Preparation method and application of spirulina platensis hangover alleviating and liver protecting peptide

Abstract

The invention discloses a preparation method and application of spirulina platensis hangover alleviating and liver protecting peptide, and belongs to the technical field of biology. According to the present invention, the active peptide with ethanol dehydrogenase activation and anti-oxidation dual functions is extracted from spirulina, has high DPPH free radical scavenging activity (86.18%), ABTS free radical scavenging activity (80.07%), in vitro ADH activation rate (126.80%) and small molecule peptide content (96.02%), and has good protection effect on early liver injury. Wherein the ADH activation rate of the key active peptide HPGIP reaches 176.06%, and the clearance rates of DPPH and ABTS are 93.36% and 87.12% respectively. The anti-alcoholism and liver-protecting effects are evaluated from multiple dimensions of metabolism promotion, oxidation inhibition and anti-inflammatory regulation, and a theoretical basis and a technical path are provided for developing a new generation of efficient and safe liver-protecting functional food.

Description

Technical Field

[0001] This invention relates to a method for preparing and applying a Spirulina platensis hangover-relieving and liver-protecting peptide, belonging to the field of biotechnology. Background Technology

[0002] Alcohol, a substance widely consumed globally, has drawn significant international attention due to its public health impact. Improper alcohol consumption can damage the liver, leading to various diseases. Research into substances with hepatoprotective effects is crucial for the prevention and treatment of alcoholic liver injury. Existing chemically synthesized drugs generally carry risks of toxic side effects and lack effective interventions targeting disease progression. Against this backdrop, the development of natural hepatoprotective agents has become a research hotspot. Due to their natural origin, high biocompatibility, and multi-target regulatory properties, they exhibit unique advantages in the field of hangover relief and liver protection.

[0003] Spirulina, as a natural bioactive substance, possesses proteins with significant bioactivity advantages. In recent years, research on spirulina bioactive peptides has shown breakthroughs in multiple fields, demonstrating significant functions in antioxidation, anti-inflammation, immune regulation, and metabolic regulation. However, its effectiveness in relieving hangovers and protecting the liver remains unknown. Therefore, further in-depth exploration of its specific mechanisms of action and efficacy verification is urgently needed. Based on this, this invention focuses on spirulina peptides, using multi-omics techniques to screen and identify peptides with hangover-relieving and liver-protecting effects, aiming to expand the high-value applications of spirulina. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention provides a Spirulina platensis hangover-relieving and liver-protecting peptide and its preparation method, aiming to solve the technical problem of how to obtain an active polypeptide with hangover-relieving and liver-protecting effects from Spirulina.

[0005] The first technical solution provided by the present invention is an active peptide of Spirulina platensis, wherein the amino acid sequence of the active peptide of Spirulina platensis is HGPIP, SMP, IPGI or IGIP.

[0006] This invention also provides a method for preparing the above-mentioned Spirulina platensis active peptides, the method comprising the following steps: (1) Mix the spirulina powder with distilled water, stir, and then use ultrasound to break up the cell walls; (2) Centrifuge the cell wall disruption liquid obtained in step (1), take the supernatant, freeze dry to obtain spirulina protein, and store at 0~15℃; (3) Take 2-6g of the spirulina protein obtained in step (2) and dissolve it in deionized water. Adjust the temperature and pH of the solution to the optimal conditions for the enzyme, add the protease, react for a period of time, and then use a water bath to inactivate the enzyme and stop the enzymatic hydrolysis. (4) Centrifuge the enzymatic hydrolysate obtained in step (3), take the supernatant, and obtain the spirulina enzymatic hydrolysate.

[0007] (5) The spirulina hydrolysate obtained in step (4) is subjected to ultrafiltration, the filtrate is collected, concentrated by rotary evaporation, and freeze-dried to obtain spirulina hydrolysate freeze-dried powder, which is stored at -20℃ for later use.

[0008] (6) The amino acid sequence of the ultrafiltration fraction obtained in step (5) was identified by ultra-high performance liquid chromatography and linear ion trap-Orbitrap tandem mass spectrometry. (7) The peptides obtained from amino acid sequence identification were pre-screened under the following conditions: peptide score (>25), relative abundance (>200w), and peptide length (<10). Computer-aided simulations were used to study the potential biological activity, free radical scavenging activity, chelating ability score, toxicity, and resistance to gastrointestinal digestive enzymes of the peptides. Target peptides with potential hangover relief and liver protection functions were screened out. (8) The target peptides screened in step (7) are synthesized in vitro, and their in vitro activity is tested, including ADH activation rate and antioxidant capacity.

[0009] In one embodiment, in step (1), the mass ratio of spirulina powder to water is 1:10 to 1:30, and the mixture is stirred and ultrasonically broken down. The ultrasonic power is 400 to 600 W and the ultrasonic time is 18 to 25 min.

[0010] In one embodiment, in step (2), the cell wall-breaking liquid obtained in step (1) is centrifuged at a temperature of 0-10°C, a speed of 6000-9000 r / min, and a time of 18-25 min, preferably 8000 r / min for 20 min. The supernatant is then taken and freeze-dried to obtain spirulina protein.

[0011] In one embodiment, in step (3), the spirulina protein obtained in step (2) is dissolved in deionized water, the temperature of the solution is adjusted to 40-60℃ and the pH to 7-8, protease is added, and after reacting for 2-6 hours, the enzyme is inactivated by water bath for 8-10 minutes, and the enzymatic hydrolysis is stopped. The water bath temperature is 100℃.

[0012] In one embodiment, in step (4), the centrifugation speed is 6000~9000 r / min and the centrifugation time is 15~25 min.

[0013] In one embodiment, in step (5), the pressure is set to 0.2 MPa. First, 20 g / L of spirulina hydrolysate is passed through a microfiltration membrane with a pore size of 0.45 μm, and then the filtrate is further purified through a microfiltration membrane with a pore size of 0.22 μm. Finally, it is filtered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa at a filtration pressure of 0.7 MPa. The filtrate is collected, concentrated by rotary evaporation, and freeze-dried to obtain lyophilized spirulina hydrolysate powder, which is stored at -20°C for later use.

[0014] In one embodiment, in step (6), ultrafiltration components are identified using ultra-high performance liquid chromatography and linear ion trap-Orbitrap tandem mass spectrometry. Sample pretreatment: Accurately weigh 3 mg of sample, add 300 μL of ultrapure water and shake to dissolve. Take 10 μL of the dissolved sample and dilute it 10 times with 90 μL of ultrapure water. Measure the sample concentration using NanoDrop. Take 50 μg of the solution sample and make up the volume to 100 μL with ultrapure water. Add 1 μL of 1M DTT to make the final DTT concentration 10 mmol / L, and reduce it in a 56℃ water bath for 1 h; add 2 μL of 1M IAM solution to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min; add 1 μL of 1M DTT solution to make the final DTT concentration 20 mmol / L to neutralize unreacted IAM. Desalt using C18 stage-tip and dry under vacuum at 45℃. LC-MS / MS Detection: After the sample entered the liquid chromatography system, it was first separated using a Reprosil-Pur 120C18-AQ analytical column (150 μm × 170 mm, 1.9 μm). The mobile phase was: A: 0.1% formic acid aqueous solution; B: 0.1% formic acid aqueous solution + 80% acetonitrile, with a flow rate of 600 nL / min and an elution program of: 0–2 min, 4–8% B; 2–45 min, 8–28% B; 45–55 min, 28–40% B; 55–56 min, 40–95% B; 56–66 min, 95% B. The peptide fractions separated by liquid chromatography were then sequentially introduced into the mass spectrometry system, where electrospray ionization (ESI) was used to convert the peptide fractions into charged ions. The primary mass spectrometry was performed in positive ion full scan mode, with a range of 100–1500 m / z, an accumulation time of 100 ms, and a resolution of 70,000. The top 20 components with the highest signal intensity were selected and dissociated through collision-induced fragmentation to generate fragment ions. A data-dependent scanning mode was used with a resolution of 17,500 nm, a normalized fragmentation energy of 28 μM, and a cumulative time of 50 ms. The raw mass spectrometry files were retrieved from the target protein database using software.

[0015] In one embodiment, in step (7), the peptides obtained by amino acid sequence identification are pre-screened. The screening conditions are peptide score (>25), relative abundance (>200w), and peptide length (<10). The potential biological activity of the screened peptides is predicted by computer-aided simulation. PeptideRanker software (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict potentially bioactive peptide fragments; AnOxPePred software (http: / / services.healthtech.dtu.dk / services / AnOxPePred-1.0 / ) was used to predict potentially antioxidant peptide fragments; ToxinPred software (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) was used to screen for peptides with therapeutic potential and no toxicity; ExPASy Peptide Cutter software (http: / / web.expasy.org / peptidecutter / ) was used to simulate the breakdown process of peptides in the gastrointestinal environment, with chymotrypsin, pepsin (corresponding to pH=1.3 and pH>2, respectively), and trypsin selected as references.

[0016] In one embodiment, in step (8), a target peptide with a purity of 95% is synthesized, and the antioxidant activity and in vitro ADH activation rate of the synthesized peptide are detected.

[0017] The second technical solution provided by this invention is a method for preparing Spirulina platensis enzymatic hydrolysate, the method comprising the following steps: (1) Mix the spirulina powder with distilled water, stir, and then use ultrasound to break up the cell walls; (2) Centrifuge the cell wall disruption liquid obtained in step (1), take the supernatant, freeze dry to obtain spirulina protein, and store at 0~15℃; (3) Take 2-6g of the spirulina protein obtained in step (2) and dissolve it in deionized water. Adjust the temperature and pH of the solution to the optimal conditions for the enzyme, add the protease, react for a period of time, and then use a water bath to inactivate the enzyme and stop the enzymatic hydrolysis. (4) Centrifuge the enzymatic hydrolysate obtained in step (3), take the supernatant, and obtain the spirulina enzymatic hydrolysate.

[0018] In one embodiment, in step (1), the mass ratio of spirulina powder to water is 1:10 to 1:30, and the mixture is stirred and ultrasonically broken down. The ultrasonic power is 400 to 600 W and the ultrasonic time is 18 to 25 min.

[0019] In one embodiment, in step (2), the cell wall-breaking liquid obtained in step (1) is centrifuged at a temperature of 0-10°C, a speed of 6000-9000 r / min, and a time of 18-25 min, preferably 8000 r / min for 20 min. The supernatant is then taken and freeze-dried to obtain spirulina protein.

[0020] In one embodiment, in step (3), the spirulina protein obtained in step (2) is dissolved in deionized water, the temperature of the solution is adjusted to 40-60℃ and the pH to 7-8, protease is added, and after reacting for 2-6 hours, the enzyme is inactivated by water bath for 8-10 minutes, and the enzymatic hydrolysis is stopped. The water bath temperature is 100℃.

[0021] In one embodiment, in step (4), the centrifugation speed is 6000~9000 r / min and the centrifugation time is 15~25 min.

[0022] The third technical solution provided by the present invention is a Spirulina platensis enzymatic hydrolysate prepared by the method described in the second technical solution, wherein the Spirulina platensis enzymatic hydrolysate contains one or more Spirulina platensis active peptides with amino acid sequences of HGPIP, SMP, IPGI and IGIP.

[0023] The fourth technical solution provided by the present invention is a product containing Spirulina platensis active peptides with amino acid sequences such as HGPIP, SMP, IPGI and / or IGIP.

[0024] The fifth technical solution provided by this invention is the application of the Spirulina platensis active peptide described in the first technical solution, or the method described in the second technical solution, or the Spirulina platensis enzymatic hydrolysate described in the third technical solution, or the product described in the fourth technical solution, in the preparation of a drug for relieving hangovers and protecting the liver.

[0025] The sixth technical solution provided by the present invention is the application of the Spirulina platensis active peptide described in the first technical solution, or the method described in the second technical solution, or the Spirulina platensis enzymatic hydrolysate described in the third technical solution, or the product described in the fourth technical solution, in the preparation of a medicine for relieving, reducing and / or treating alcohol-induced liver damage.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This study extracted active peptides with dual functions of alcohol dehydrogenase activation and antioxidant from Spirulina, providing reference and guidance for the deep processing of Spirulina and the development of drugs with hangover relief and liver protection functions.

[0027] (2) The Spirulina hydrolysate obtained in this invention has high DPPH free radical scavenging activity (86.18%), ABTS free radical scavenging activity (80.07%), in vitro ADH activation rate (126.80%) and small molecule peptide content (96.02%), and shows good protective effect against early liver injury.

[0028] (3) The key active peptides of the spirulina protein obtained in this invention were identified and their synthesis verified. The key active peptide HPGIP was obtained, with an ADH activation rate of 176.06%, which is the highest value reported so far. At the same time, HPGIP has high antioxidant activity, with DPPH and ABTS scavenging rates of 93.36% and 87.12%, respectively. Compared with existing studies, the breakthrough of this invention lies in the first discovery that the peptide HPGIP has both super ADH activation and free radical scavenging capabilities, and its activity far exceeds that of traditional plant / animal-derived peptides.

[0029] (4) This invention optimizes the hydrolysis process of spirulina protein through multi-index screening and obtains target molecular weight peptides by ultrafiltration separation; and establishes a three-level evaluation system of “in vitro ADH activation-acute alcohol poisoning model-chronic liver injury model” to evaluate the liver protection efficacy from multiple dimensions of metabolism promotion, oxidation inhibition and anti-inflammatory regulation, providing a theoretical basis and technical path for the development of a new generation of highly efficient and safe liver protection functional foods. Attached Figure Description

[0030] Figure 1 Flowchart for modeling early ALD in mice. Detailed Implementation

[0031] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] In this invention, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, wherein each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0034] In this invention, polypeptides are represented by consecutive single-letter abbreviations of IUPAC. For example, Spirulina hangover relief and liver protection peptide HPGIP represents a polypeptide with a sequence composition of histidine-proline-glycine-isoleucine-cysteine.

[0035] The following embodiments involve test methods: (1) Determination of DPPH (diphenol) free radical scavenging ability. 100 μL of sample and 100 μL of DPPH working solution were added to 96-well plates, with final sample concentrations of 0.5, 1, 2.5, 5, and 10 mg / mL, respectively. The microplate reader was preheated to 37℃, and the reaction was carried out for 30 min in the dark. The absorbance was measured at 517 nm. The blank control used the sample solvent. The formula for calculating the DPPH free radical scavenging rate is: DPPH free radical scavenging rate (%) = (A 空白 -A 样品 )÷A 样品 ×100 In the above formula, A 空白 The absorbance value represents the control group; while A 样品 This indicates the absorbance after adding different sample solutions.

[0036] (2) The ABTS free radical scavenging ability was determined using the ABTS free radical scavenging activity test kit. For the interference-free control group, an equal amount of PBS diluted with water was used as a reference. A linear regression graph from low to high concentration was generated using Trolox reference material. This graph covered the range of 0.15-0.9 mM and was used as the sample absorbance value to input into the corresponding standardized equation to calculate the mass fraction of the corresponding standard compound. The final result was converted into TEAC form containing millimoles per gram and displayed.

[0037] (3) In vitro ADH (alcohol dehydrogenase) activation rate determination. The procedure was performed according to the ADH kit instructions. The sample solution (50 μL) was mixed with the working solution (150 μL). After incubation at 37°C for 5 min, ADH solution (0.2 U / mL, 50 μL) was added to initiate the reaction. The absorbance was measured using a microplate reader at a wavelength of 340 nm, measured every minute for 10 min. Distilled water was used instead of ADH solution in the background group, distilled water in the control group instead of the sample solution, and distilled water in the blank group instead of both ADH solution and sample solution. The initial reaction rate and ADH activation rate were calculated. The formulas for calculating the initial reaction rate and ADH activation rate are as follows: Initial reaction rate = ΔA 340 / Δ minutes

[0038] (4) Determination of serum-related biochemical indicators: After the whole blood sample was left to stand at 4℃ for 8 h, it was centrifuged at 2000 r / min for 20 min under the same temperature conditions. The supernatant was taken and the serum sample was taken to determine the serum indicators of mice according to the instructions of the Nanjing Jiancheng Biotechnology Co., Ltd. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) assay kit.

[0039] (5) Determination of liver-related biochemical indicators: Weigh a small piece of liver tissue and add 4℃ physiological saline at a ratio of 1:9 (g: mL) to make a 10% liver tissue homogenate. After centrifugation at 3000 r / min and 4℃ for 10 min, take the supernatant and determine the concentration of ADH, ALDH, MDA, SOD, GSH and total protein in mouse liver according to the kit instructions.

[0040] Raw materials used in the examples: Spirulina powder, originating from Chenghai Lake, Yunnan; Haiwang Jinzun tablets, Shenzhen Haiwang Group Co., Ltd.; Hongxing Erguotou (56% vol), Hongxing Co., Ltd.; Alcoholic liquid feed, Nantong Trofi Feed Technology Co., Ltd.; Alkaline protease, neutral protease, pepsin, trypsin, papain, flavor protease, bromelain, as well as DPPH and ABTS test kits, Shanghai Yuanye Biotechnology Co., Ltd.; PierceBCA protein test kit, Thermo Fisher Scientific (China) Co., Ltd.; 17 amino acid standards, 5 molecular weight standards, alcohol dehydrogenase, chromatographic grade acetonitrile, formic acid, ammonium bicarbonate (NH4HCO3), analytical grade dithiothreitol (DTT) and iodoacetamide (IAM), Sigma-Aldrich, USA; ALT, AST, triglycerides, total cholesterol, alcohol dehydrogenase, aldehyde dehydrogenase, superoxide dismutase, and glutathione assay kits, Nanjing Jiancheng Bioengineering Institute; Malondialdehyde test kit, Shanghai Beyotime Biotechnology Co., Ltd. Beijing Vital River Laboratory Animal Technology Co., Ltd. purchased 120 SPF-grade male C57BL / 6J mice, 8 weeks old, weighing between 20-22 g. These mice were raised at a temperature of (23 ± 2)℃, a humidity of 50%~60%, and a 12h:12h day-night cycle, with access to water at any time. The ethical approval number for the animal experiment was JN.NO20250113C060022

[004] , approved by the Animal Experiment Ethics Review Committee of Jiangnan University.

[0041] Example 1 Weigh 10 g of spirulina powder and mix thoroughly with 200 mL of distilled water. Stir and then perform ultrasonic cell disruption at a power of 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, adjust the temperature to 50℃ and pH to 7.5, add 0.75 g of flavor protease, and hydrolyze for 4 h. Then, inactivate the enzyme by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0042] Comparative Example 1: (Changing the type of protease) Weigh 10 g of spirulina powder and mix thoroughly with 200 mL of distilled water. Stir and then perform ultrasonic cell disruption at a power of 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, adjust the temperature to 37℃ and pH to 3.0, add 1 g of pepsin, and hydrolyze for 4 h. Then, inactivate the enzyme by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0043] Comparative Example 2: (Changing the type of protease) Weigh 10 g of spirulina powder and mix thoroughly with 200 mL of distilled water. Stir and then sonicate to disrupt the cell wall using 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, adjust the temperature to 37℃ and pH to 10.0, add 1 g of alkaline protease, and hydrolyze for 4 h. Then, inactivate the enzyme by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0044] Comparative Example 3: (Changing the hydrolysis time of flavor protease) Weigh 10 g of spirulina powder and mix thoroughly with 200 mL of distilled water. Stir and then perform ultrasonic cell disruption at a power of 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, then adjust the optimal conditions for various enzymes to 50℃ and pH 7.5. Add 0.75 g of flavor protease, and after 2 h of enzymatic hydrolysis, inactivate the enzymes by bathing at 100℃ for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0045] Comparative Example 4: (Changing the hydrolysis time of flavor protease) Weigh 10 g of spirulina powder and mix thoroughly with 200 mL of distilled water. Stir and then perform ultrasonic cell disruption at a power of 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, then adjust the optimal conditions for various enzymes to 50℃ and pH 7.5. Add 0.75 g of flavor protease, and after 6 h of enzymatic hydrolysis, inactivate the enzymes by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0046] Comparative Example 5: (Changing the cell wall breaking method) Grinding and cell wall disruption. Weigh 10 g of spirulina powder, grind for 30 min, then add 200 mL of water and mix thoroughly. Collect the cell wall disruption liquid, centrifuge at 4℃ and 8000 r / min for 20 min, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, then adjust the optimal conditions for various enzymes to 50℃ and pH 7.5, add 0.75 g of flavor protease, and hydrolyze for 1 h. After hydrolysis, inactivate the enzymes by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Samples were taken, and the antioxidant activity of the spirulina protein hydrolysate was compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0047] Comparative Example 6: (Changing the cell wall breaking method) Enzymatic hydrolysis was performed to break down the cell walls. 10 g of Spirulina was added to 200 mL of distilled water, along with 2% cellulase. The pH was set to 5.5 and the temperature to 50℃, and enzymatic hydrolysis was carried out for 5 h. The broken-down liquid was collected, centrifuged at 8000 r / min for 20 min at 4℃, and the supernatant was collected. The supernatant was then freeze-dried to obtain Spirulina protein, which was stored at 4℃ for later use. 3 g of Spirulina protein was dissolved in deionized water, and the optimal conditions for various enzymes were adjusted to 50℃ and pH 7.5. 0.75 g of flavor protease was added, and enzymatic hydrolysis was performed for 1 h. The enzymes were then inactivated by a 100℃ water bath for 10 min to stop the hydrolysis. The hydrolysate was centrifuged at 8000 r / min for 20 min, and the supernatant was obtained as the Spirulina protein hydrolysate. Samples were taken, and the antioxidant activities of the Spirulina protein hydrolysate were compared: DPPH free radical scavenging capacity and ABTS free radical scavenging rate were determined. ADH activation rate was also measured. The results are shown in Table 1.

[0048] Table 1. Antioxidant detection results of Spirulina platensis enzymatic hydrolysates from Example 1 and Comparative Examples 1-6

[0049] Experimental Example 2: Synthesis and Activity Verification of the Target Peptide Take 10 g of spirulina powder, mix thoroughly with 200 mL of distilled water, stir, and then perform ultrasonic cell disruption at a power of 500 W for 20 min. Collect the disrupted liquid, centrifuge at 8000 r / min for 20 min at 4℃, collect the supernatant, freeze-dry to obtain spirulina protein, and store at 4℃ for later use. Dissolve 3 g of spirulina protein in deionized water, adjust the temperature to 50℃ and pH to 7.5, add 0.75 g of flavor protease, and hydrolyze for 4 h. Then, inactivate the enzyme by bathing in a 100℃ water bath for 10 min to stop the hydrolysis. Centrifuge the hydrolysate at 8000 r / min for 20 min, and the supernatant is the spirulina protein hydrolysate. Set the pressure to 0.2 MPa, first pass the 20 g / L spirulina protein hydrolysate through a microfiltration membrane with a pore size of 0.45 μm, and then further purify the filtrate through a microfiltration membrane with a pore size of 0.22 μm. Finally, the solution was filtered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa at a pressure of 0.7 MPa. The filtrate was collected, concentrated by rotary evaporation, and freeze-dried to obtain lyophilized spirulina hydrolysate powder, which was stored at -20°C for later use. The amino acid sequences of the ultrafiltration fraction were identified using ultra-high performance liquid chromatography (UHPLC) and linear ion trap-Orbitrap tandem mass spectrometry (LIS-MS / MS). The amino acid sequences of the fraction treated with the ultrafiltration membrane (<3 kDa) were identified using LC-MS / MS. A total of 9672 peptides were obtained by matching the spirulina protein database. These 9672 peptides were pre-screened based on peptide score (>25), relative abundance (>200w), and peptide length (<10), resulting in 838 peptides. The potential biological activities, free radical scavenging activities, chelating capacity scores, toxicity, and resistance to gastrointestinal digestive enzymes of these 838 peptides were studied using computer-aided simulations. Finally, 13 promising peptides were screened out. Detailed results are shown in Table 2.

[0050] Table 2. Computer-aided analysis of peptide sequences

[0051] Thirteen peptides from Table 2 were selected for in vitro synthesis with a purity of 95%. The in vitro activity of these 13 peptides was tested, including ADH activation rate and antioxidant capacity. The specific results are shown in Table 3.

[0052] Table 3. In vitro activity of the synthesized peptides

[0053] Note: Different letters in the same column indicate statistically significant differences between groups (P<0.05).

[0054] In vitro experiments validated the dual ADH activation and antioxidant activities of 13 Spirulina-derived synthetic peptides, revealing their significant potential for alcohol detoxification and liver protection. Four peptides showed DPPH and ABTS clearance rates exceeding 80%. Data showed that HPGIP, SPLP, SMP, and SPFP peptides exhibited superior overall activity: HPGIP demonstrated an ADH activation rate as high as 176.05%, while its DPPH and ABTS clearance rates reached 93.36% and 87.12%, respectively, far exceeding the performance of squid peptides (ADH 122.71%) and corn peptides (ADH 90.12%) reported in the literature. Observation of the composition of several highly active peptides revealed that their amino acid sequences contained proline, glycine, and leucine, which may be the key reason for their high activity. ADH activation rate and antioxidant activity showed a non-perfect positive correlation, as seen with HPGIP showing high levels of both, while SPLP exhibited stronger antioxidant activity but slightly lower ADH activation, suggesting a possible differentiation in the molecular mechanisms underlying these two types of activity. Compared with existing research, the breakthrough of this invention lies in the first discovery that the peptide HPGIP has both super ADH activation and free radical scavenging capabilities, and its activity far exceeds that of traditional plant / animal-derived peptides.

[0055] Example 3: Verification of the hangover-relieving effect of spirulina polypeptides To evaluate the hepatoprotective and alcohol-detoxifying effects of the spirulina active peptides obtained in this invention, an animal experiment was conducted. One hundred mice were selected and acclimatized for 7 days in a temperature-controlled environment (22 ± 2℃). They were then randomly divided into 10 groups (n=10) according to body weight. The positive control group was administered 400 mg / kg BW via gavage, while the blank control group and alcohol model group received an equal volume of physiological saline. The seven experimental groups were administered 400 mg / kg BW of liquid containing different spirulina active peptides via gavage. After 30 min of pretreatment, except for the blank group, the other groups were administered 56% (v / v) Hongxing Erguotou ethanol solution (9.8 mL / kg BW), while the blank group received an equal volume of physiological saline simultaneously. The righting reflex test was used to detect the mice's behavioral response after drinking alcohol, and a timer was started immediately after the mice consumed the alcohol. After the last gavage, the activity of mice in each group was observed. Except for the control group, mice in the other groups were gently placed back-down in their cages. If a mouse maintained this supine position for more than 30 seconds, the righting reflex was considered to have disappeared. If the mouse could turn over twice within 60 seconds, the righting reflex was considered to have recovered. The number of mice that became intoxicated, the number of mice that died, the time of disappearance of the righting reflex, and the time of recovery of the righting reflex were observed and recorded. The results showed that the spirulina active peptide HPGIP had the best hangover-relieving effect, effectively increasing the latency of the righting reflex to 74.28% and reducing the duration of sleep to 65.24 ± 12.84 min (54.36% less than the model group). This improvement even exceeded that of the positive control (89.45 ± 28.81 min).

[0056] The activity of ADH and ALDH in the liver of mice with early-stage ALD was examined. The results showed that ADH and ALDH activities were significantly increased compared to the model group (P<0.05), and the Spirulina bioactive peptide HPGIP increased ADH and ALDH activities in mouse liver by 57.62% and 90.47%, respectively, showing the greatest increase. ADH and ALDH play important roles in alcohol metabolism, and intervention with Spirulina protease hydrolysate significantly increased the activity of ADH and ALDH in mouse liver, promoting alcohol metabolism. The results are shown in Table 4.

[0057] Table 4. Results of verification of the hangover-relieving function of different Spirulina active peptides

[0058] Example 4: Verification of Liver-Protecting Efficacy To verify the hepatoprotective effect of spirulina bioactive peptides, 100 mice were selected and, after 7 days of acclimatization, randomly divided into 10 groups of 10 mice each. An early ALD model was established in mice subjected to chronic alcohol feeding followed by acute alcohol gavage. Except for the control group and the model group, each group received intervention with positive samples at a fixed time daily. The control group and the alcohol model group received an equal dose of physiological saline. The procedure is as follows: Figure 1 As shown.

[0059] On day 24, mice in each group were administered 400 mg / kg BW Haiwang Jinzun and seven different spirulina active peptides by gavage. The control group and model group were administered an equal volume of physiological saline. 30 minutes later, all mice except the control group were administered 12 mL / kg BW 45% ethanol solution by gavage. The control group was administered maltodextrin with the same caloric content by gavage. All mice were then fasted for 9 hours and subsequently anesthetized. Serum: Blood from mice was collected via the retroocular venous plexus into sterile centrifuge tubes and allowed to stand at room temperature. The blood was then centrifuged at 3500 r / min for 10 min at 4°C. The resulting serum sample was stored at -80°C for subsequent biochemical analysis. After blood collection, the mice were dissected at the neck, and the liver was removed. The liver was then weighed to calculate the liver index. A portion of the liver weight / body weight was fixed in formalin for pathological section analysis, while the remainder was stored in liquid. This liquid was rapidly frozen in nitrogen and stored at -80°C for subsequent biochemical analysis.

[0060] Liver-related biochemical indicators were measured, including the concentrations of ADH, ALDH, MDA, SOD, GSH, and total protein in mouse liver. The levels of ALT and AST in the serum of mice with early-stage ALD were measured. By detecting serum ALT and AST levels, the repair effect of spirulina bioactive peptides on alcoholic liver injury was systematically evaluated. The results are shown in Table 5.

[0061] Table 5. Results of verification of the hepatoprotective function of different Spirulina bioactive peptides

[0062] Compared to the untreated control group, the levels of ALT and AST activities in the blood of mice affected by alcohol were significantly increased (P<0.05), indicating a deeper degree of liver damage. After administration of spirulina active peptides, a decreasing trend in ALT and AST activities in the blood of mice was observed (P<0.05). Specifically, administration of HPGIP effectively reduced serum ALT (43.63%) and AST (40.27%) activities, bringing their levels close to and lower than those of the control group without any intervention, which was statistically significant (P<0.05).

[0063] This study aimed to understand the antioxidant protective effects of spirulina bioactive peptides against alcohol-induced liver damage by analyzing oxidative stress markers (such as MDA, SOD, and GSH) in the liver. Experimental data showed that compared to the untreated control group, alcohol-induced mouse hepatocyte SOD activity decreased by 21.84% (P<0.05), while MDA concentration increased by 85.68% (P<0.05). Compared to the alcohol-induced model group, liver damage was alleviated in both the positive control group and groups treated with different spirulina bioactive peptides. In particular, after using HPGIP spirulina bioactive peptides, the relative values ​​of SOD activity and GSH content were significantly increased compared to the alcohol model group, by 18.04% and 58.78%, respectively, and the MDA concentration decreased by 39.26% (P<0.05). HPGIP can stimulate the enhancement of ADH and ALDH activity to promote alcohol metabolism. During this process, the reduced alcohol concentration may lead to the inhibition of CYP2E1, thereby slowing down the generation of reactive oxygen species. Meanwhile, the decrease in acetaldehyde concentration will also help to mitigate ROS accumulation to some extent. Furthermore, HPGIP is rich in acidic and nonpolar amino acids (such as Gln, Asn, and His). The hydrolysis products of these proteins can act as effective free radical scavengers or metal ion reducing agents, while nonpolar amino acids exhibit strong free radical scavenging capabilities through direct electron transfer. This suggests that SPs may work by fundamentally weakening and efficiently removing free radicals, not only accelerating alcohol decomposition but also enhancing the resistance of mouse liver to oxidation, successfully inhibiting excessive lipid oxidation in mouse hepatocytes, and significantly reducing oxidative damage to hepatocytes, ultimately leading to a decrease in the activity of converting enzymes such as AST and ALT in the blood.

[0064] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A Spirulina platensis active peptide, characterized in that, The amino acid sequence of the active peptide from Spirulina platensis is HGPIP, SMP, IPGI, or IGIP.

2. A method for preparing enzymatic hydrolysate of Spirulina platensis, characterized in that, The method includes the following steps: (1) Mix the spirulina powder with distilled water, stir, and then use ultrasound to break up the cell walls; (2) Centrifuge the cell wall disruption liquid obtained in step (1), take the supernatant, and freeze-dry it to obtain spirulina protein; (3) Take 2-6g of the spirulina protein obtained in step (2) and dissolve it in deionized water. Adjust the temperature and pH of the solution to the optimal conditions for the enzyme, add the protease, react for a period of time, and then use a water bath to inactivate the enzyme and stop the enzymatic hydrolysis. (4) Centrifuge the enzymatic hydrolysate obtained in step (3), take the supernatant, and obtain the spirulina enzymatic hydrolysate.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass ratio of spirulina powder to water is 1:10 to 1:

30. Stir, then use ultrasound to break up the cell walls. The ultrasonic power is 400 to 600W and the ultrasonic time is 18 to 25 minutes.

4. The preparation method according to claim 2, characterized in that, In step (2), the cell wall-breaking liquid obtained in step (1) is centrifuged at a temperature of 0~10℃, a speed of 6000~9000r / min, and a time of 18~25min, preferably 8000r / min for 20min. The supernatant was collected and freeze-dried to obtain spirulina protein.

5. The preparation method according to claim 2, characterized in that, In step (3), the spirulina protein obtained in step (2) is dissolved in deionized water, the temperature of the solution is adjusted to 40-60℃ and the pH to 7-8, protease is added, and after reacting for 2-6 hours, the enzyme is inactivated by water bath for 8-10 minutes, and the enzymatic hydrolysis is stopped. The water bath temperature is 100℃.

6. The preparation method according to claim 2, characterized in that, In step (4), the centrifugation speed is 6000~9000 r / min and the centrifugation time is 15~25 min.

7. A Spirulina platensis enzymatic hydrolysate prepared by the method according to any one of claims 2 to 6, characterized in that, The Spirulina platensis enzymatic hydrolysate contains one or more Spirulina platensis bioactive peptides with amino acid sequences of HGPIP, SMP, IPGI, and IGIP.

8. A product characterized in that, The product contains Spirulina platensis active peptides with amino acid sequences such as HGPIP, SMP, IPGI and / or IGIP.

9. The use of the Spirulina platensis active peptide according to claim 1, or the method according to any one of claims 2 to 6, or the Spirulina platensis enzymatic hydrolysate according to claim 7, or the product according to claim 8 in the preparation of a medicine for relieving hangovers and protecting the liver.

10. The use of the Spirulina platensis active peptide of claim 1, or the method of any one of claims 2 to 6, or the Spirulina platensis enzymatic hydrolysate of claim 7, or the product of claim 8 in the preparation of a medicament for relieving, reducing and / or treating alcohol-induced liver injury.

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