe2. This indicates that the Chlorella enzymatic hydrolysate component with better pancreatic lipase inhibition activity can be obtained after separation by Sephadex G-25 gel chromatography column.

[0071]

[0072] Example 4: Isolation, purification, and sequence identification of peptide components from protein hydrolysates of mutagenic Chlorella strains.

[0073] The fraction Pe1, which was purified from gel filtration chromatography and had high inhibition of pancreatic lipase activity, was identified as a peptide by RPLC-MS. The mass spectrometry data was analyzed by PEAKS software to identify the peptide sequence. The specific steps were as follows: (1) First, the sample was desalted using the ZipTip C18 method. The sample was dissolved in 0.1% trifluoroacetic acid (TFA). The TIP head was rinsed 10 times with a mixture of 50 μL of 60% acetonitrile (ACN) (abbreviated as 60% ACN) and 0.1% TFA (abbreviated as 0.1% TFA). Then, it was washed 10 times with 10 μL of 0.1% TFA. The sample was aspirated into the TIP head and discharged. This operation was repeated 20 times. Then wash 5 times with 10 μL 0.1% TFA, and finally elute the peptide with a mixture of 10 μL 60% ACN and 0.1% TFA, and vacuum dry; (2) The RPLC-MS detection steps are as follows: dissolve the peptide in 20 μL of dissolving solution (0.1% formic acid), vortex, centrifuge at 4℃ and 17000 rpm for 20 min, and take the supernatant for mass spectrometry identification. The chromatographic conditions are as follows: the chromatographic column used is PepMap RSLC C18, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile solution, and the database is further searched using PEAKS software to obtain the purified peptide sequence. Figure 5 The total ion chromatogram results show that the Pe1 component was well separated, with a total of 7358 polypeptide sequences identified. The molecular weights of the polypeptides were mainly distributed between 0.3 and 1.0 kDa. Figure 5 (b) and mainly concentrated between 5-17 amino acids, with peptides of 8 amino acids accounting for 12.08% and peptides of 7 amino acids accounting for 12.04%. Figure 5 c).

[0074] Example 5: Screening of pancreatic lipase activity inhibitory peptides induced by mutagenesis in Chlorella strains

[0075] (1) Computer-aided screening: Among the identified peptide sequences, screening was first performed based on a confidence level (ALC) greater than 95%, followed by prediction of bioactivity and physicochemical properties. The Peptide Ranker tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict and score the bioactivity of peptides, with a Peptide Ranker score threshold of 0.5. The AllerTOP tool (https: / / ddg-pharmfac.net / allertop_test / method_description / ) was used to assess peptide sensitization. The ToxinPred tool (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to assess peptide toxicity. The Innovagen tool (http: / / www.innovagen.com / proteomics-tools) was used to predict peptide solubility and isoelectric point. Based on the confidence level assessment, 108 peptide sequences with a confidence level of over 95% were selected from the 7358 peptides in Example 4. Using Peptide Ranker analysis, 32 peptides with potential biological activity were further screened from these 108 peptides. AllerTOP, ToxinPred, and Innovagen tools were then used to predict the physicochemical properties of these 32 peptides, including sensitization, toxicity, solubility, and isoelectric point. Finally, 12 non-toxic, non-sensitizing, and well-soluble peptides were selected (as shown in Table 2).

[0076] Table 2. Amino acid sequences and predicted physicochemical properties of peptides identified by mutagenesis of Chlorella.

[0077]

[0078] (2) Molecular docking technology screening: The 12 peptides obtained by computer-aided screening were further screened using molecular docking technology, specifically: 1) Receptor protein preparation: The crystal structure of pancreatic lipase (PDB number: 1LPB) was obtained from the PDB database (https: / / www.rcsb.org). The protein was optimized using the pymol molecular visualization system to remove bound small molecule ligands, redundant protein subunits, and irrelevant components such as water of crystallization. Subsequently, hydrogen atoms were added to the protein using AutoDocker Tools software, and finally, a receptor file (pdbqt format) that meets the docking requirements was generated; 2) Ligand preparation: The two-dimensional structure of the peptide was constructed using Chem Draw chemical drawing software, and molecular mechanics optimization was performed using the Chem 3D module to obtain a stable three-dimensional conformation. Then, the ligand molecular file (pdbqt format) was finally output using AutoDocker Tools software to supplement hydrogen atoms, combine nonpolar hydrogens, and retain the default torsion bond settings; 3) Molecular docking: After loading the receptor and ligand files in AutoDocker Tools software, the docking parameters were set. The spatial coordinates of the active site were determined and the volume parameters of the binding cavity were adjusted. A semi-flexible docking mode was selected, generating 25 binding conformations for each ligand molecule. Other parameters were set to system preset values. After docking, the complex system with the lowest binding free energy was analyzed using Discovery Studio software to study the interaction between the ligand and the amino acid residues at the receptor active site. The results are shown in Table 3.

[0079] Table 3. Molecular docking of identified peptides from mutagenic Chlorella with pancreatic lipase-encoded proteins.

[0080]

[0081] Table 3 shows that five peptides, AGVAGPK, FVDLLK, DRGEGR, SGLDGAK, and TGPLGSR, can dock with pancreatic lipase, with binding energies of -5.15, -2.39, -4.50, -4.94, and -5.60 kcal / mol, respectively. Visual analysis of the docking results using Discovery Studio software revealed that pancreatic lipase forms hydrogen bonds with peptide AGVAVPK at PHE77, ASP79, CYS237, CYS238, LYS239, PHE258, ALA259, and CYS261 sites; with FVDLLK at ASP205 and LYS238 sites; with QGPVGRS at PRO46, ASN47, LYS91, and LYS95 sites; and with SGLDGAK at ASN261. 12. Hydrogen bonds were formed at LEU213, LYS238, LYS239, GLN244, PHE258, and ASN262, and hydrogen bonds were formed with TGPLGSR at ASN212, LYS238, GLN244, PHE258, and ALA259. This indicates that the five polypeptides AGVAGPK, FVDLLK, DRGEGR, SGLDGAK, and TGPLGSR have a strong binding affinity to pancreatic lipase, thereby inhibiting pancreatic lipase activity and reducing the body's absorption of fat.

[0082] (3) Screening of Chlorella peptides by docking the matrix metalloproteinase-9 (MMP-9) protein, a core gene for obesity development, with the ligands of the above-mentioned five Chlorella pancreatic lipase inhibitory peptides: Based on the molecular docking method described in step (2) above, the above-mentioned five Chlorella pancreatic lipase inhibitory peptide ligands were docked with the receptor of the MMP-9 protein, a core gene for obesity development, to further screen Chlorella pancreatic lipase inhibitory peptides. The crystal structure of the protein encoded by the core gene for obesity was downloaded from the PDB database. The relevant information was MMP-9 (PDB ID: 1L6J), and docking was performed one by one with the five potential Chlorella pancreatic lipase inhibitory peptides identified above. The results are shown in Table 4.

[0083] Table 4. Mutagenic docking of peptides identified in Chlorella vulgaris with the protein encoded by MMP-9, a core gene for obesity development.

[0084]

[0085] As shown in Table 4, among the five peptides, only AGVAVPK, SGLDGAK and TGPLGSR successfully docked with the protein encoded by the MMP-9 gene, with binding energies of -3.87, -1.98 and -1.79 kcal / mol, respectively, all of which are lower than -1.2 kcal / mol. The docking results were then imported into Discovery Studio for visualization analysis. It was found that the core obesity gene MMP-9 formed hydrogen bonds with the peptide AGVAVPK at ASP249, ARG265, ASP331, and SER333 sites; with SGLDGAK at LEU41, TRP85, and LYS239 sites; and with TGPLGSR at LYS239, GLN244, ASP247, and ARG265 sites. This indicates that MMP-9 has a strong binding affinity to the AGVAVPK, SGLDGAK, and TGPLGSR proteins, suggesting that the potential target of these three pancreatic lipase inhibitory peptides is MMP-9. This suggests that these three peptides may regulate the expression of MMP-9 protein in obese cells, thereby achieving a lipid-lowering effect.

[0086] Example 6: Screening of Chlorella heptapeptide TGPLGSR by inhibiting lipase activity

[0087] Based on the pancreatic lipase activity inhibition assay method described in step (4) of Example 3, the above three peptides AGVAVPK, SGLDGAK, and TGPLGSR were applied to pancreatic lipase to investigate their inhibitory activity against pancreatic lipase. The IC50 was analyzed using Origin 2024 software to generate graphs. 50 Value. Further, by preparing different concentrations of polypeptide solutions and mixing them with 1 mg / mL pancreatic lipase solution, incubating at 37℃ for 30 min, adding different concentrations of PNPB (1, 2.5, 5, 7.5, 10 mM) for reaction, measuring absorbance values ​​every minute, calculating reaction rate (V), and plotting straight lines at different concentrations using the Linerweave-Burk double reciprocal method, the type of inhibition of pancreatic lipase by Chlorella polypeptide was determined by formula (3).

[0088]

[0089] Where v0 is the initial velocity of the enzyme-catalyzed reaction, in mol / (L·s); V max The maximum reaction rate is expressed in mol / (L·s); K m [S] is the Michaelis constant, in mol / L; [S] is the substrate concentration, in mol / L.

[0090] Depend on Figure 6It was found that among the three peptides, TGPLGSR peptide exhibited the most significant inhibitory activity against pancreatic lipase, with an inhibition rate as high as 84.63%. The half-maximal inhibitory concentrations (WMCs) of AGVAVPK, SGLDGAK, and TGPLGSR peptides were 28.49 mg / mL, 35.89 mg / mL, and 3.83 mg / mL, respectively, indicating that TGPLGSR showed excellent inhibitory activity, and its WMC was significantly lower than that of the other two peptides. Figure 7 As shown in the figure. Furthermore, with increasing concentration of the peptide TGPLGSR, the maximum reaction rate (VT) increases. max ) remains unchanged, and the Michaelis constant (K) m The increase in ) indicates that the inhibition mode of the peptide TGPLGSR is competitive inhibition ( Figure 8 (As shown).

[0091] Furthermore, compared with other reported microalgal-derived protein peptides (as shown in Table 5), the heptapeptide TGPLGSR of this invention has a lower binding energy to the pancreatic lipase-encoded protein molecule, indicating that it has higher pancreatic lipase inhibitory activity. Moreover, its hydrogen bond binding sites with the pancreatic lipase-encoded protein molecule differ from other protein peptides, specifically: LPLL binds to pancreatic lipase at CYS238, CYS262, GLN239, ASN263, ASN213, LYS233, VAL260, and P... A hydrogen bond is formed at HE259; FLGF forms a hydrogen bond with pancreatic lipase at GLN239; YPFW forms hydrogen bonds with pancreatic lipase at PHE259, CYS238, GLN239, VAL260, and PHE259; YYGR forms hydrogen bonds with pancreatic lipase at ALA208, VAL260, CYS262, CYS238, LEU214, and ASP206; FLQR forms a hydrogen bond with pancreatic lipase at GLN22. Furthermore, the secondary spatial conformation of the heptapeptide TGPLGSR of this invention, compared to other reported microalgal-derived protein peptides, shows that… Figure 9 As shown in Table 5, the total number of hydrogen bonds and the number of parallel β-sheet hydrogen bonds of the heptapeptide TGPLGSR of the present invention are 40 and 36, respectively, which are more than those of other microalgae-derived protein peptides. This indicates that the heptapeptide TGPLGSR of the present invention has a more stable structure and higher activity than other algae-derived protein peptides.

[0092] Table 5. Docking and secondary structure spatial conformation of microalgal protein peptides from different sources with pancreatic lipase-encoded proteins.

[0093]

[0094] The protein peptides represented by ae are all from the reported literature "Lin ML, Zeng QH, Yang JX, Huang JY, Zhou MS, Lin ZH, Zhang ZH, Xu FY, Wang JJ. Spirulina derived peptides: Inhibition mechanism on pancreatic lipase and impact on lipid-lowering effects of Caenorhabditis elegans. International Journal of Biological Macromolecules, 2025, 319: 145403".

[0095] Example 7: Analysis of the effect of Chlorella heptapeptide TGPLGSR on the differentiation of mouse 3T3-L1 preadipocytes

[0096] (1) Culture of mouse 3T3-L1 preadipocytes: 3T3-L1 preadipocytes were taken from liquid nitrogen and thawed slowly by shaking in a 37°C water bath. The completely thawed cell suspension was transferred to a culture dish containing complete culture medium (90% DMEM and 10% fetal bovine serum) and cultured in a 37°C, 5% CO2 incubator. The culture medium was replaced after 24 h, and the cell growth status was observed. When the cells reached 80-90% confluence in the culture dish, they were passaged. The culture medium was then removed, and the cells were washed 1-2 times with preheated PBS buffer. 1 mL of trypsin-EDTA digestion solution preheated to 37°C was added, and the cells were incubated at 37°C for 1-3 min. The culture flask was inverted and the cells were left to stand for 10-30 s. The cell morphology was observed. When more than 80% of the cells became rounded, the cells were gently tapped to detach from the flask. A small amount of complete culture medium was added to stop the digestion, and then 8-10 mL of complete culture medium was added. After shaking well, half of the cell suspension was transferred to a new culture flask for continued culture.

[0097] (2) Construction of mouse 3T3-L1 mature adipocyte model: The density of 3T3-L1 preadipocytes was adjusted to 2×10 5Adipocytes were seeded at a density of 1 mL / well in 12-well plates and cultured at 37°C in a 5% CO2 incubator until 95-100% confluence. The culture medium was then removed, and the cells were cultured in induction medium for 48 h, with the medium changed every 48 h for approximately 10 days. Adipocyte maturation was observed in four groups: control group (treated with a specific inducer for 12-14 days); low-dose group (treated with 1.6 mg / mL of TGPLGSR in combination with the inducer until induction was complete); medium-dose group (treated with 3.2 mg / mL of TGPLGSR in combination with the inducer until induction was complete); and high-dose group (treated with 4.8 mg / mL of TGPLGSR in combination with the inducer until the entire induction phase was completed).

[0098] (3) Oil Red O staining of Chlorella heptapeptide TGPLGSR in 3T3-L1 cells: Saturated Oil Red O stock solution was mixed with distilled water at a volume ratio of 6:4, shaken to mix, and placed in a 4℃ refrigerator for 12 h. The next day, the solution was initially filtered using qualitative filter paper, and then filtered a second time after standing at 4℃ for 24 h. The resulting solution was Oil Red O working solution. The 12-well plates after induction were removed, the culture medium was discarded, and the cells were washed twice with PBS buffer. Universal tissue fixative was added and the cells were fixed at room temperature for 20 min. The fixative was discarded, and the cells were washed twice with PBS. A small amount of 60% isopropanol was added to each well, covering the cells for about 15-20 s, and then aspirated. Oil Red O working solution was added again, and the cells were stained at room temperature in the dark for 30 min. After staining, the staining solution was removed, and the cells were washed with 60% isopropanol for 3-5 s and then washed three times with pure water for 5 min each time. After staining, PBS was added to cover the cells and observed under a microscope.

[0099] Depend on Figure 10 The results showed that, compared with the control group, the low, medium, and high doses of Chlorella heptapeptide TGPLGSR significantly reduced the intracellular lipid content of differentiated cells, and this reduction effect was dose-dependent. Further analysis of the lipid droplet positive area ratio revealed that in the untreated group, the lipid droplet area ratio was as high as 78.05%, while in the groups treated with 1.6, 3.2, and 4.8 mg / mL TGPLGSR, the Oil Red O staining lipid droplet area ratios were 56.24%, 37.31%, and 29.75%, respectively, significantly reducing the Oil Red O staining level to 72%, 47%, and 38% of the control group. This indicates that Chlorella heptapeptide TGPLGSR at concentrations of 1.6, 3.2, and 4.8 mg / mL can significantly reduce the differentiation of 3T3-L1 preadipocytes.

[0100] Example 8: Analysis of the effects of Chlorella heptapeptide TGPLGSR on TG and TC content in mouse 3T3-L1 preadipocytes

[0101] Mature 3T3-L1 adipocytes were collected, mixed with RIPA lysis buffer and a triplet of enzyme inhibitors, and homogenized until completely lysed. The mixture was then centrifuged at 12000 rpm for 5 min, and the supernatant was collected. Following the kit instructions, the total cholesterol (TC) and triglyceride (TG) levels in each group of cells were measured using the total cholesterol and triglyceride assay kits, respectively. Figure 11 The results showed that, after complete differentiation, the TC and TG contents in 3T3-L1 preadipocytes treated with the peptide TGPLGSR were significantly lower than those in the control group. The TC and TG contents in the low, medium and high dose treatment groups were reduced by 9.85%, 21.01% and 36.55% and 18.42%, 34.60% and 52.79% respectively compared with the control group, indicating that Chlorella heptapeptide TGPLGSR can effectively inhibit the accumulation of TC and TG in 3T3-L1 mature adipocytes.

[0102] Example 9: Analysis of the effects of Chlorella heptapeptide TGPLGSR on related genes in mouse 3T3-L1 preadipocytes

[0103] (1) Cell pretreatment: Successfully differentiated 3T3-L1 preadipocytes were taken, the culture medium was removed, and the cells were washed with PBS. After digesting the cells with a cell scraper or trypsin, the cells were centrifuged at 12,000 rpm for 5 min and collected.

[0104] (2) Total RNA extraction: Add 1 mL of RNAiso PULS reagent to the cells, shake for 30 s and let stand for 5 min; add 200 μL of chloroform, mix and shake and let stand for 5 min; after centrifugation, take the supernatant, add an equal volume of propanol, mix and let stand for 5 min; centrifuge again, remove the supernatant, add 1 mL of 75% ethanol, mix and let stand at room temperature for 10 min; centrifuge to precipitate RNA, remove ethanol, let stand for 5 min and then dissolve the precipitate with 50 μL of DEPC water.

[0105] (3) cDNA synthesis: Prepare the cDNA synthesis reaction system according to Table 6, vortex to mix, and briefly centrifuge. Incubate at 42℃ for 15 min, then at 85℃ for 5 min. After incubation, briefly centrifuge and store at -20℃ for later use.

[0106] (4) Real-time quantitative PCR reaction: Prepare the qPCR reaction system according to Table 7 to start the real-time quantitative PCR reaction. The reaction program is as follows: pre-denaturation at 95℃ for 5 min; hold at 95℃ for 15 s; cool to 60℃ and hold for 30 s, repeat 40 times; hold at 60℃ for 2 min. Analyze the amplification curve and melting curve of the amplification products, and use 2 -△△CtThe method for calculating the fold change in gene expression is as follows: First, the difference in cycle number ΔCt between samples is calculated using the formula ΔCt = Ct (target gene) - Ct (internal reference gene). Second, the ΔΔCt value is calculated to compare the difference between the experimental group and the control group using the formula ΔΔCt = ΔCt (experimental group) - ΔCt (control group). Finally, the relative fold change in gene expression is calculated using the formula 2. -△△Ct The results indicate the fold change in gene expression levels in the experimental group relative to the control group.

[0107] Depend on Figure 12 The results showed that the relative expression levels of FAS in the low, medium, and high-dose treatment groups were significantly lower than those in the control group, decreasing by 29.3%, 75.3%, and 75.7%, respectively. However, there was no significant difference in the relative expression levels of FAS between the medium and high-dose treatment groups. This may be because FAS expression becomes insensitive to dose changes after reaching a certain threshold. This finding indicates that the addition of Chlorella heptapeptide TGPLGSR inhibited FAS expression. A similar phenomenon was observed in the expression of acetyl-CoA carboxylase (ACC). The relative expression levels of ACC in the low, medium, and high-dose treatment groups were significantly lower than those in the control group, decreasing by 48.7%, 77.6%, and 82.8%, respectively. Therefore, Chlorella heptapeptide TGPLGSR can reduce lipid levels in mouse 3T3-L1 preadipocytes by decreasing FAS and ACC expression.

[0108] Table 6 cDNA Synthesis Reaction System

[0109]

[0110] Table 7 qPCR reaction system

[0111]

[0112] Example 10: Analysis of the effects of Chlorella heptapeptide TGPLGSR on MMP-9 and related pathways in mouse 3T3-L1 preadipocytes

[0113] (1) Preparation and concentration determination of protein supernatant from mouse 3T3-L1 preadipocytes treated with Chlorella heptapeptide TGPLGSR: 3T3-L1 mature adipocytes were collected, and RIPA lysis buffer and enzyme inhibitor triplet were added and mixed. The mixture was then homogenized until the tissue was completely broken and homogeneous. The mixture was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected for later use. The protein concentration was determined using a BCA kit. Solution A and solution B were mixed at a volume ratio of 50:1 and reacted with standard or diluted protein extract. The mixture was incubated at 37°C for 30 min, and the absorbance was measured at 450 nm to calculate the protein concentration.

[0114] (2) Gel pouring: Prepare 10 mL of 12% separating gel, add TEMED and ammonium persulfate (APS), mix well, pour the gel to 2-3 mm below the comb, block with isopropanol, let stand for 45 min, and discard the deionized water. Prepare 5 mL of 5% stacking gel, add TEMED and APS, mix, pour the gel to the top of the glass plate, insert the comb, and let stand for 20 min. Remove the comb, fix the gel in the electrophoresis tank, and pour in the buffer solution.

[0115] (3) Electrophoresis: Mix the loading buffer and protein solution in equal volumes to prepare the loading solution, ensuring a final protein concentration of 1 mg / mL. Heat at 100℃ for 5 min, cool in an ice bath, and centrifuge. Add 20 μL of loading solution to each well, leaving one well for 10 μL of pre-stained marker. Add buffer to the electrophoresis tank and electrophores at a constant voltage of 70 V for about 30 min. When bromoflavin enters the separating gel, switch to 90 V until the indicator is 0.5 cm from the bottom of the gel. Turn off the power and remove the gel.

[0116] (4) Transferred proteins and immunoassay: Before electrophoresis, the PVDF membrane was soaked in methanol for 15 s, rinsed with ddH2O (double-distilled water, i.e., water purified by two distillations) for 2 min, and soaked in transfer buffer for 5 min. The gel was pried open, trimmed, and then placed in the buffer for equilibration for 15 min. The transfer "sandwich" was made in sequence, avoiding air bubbles. The positive and negative electrodes were connected, transfer buffer was added, and the membrane was transferred at a constant current of 200 mA in ice water for 70 min. After transfer, the PVDF membrane was removed and blocked in 5% BSA solution for 2 h. The membrane was washed with TBST solution for 5 min, and repeated 3 times. The primary antibody was added and incubated at 4℃ for 12 h. After washing with TBST, horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 2 h. The membrane was washed with TBST for 15 min, and repeated 5 times. The membrane was reacted with chemiluminescent reagent for 2 min and then placed in a BLT imaging system for photographic capture. The gray value was calculated using IPP 6.0.

[0117] (5) Detection of cellular inflammatory factor expression: The expression of relevant cellular inflammatory factors was detected using the Mouse IL6 ELISA kit and the Mouse TNFα ELISA kit according to the instructions. Cellular proteins were extracted and quantified according to step (1). After equilibrating the aluminum foil bag at room temperature for 20 min, the required strips were removed. Six standard wells and four sample wells were prepared. 50 μL of different concentrations of standard were added to the standard wells, and three replicates were set. 50 μL of the sample to be tested was added to the sample wells, and three replicates were set. Then, 100 μL of horseradish peroxidase-labeled detection antibody was added to each well, the plate was sealed with a sealing membrane, and incubated at 37℃ for 30 min. After incubation, the liquid was discarded, the plate was patted dry, each well was filled with washing buffer, and the plate was washed 5 times after standing for 1 min. 50 μL of substrate A and 50 μL of substrate B were added to each well, and the plate was incubated at 37℃ in the dark for 10-15 min. Finally, 50 μL of stop solution was added to all wells, and the OD value was measured at 450 nm.

[0118] Depend on Figure 13 It was observed that the expression level of MMP-9 protein in mouse 3T3-L1 preadipocytes significantly decreased from 3.7 in the control group to 2.29, 1.71, and 1.06, respectively, representing reductions of 37.3%, 53.3%, and 71.2%, and exhibiting a clear dose-dependent effect. This indicates that the chlorella heptapeptide TGPLGSR has a significant inhibitory effect on MMP-9 protein expression in adipocytes. With increasing chlorella heptapeptide dosage, the phosphorylation level of p-AMPK in mouse cells significantly increased, but the total AMPK protein level remained unchanged. Simultaneously, the expression level of PNLIP gradually decreased with increasing heptapeptide concentration. Figure 14The results showed that the levels of inflammatory cytokines TNF-α and IL-6 in the supernatant of 3T3-L1 cells treated with Chlorella heptapeptide decreased significantly with increasing heptapeptide dosage. These findings indicate that reduced MMP-9 protein expression in 3T3-L1 preadipocytes inhibits the expression of inflammatory cytokines TNF-α and IL-6. Furthermore, as the inflammatory response weakens, AMPK phosphorylation levels significantly increase, thereby inhibiting lipid production. In addition, the expression level of PNLIP, a key gene in lipid synthesis, was also significantly reduced, further demonstrating that the heptapeptide TGPLGSR can significantly reduce intracellular lipid production in mouse 3T3-L1 preadipocytes. Therefore, it is speculated that Chlorella heptapeptide TGPLGSR reduces the release of inflammatory factors (such as TNF-α and IL-6) by targeting and inhibiting the expression of MMP-9, thereby relieving the inhibition of AMPK by inflammation and activating its phosphorylation. The activated AMPK inhibits the synthesis of endogenous fatty acids by downregulating the expression of ACC and FAS, and at the same time downregulates the expression of the gene PNLIP to reduce the hydrolysis and absorption of exogenous food fat. Through the dual inhibition of endogenous and exogenous lipids, it ultimately reduces lipid accumulation and achieves the effect of weight loss.

[0119] Comparative Example 1: Comparison of protein content in Chlorella strains obtained by single ultraviolet irradiation mutagenesis or single electric shock mutagenesis

[0120] Steps: The remaining mutation parameters and procedures are the same as in Embodiment 1 of this invention.

[0121] The difference is that the wild-type Chlorella strain is subjected to only ultraviolet irradiation (parameters same as in Example 1) and not to electric shock; or it is subjected to only electric shock (parameters same as in Example 1) and not to ultraviolet irradiation.

[0122] According to the protein content determination method in Example 1, the final protein content of the Chlorella strains obtained by single UV irradiation mutagenesis or single electric shock mutagenesis was 30-35% and 42-47%, respectively. The experimental results show that the protein content of the composite mutagenesis strain (71-75%) is significantly higher than that of any single mutagenesis, confirming that physical composite mutagenesis produces a synergistic effect and is a prerequisite for obtaining highly active peptide raw materials.

[0123] Comparative Example 2: Comparison of protein extraction rate and separation / purification using a single method in mutagenic Chlorella strains

[0124] Steps: Use the same batch of mutagenic algal powder obtained by the present invention (algal powder acquisition and protein extraction, separation and purification are the same as in Example 2).

[0125] The differences are as follows: 1) Ethanol extraction only, omitting the subsequent cellulosic hydrolysis and physical cell wall disruption steps (A); 2) Or cellulosic hydrolysis only, omitting the ethanol extraction and physical cell wall disruption steps (B); 3) Or physical cell wall disruption only, omitting the ethanol extraction and cellulosic hydrolysis steps (C); 4) Or ethanol extraction and cellulosic hydrolysis, omitting the physical cell wall disruption steps (D); 5) Or ethanol extraction and physical cell wall disruption, omitting the cellulosic hydrolysis steps (E); 6) Or cellulosic hydrolysis and physical cell wall disruption, omitting the ethanol extraction steps (F).

[0126] Depend on Figure 15 The results showed that the crude protein content and purified protein content were 8% and 12% after ethanol extraction (A), 10% and 14% after cellulase hydrolysis (B), 18% and 26% after physical cell wall disruption (C), 14% and 19% after ethanol extraction and cellulase hydrolysis (D), 20% and 28% after ethanol extraction and physical cell wall disruption (E), and 34% and 41% after cellulase hydrolysis and physical cell wall disruption (F). These results indicate that the synergistic process of "ethanol pre-extraction + enzymatic hydrolysis + physical cell wall disruption" in this invention can release proteins more efficiently and completely, providing a higher quality substrate for subsequent enzymatic hydrolysis. This demonstrates that the combination is not simply an addition of steps, but rather a synergistic effect produced by the treatment of the Chlorella cell wall structure.

[0127] Comparative Example 3: Comparison of different enzymatic hydrolysis methods for Chlorella proteins

[0128] Step: Use the same batch of high-purity Chlorella protein as in this invention.

[0129] The differences are as follows: neutral protease was used for enzymatic hydrolysis at pH 7.0 and 50℃; pepsin was used for enzymatic hydrolysis at pH 2.0 and 37℃; trypsin was used for enzymatic hydrolysis at pH 7.0 and 37℃; and papain was used for enzymatic hydrolysis at pH 9.5 and 50℃. The experimentally determined degrees of protein hydrolysis by pepsin, trypsin, neutral protease, and papain were 15.86%, 21.49%, 22.79%, and 17.32%, respectively. Figure 16 As shown in Figure A), the inhibition rates of the enzymatic hydrolysates on pancreatic lipase activity were 12.57%, 25.06%, 15.59%, 28.88%, and 19.71%, respectively. Figure 16 (As shown in B). The results indicate that using alkaline protease produces a mixture of peptides with significantly higher inhibitory activity, suggesting that protease selection is crucial for releasing the target active fragment.

[0130] Comparative Example 4: Comparison of the activities of different isolated, purified, and screened components of Chlorella protein enzymatic hydrolysis products.

[0131] Steps: Use the same batch of alkaline protease hydrolysate.

[0132] The differences are as follows: the D-1 (MW < 3 kD), D-2 (MW = 3~5 kD), D-3 (MW = 5~10 kD) and D-4 (MW > 10 kD) fractions obtained by ultrafiltration; the Pe1 and Pe2 fractions obtained by Sephadex G-25 chromatography of the D-1 (MW < 3 kD) fraction; the heptapeptide TGPLGSR obtained by screening the Pe1 fraction; and the positive control groups simvastatin and orlistat.

[0133] The experiments showed that the inhibition rates of pancreatic lipase activity by fractions D-2, D-3, and D-4 were -3.99%, 11.62%, and 25.58%, respectively, all lower than the inhibition rate of fraction D-1 (29.31%). Furthermore, the Pe2 fraction obtained by chromatographic purification of fraction D-1 showed an inhibition rate of 39.26% on pancreatic lipase activity, higher than fraction D-1, but significantly lower than the Pe2 fraction's inhibition rate of 67.51%. The heptapeptide TGPLGSR fraction obtained from screening fraction Pe2 showed an inhibition rate of 84.63%. Compared with the clinical positive control group of simvastatin and orlistat, the Pe2 fraction's inhibition rate was significantly higher than simvastatin (30.54%) and roughly equivalent to orlistat (90.91%) (Table 8). The experimental results indicate that the specific sequence of "ultrafiltration (initial screening by molecular weight), followed by gel chromatography (further subdivision by properties), and regenerative immunoassay (fine screening)" can reduce impurity interference, optimize purification efficiency, and more efficiently enrich the target active peptides.

[0134] Table 8. Effects of different isolated, purified, and screened components of Chlorella alkaline protease hydrolysate on pancreatic lipase inhibition rate.

[0135]

Claims

1. A Chlorella heptapeptide TGPLGSR with multi-target lipid-lowering activity, characterized in that, The amino acid sequence of the Chlorella heptapeptide TGPLGSR is shown in SEQ ID NO: 1.

Images