WO2023020290A1 - Polypeptide derived from dry-cured ham and capable of allevating alcoholic liver damage, and preparation method therefor - Google Patents
Polypeptide derived from dry-cured ham and capable of allevating alcoholic liver damage, and preparation method therefor Download PDFInfo
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- WO2023020290A1 WO2023020290A1 PCT/CN2022/110215 CN2022110215W WO2023020290A1 WO 2023020290 A1 WO2023020290 A1 WO 2023020290A1 CN 2022110215 W CN2022110215 W CN 2022110215W WO 2023020290 A1 WO2023020290 A1 WO 2023020290A1
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- dry
- alcoholic liver
- cured ham
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- alleviating
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention belongs to the technical field of functional polypeptide preparation, and relates to a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and a preparation method thereof.
- Alcoholic liver disease is a devastating liver disease caused by heavy, long-term alcohol consumption. Its pathological features are mainly fat accumulation in the liver, leading to fatty liver, liver fibrosis and cirrhosis. ALD is a major cause of increased disease-related morbidity and mortality in liver injury, accounting for 5.9% of all global deaths from the disease. Although the focus of research in recent years has begun to focus on chronic alcoholic liver injury, the pathogenesis of ALD has not yet been clarified. Pathogen molecule-associated lipopolysaccharide-mediated inflammation in hepatocytes. Among them, regulation of intestinal homeostasis and repair of oxidative stress damage have played important roles in potential targets for the treatment of ALD.
- Dry-cured ham is popular among meat products in many countries. During the long ripening process of dry-cured ham, up to 10% of the protein in the ham is hydrolyzed into polypeptides, which can have special biological effects on the human body. For example, peptides isolated from Spanish Parma ham have antihypertensive, anti-2-diabetic, antioxidant, anti-inflammatory and antibacterial activities. Previous studies have screened and identified a large number of natural antioxidant peptides in Zhejiang Jinhua ham (JHP) from China. However, the current research on JHP is limited to the test-tube experiments of antioxidants, and there is no report on whether there is a polypeptide that relieves ALD in JHP.
- JHP Zhejiang Jinhua ham
- bioactive peptides are more stable, safer and easier to absorb than antibiotics in the treatment of alcoholic liver injury.
- Exogenous bioactive peptides such as mushroom polysaccharide peptides, versicolor polysaccharide peptides, ganoderma lucidum polysaccharide peptides, kelp polysaccharide peptides, and corn peptides can reduce liver damage.
- the peptide mainly derived from dry-cured ham myosin is closely related to the alleviation of alcoholic liver injury, and its application in liver disease protection has a good prospect.
- the object of the present invention is to provide a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and a preparation method thereof.
- the technical solution provided by the present invention is: a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver damage, the amino acid sequence is: Lys-Arg-Gln-Lys-Tyr-Asp.
- the technical solution provided by the present invention is: a method for preparing a dry-cured ham-derived polypeptide that can relieve alcoholic liver damage, comprising the following steps:
- Step 1 Mix the biceps femoris of dry-cured ham with phosphate buffer, then homogenize with a homogenizer under ice bath conditions, and then centrifuge to take the supernatant to obtain dry-cured ham polypeptide extract;
- Step 2 Perform ultrafiltration on the dry-cured ham polypeptide extract, and then separate it with a gel column.
- the elution conditions are: the eluent is 0.01mol/L HCl, separated at a constant temperature, the flow rate is 0.8mL/min, and detected by 280nm ultraviolet light Fractions were measured with an automatic fraction collector, and each tube fraction was collected with an automatic fraction collector; the fractions were dried in a vacuum freeze dryer;
- Step 3 Using liquid phase separation to further separate the most effective component for alleviating alcoholic liver injury obtained in step 2, to obtain the most active polypeptide for alleviating alcoholic liver injury;
- Step 4 Using mass spectrometry to analyze the peptide with the most active alcohol-induced liver injury obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: Lys-Arg-Gln-Lys-Tyr -Asp.
- Step 3 includes dissolving the freeze-dried sample in 1 mL of distilled water, injecting it into the HPLC system, and adopting BEH C18 chromatographic column to carry out gradient elution, the flow rate is 0.3 mL/min, and the eluent A is 0.1% formic acid, The eluent B is 100% acetonitrile; the flow rate gradient is: 0-10min, 100%A, 10-22min, 30-80%B; 22-23min, 100%A; the peptide peak is detected at 280nm ultraviolet wavelength; the peptide The corresponding peaks were divided into seven fractions, which were freeze-dried; seven fractions of dry-cured ham small peptides were collected, and the activity of each small peptide in alleviating alcoholic liver injury was determined after drying.
- the dry-cured ham-derived peptide of the present invention has potential medical value for alleviating alcoholic liver injury.
- the active peptides involved in the present invention have the functions of improving intestinal flora, reducing liver lipid accumulation, and relieving liver pressure. They have the characteristics of simple structure, safety, and strong activity, and play the role of nutrition and health care. The development of new food for relieving alcoholic liver injury without toxic and side effects provides active ingredients and has broad application prospects.
- Figure 1 shows the effect of peptides obtained by size-exclusion chromatography on alcohol-induced liver damage (ALD) in male mice.
- FIG 2 shows the effects of feeding for 35 days on the expression of intestinal enzymes in mice in the blank group (CTRL), alcohol group (EtOH), alcohol+bifendate group (EtOH), and alcohol+peptide group (EtOH+JHP).
- Fig. 3A is the HPLC chart of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
- Fig. 3B is a primary mass spectrum (MS) diagram of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
- Figure 3C is a MS/MS spectrum of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
- Figure 4 shows the effect of feeding a control diet or an ethanol-containing diet (with or without JHP) on the intestinal flora of mice.
- FIG. 5 shows the effect of KRQKYD on intestinal tight junctions in mice.
- Figure 6 shows the effect of KRQKYD on the oxidative stress response of the mouse liver.
- Fig. 7 is a technical roadmap of the present invention.
- Example 1 A dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and its preparation method
- the dry-cured ham-derived polypeptide of the present invention that can relieve alcoholic liver damage has the following amino acid sequence:
- Hexapeptide Lys-Arg-Gln-Lys-Tyr-Asp; hereinafter, the hexapeptide can also be abbreviated as: KRQKYD.
- the ham-derived hexapeptide sequence for alleviating alcoholic liver damage includes the active hexapeptide sequence as the core.
- the preparation method of the dry-cured ham source of the present invention for alleviating alcoholic liver damage peptide comprises the following steps:
- the slurry obtained in step (1) is centrifuged, and the supernatant is taken. During centrifugation, the centrifuge was set at a rotational speed of 12000g; the time was 20min; and the temperature was 4°C.
- the dry-cured ham polypeptide extract was subjected to ultrafiltration, and then separated by a gel column.
- the elution conditions were: the eluent was 0.01mol/L HCl, separated at a constant temperature, and the flow rate was 0.8mL/min.
- Fractions were measured with a 280 nm UV detector (Amersham Biosciences), and fractions from each tube were collected with an automatic fraction collector. Fractions were dried in a vacuum freeze dryer for further analysis.
- the process parameters of ultrafiltration are: P/N: S02-E003-05-N, medium/rated value: mPES/3KDa, surface area: 790cm 2 .
- Step (4) is specifically: dissolve the freeze-dried sample in 1 mL of distilled water, inject it into the HPLC system, and use BEH C18 chromatographic column (1.7 ⁇ m, 2.1 ⁇ 100 mm, Waters Inc., Milford, MA, USA) to carry out gradient elution, The flow rate was 0.3 mL/min, the eluent A was 0.1% formic acid, and the eluent B was 100% acetonitrile. The flow rate gradient is: 0-10min, 100%A; 10-22min, 30-80%B; 22-23min, 100%A. Peptide peaks were detected at a UV wavelength of 280 nm. The peaks corresponding to the peptides were divided into seven parts and freeze-dried; seven dry-cured ham small peptide fractions were collected and dried to determine the activity of each small peptide in alleviating alcoholic liver injury.
- step (5) Using mass spectrometry to analyze the most active peaks for alleviating alcoholic liver damage obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: KRQKYD; the specific technical scheme of step (5) is: using Acquity (Waters Inc.) high-performance liquid chromatography system, using a reversed-phase BEH C18 column (1.7 ⁇ m, 2.1 ⁇ 100mm, Waters Inc.) to separate the most effective peaks for alleviating alcoholic liver injury. Gradient elution: flow rate 0.3mL/min, eluent a is 0.1% formic acid, and eluent b is 100% ACN.
- the flow gradient is: 0-10min, 100%a; 10-22min, 30-80%b; 22-23min, 100%a.
- the column temperature was maintained at 25°C.
- the flow goes directly to the MS/MS system for multiple reaction measurements.
- the synthetic KRQKYD method is as follows:
- cleaning resin repeat the content of step 4;
- the synthesized peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a BEH C18 column (1.7 ⁇ m, 2.1 ⁇ 100 mm, Waters Inc., Milford, MA, USA). Mobile phase is 0.1% formic acid (solvent A) and 100% acetonitrile (solvent B), and mobile phase is: 0-10min, 100% solvent A; 10-22min, 30-80% solvent B; 22-23min, 100% solvent A, flow rate 0.3ml/min. The separation process was detected at a UV wavelength of 280nm. The synthesized peptides were identified by liquid chromatography-mass spectrometry.
- the dry-cured ham-derived peptide for alleviating alcoholic liver injury of the present invention has the functions of improving intestinal flora and reducing liver cell oxidative stress damage, and is very suitable for preparing food for relieving intestinal and liver discomfort and repairing alcoholic liver injury. Medicines for liver damage.
- Dry-cured ham sources attenuate the effects of alcoholic liver damage peptide (JHP); JHP is a complex mixture of peptides with a wide range of molecular weights in male mice. Determining the ALD preventive effect of functional ingredients in 36-month dry-cured ham.
- JHP alcoholic liver damage peptide
- Component A was eluted first due to its largest molecular weight, while component I was eluted last because of its smallest molecular weight. All fractions were collected and freeze-dried. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) were measured in 194 mice fed different fractions isolated from dry-cured ham for 36 months (Fig. 1B-D). The levels of ALT (128.89U/L), AST (294.39U/L) and MDA (80.47mmol/mL) in group G were lower than those in other groups. Therefore, JHP-G has the greatest alleviating property of alcoholic liver injury. For further analysis, JHP-G was dried in a vacuum freeze dryer.
- ALT alanine aminotransferase
- AST aspartate aminotransferase
- MDA malondialdehyde
- Component G was further purified by reversed-phase high-performance liquid chromatography, which had intermolecular polarity differences. A total of 7 fractions were separated from G-1 to G-7 (A of Fig. 2). The isolated fractions were lyophilized and assayed for their activity on ALD ( Figure 2, panels B-D). The G-6 site has the greatest damage relief.
- Amino acid sequencing of G-6 was carried out by reversed-phase high-performance liquid chromatography-mass spectrometry (LC-MS/MS). Found to be from Lys-Arg-Gln-Lys-Tyr-Asp (KRQKYD).
- the Acquity (Waters Inc.) high performance liquid chromatography system was used to separate the most effective peaks for alleviating alcoholic liver injury with a reversed-phase BEH C18 column (1.7 ⁇ m, 2.1 ⁇ 100mm, Waters Inc.).
- KRQKYD affects liver oxidative stress injury in alcohol-treated mice
- ROS reactive oxygen species
- SOD superoxide dismutase
- GSH-Px glutathione oxidase
- the level of reactive oxygen species (161.21 U/mg prot) in the alcohol-treated (EtOH) group was higher than that in the blank group (94.66 U/mg prot), indicating that alcohol-treated mice produced more reactive oxygen species.
- the reactive oxygen species level in the KRQKYD treatment group was 71.19 U/mg prot, which was lower than that in the control group. This indicated that KRQKYD pretreatment significantly reduced the ROS level in the EtOH group.
- Alcohol consumption significantly decreased the levels of GSH-Px and SOD in the liver, as shown in B of Fig. 4 .
- KRQKYD pretreatment significantly improved the decline of GSH301Px and SOD activities in the EtOH group.
- the results showed that KRQKYD can reduce alcohol-induced liver oxidative stress damage by inhibiting the production of reactive oxygen species and increasing the activity of GSH-Px and SOD; the protective effect of KRQKYD on ALD may be through reducing the expression of CYP2E1 to reduce oxidative stress, and through Activation of the Nrf2/HO319 pathway enhances the oxidative defense system.
- GM was mainly composed of Verrucobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria (Fig. 5A).
- the proportions of Verrucomicrobia, Actinomycetes and Desulfovibrio were significantly decreased in the EtOH group, but increased progressively after KRQKYD pretreatment.
- Alcohol intake significantly increased the proportion of Proteus bacteria
- KRQKYD pretreatment significantly decreased the proportion of Proteobacteria in alcohol-treated mice.
- KRQKYD can increase the expression of compact protein and then affect the intestinal homeostasis
- KRQKYD pretreatment progressively increases the levels of these proteins in alcohol-treated mice.
- KRQKYD promotes components of tight junctions, enhances barrier function, and promotes intestinal integrity.
- the results showed that KRQKYD increased the expression of antimicrobial peptides and claudin and improved intestinal balance in mice.
- Figure 1 Effect of peptides obtained by gel exclusion chromatography on ALD in male mice.
- A SephadexTM 10/300 GL size exclusion chromatographic analysis of peptides in dry-cured ham;
- B-D JHP-(A-I) on alanine aminotransferase ALT in serum of male mice;
- B aspartate aminotransferase AST;
- C Effect of malondialdehyde MDA(D) concentration.
- FIG. 2 Effects of blank group (CTRL), alcohol group (EtOH), alcohol+bifendate group (EtOH), alcohol+peptide group (EtOH+JHP) on the expression of intestinal enzymes in mice fed for 35 days
- CTRL blank group
- EtOH alcohol group
- EtOH alcohol+bifendate group
- EtOH+JHP alcohol+peptide group
- A Separation of G components by high-performance liquid chromatography
- B-D G-(1-7) on alanine aminotransferase ALT (B), aspartate aminotransferase AST in male mouse serum
- C malondialdehyde MDA
- Figure 3A- Figure 3C are the MS structure identification diagrams of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD), respectively.
- Fig. 4 Effect of KRQKYD on oxidative stress in alcoholic liver of mice.
- A liver reactive oxygen species level
- B liver antioxidant enzyme activity level
- C HO-1, Nrf2 and CYP2E1 gene expression levels in liver
- D-E liver tissue HO-1, Nrf2, CYP2E1 protein expression level.
- Figure 5 Effects of 35 days on the gut microbiota of mice fed a control diet or an ethanol-containing diet (with or without JHP).
- A Relative abundance at the level of gut bacterial phylum
- B Relative abundance at the level of gut bacterial genus.
- Figure 6 Effect of KRQKYD on intestinal barrier homeostasis in mice.
- A Quantitative RT-PCR detection of tight junction-1 (ZO-1), claudin-1 (Claudin-1), claudin (Occludin) mRNA expression levels;
- B-C western blot detection of ZO-1, Claudin-1 , Occludin expression level.
- Example 2 A dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and its preparation method
- a dry-cured ham-derived polypeptide that can relieve alcoholic liver injury the amino acid sequence is: Lys-Arg-Gln-Lys-Tyr-Asp.
- the technical solution provided by the present invention is: a method for preparing a dry-cured ham-derived polypeptide that can relieve alcoholic liver damage, comprising the following steps:
- Step 1 Mix the biceps femoris of dry-cured ham with phosphate buffer, then homogenize with a homogenizer under ice bath conditions, and then centrifuge to take the supernatant to obtain dry-cured ham polypeptide extract;
- Step 2 Perform ultrafiltration on the dry-cured ham polypeptide extract, and then separate it with a gel column.
- the elution conditions are: the eluent is 0.01mol/L HCl, separated at a constant temperature, the flow rate is 0.8mL/min, and detected by 280nm ultraviolet light Fractions were measured with an automatic fraction collector, and each tube fraction was collected with an automatic fraction collector; the fractions were dried in a vacuum freeze dryer;
- Step 3 Using liquid phase separation to further separate the most effective component for alleviating alcoholic liver injury obtained in step 2, to obtain the most active polypeptide for alleviating alcoholic liver injury;
- Step 4 Using mass spectrometry to analyze the peptide with the most active alcohol-induced liver injury obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: Lys-Arg-Gln-Lys-Tyr -Asp.
- Step 3 includes dissolving the lyophilized sample in 1 mL of distilled water, injecting it into the HPLC system, and performing gradient elution with a BEH C18 chromatographic column at a flow rate of 0.3 mL/min, eluent A is 0.1% formic acid, and eluent B is 100 % acetonitrile; the flow rate gradient is: 0-10min, 100%A, 10-22min, 30-80%B; 22-23min, 100%A; the peptide peak is detected at 280nm ultraviolet wavelength; the peak corresponding to the peptide is divided into seven The seven parts were freeze-dried; seven dry-cured ham small peptide fractions were collected, and the activity of each small peptide in alleviating alcoholic liver injury was determined after drying.
- the hexapeptide can also increase the relative abundance of Desulfobacteria in the intestine (from 5.2 ⁇ 0.04% to 7.1 ⁇ 0.02%) and reduce the relative abundance of Deuterobacteria (from 66.5 ⁇ 0.02% to 57.3 ⁇ 0.01%), etc. function, compared with alcohol-impaired gut, can increase the tight junction of intestinal epithelial cells, improve claudin (18.2 ⁇ 0.2% up-regulation), claudin-1 (15.8 ⁇ 0.3% up-regulation) and tight junction-1 (22.5% up-regulation ⁇ 0.3%) protein expression, reduce liver inflammation cascade reaction and can be used for the preparation of functional food or medicine.
- the peptide derived from dry-cured ham for alleviating alcoholic liver injury up-regulates the expression of Nrf2/HO-1 antioxidant defense system, reduces the oxidative stress damage of liver cells, and has the function of inhibiting the generation of active oxygen, so it is very suitable as a
- the main functional ingredients are used to prepare food or medicine for alleviating alcoholic liver damage.
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Abstract
A polypeptide derived from dry-cured ham and capable of alleviating alcoholic liver damage, and a preparation method therefor, wherein the amino acid sequence is Lys-Arg-Gln-Lys-Tyr-Asp. The active peptide has the functions of improving intestinal flora, reducing lipid accumulation in the liver and alleviating hepatic pressure at the same time; has the characteristics of a simple structure, being safe, having high activity, etc.; plays a role in nutrition and health care; is expected to provide effective components for developing new non-toxic food products without side effects for alleviating alcoholic liver damage; and has broad application prospects.
Description
本发明属于功能性多肽制备技术领域，涉及一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法。The invention belongs to the technical field of functional polypeptide preparation, and relates to a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and a preparation method thereof.
酒精性肝病(ALD)是一种由长期大量饮酒引起的破坏性肝脏疾病。其病理特点主要是肝内脂肪堆积，导致脂肪肝、肝纤维化和硬化。ALD是肝损伤疾病相关发病率和死亡率提高的主要原因，占全球因疾病死亡总人数的5.9％。虽然近年来研究重点开始聚焦于慢性酒精性肝损伤，但是ALD的发病机制尚未明确，可能的机制包括肠道稳态失衡，活性氧介导的氧化应激损伤，肝细胞脂质代谢紊乱和由病原体分子相关的脂多糖介导的肝细胞炎症。其中，调节肠道稳态和修复氧化应激损伤在治疗ALD的潜在靶点中发挥了重要作用。Alcoholic liver disease (ALD) is a devastating liver disease caused by heavy, long-term alcohol consumption. Its pathological features are mainly fat accumulation in the liver, leading to fatty liver, liver fibrosis and cirrhosis. ALD is a major cause of increased disease-related morbidity and mortality in liver injury, accounting for 5.9% of all global deaths from the disease. Although the focus of research in recent years has begun to focus on chronic alcoholic liver injury, the pathogenesis of ALD has not yet been clarified. Pathogen molecule-associated lipopolysaccharide-mediated inflammation in hepatocytes. Among them, regulation of intestinal homeostasis and repair of oxidative stress damage have played important roles in potential targets for the treatment of ALD.
在许多国家，肉制品中的干腌火腿都很受欢迎。在干腌火腿漫长的成熟过程中，火腿中多达10％的蛋白质被水解成多肽，这些多肽可以对人体产生特殊的生物学效应。例如从西班牙帕尔玛火腿中分离得到的多肽具有抗高血压、抗2-型糖尿病、抗氧化、抗炎和抗菌的活性。先前已有研究对中国的浙江金华火腿(JHP)中大量天然抗氧化肽进行了筛选和鉴定。然而，目前JHP的研究局限于抗氧化剂的试管实验，对JHP中是否存在缓解ALD的多肽尚未见报道。虽然有证据表明，增加合成抗氧化剂(美他多辛)、抗生素(阿莫西林克拉维酸、利福昔明、环丙沙星)和免疫抑制剂(利隆纳普)的应用可抑制ALD的发病过程，但是在治疗酒精性肝损伤方面生物活性肽相比抗生素更稳定、更安全也更易吸收。外源生物活性肽如蘑菇多糖肽、云芝多糖肽、灵芝多糖肽、海带多糖肽、玉米肽等均可减轻肝脏损伤。其中以干腌火腿肌球蛋白为主要源的多肽与缓解酒精性肝损伤关系紧密，在肝病保护中的应用具有良好前景。Dry-cured ham is popular among meat products in many countries. During the long ripening process of dry-cured ham, up to 10% of the protein in the ham is hydrolyzed into polypeptides, which can have special biological effects on the human body. For example, peptides isolated from Spanish Parma ham have antihypertensive, anti-2-diabetic, antioxidant, anti-inflammatory and antibacterial activities. Previous studies have screened and identified a large number of natural antioxidant peptides in Zhejiang Jinhua ham (JHP) from China. However, the current research on JHP is limited to the test-tube experiments of antioxidants, and there is no report on whether there is a polypeptide that relieves ALD in JHP. Although there is evidence that increased use of synthetic antioxidants (metadoxine), antibiotics (amoxicillin-clavulanic acid, rifaximin, ciprofloxacin) and immunosuppressants (lironaprol) suppress the progression of ALD However, bioactive peptides are more stable, safer and easier to absorb than antibiotics in the treatment of alcoholic liver injury. Exogenous bioactive peptides such as mushroom polysaccharide peptides, versicolor polysaccharide peptides, ganoderma lucidum polysaccharide peptides, kelp polysaccharide peptides, and corn peptides can reduce liver damage. Among them, the peptide mainly derived from dry-cured ham myosin is closely related to the alleviation of alcoholic liver injury, and its application in liver disease protection has a good prospect.
发明内容Contents of the invention
本发明的目的在于提供一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法。The object of the present invention is to provide a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and a preparation method thereof.
为实现上述目的及其他相关目的，本发明提供的技术方案是：一种可缓解酒精性肝损伤的干腌火腿源多肽，氨基酸序列为：Lys-Arg-Gln-Lys-Tyr-Asp。To achieve the above and other related purposes, the technical solution provided by the present invention is: a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver damage, the amino acid sequence is: Lys-Arg-Gln-Lys-Tyr-Asp.
为实现上述目的及其他相关目的，本发明提供的技术方案是：一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法，包括下列步骤：In order to achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a method for preparing a dry-cured ham-derived polypeptide that can relieve alcoholic liver damage, comprising the following steps:
步骤1：将干腌火腿的股二头肌与磷酸盐缓冲液混合，然后于冰浴条件下用匀浆机进行均 质，接着离心取上清，得到干腌火腿多肽提取物；Step 1: Mix the biceps femoris of dry-cured ham with phosphate buffer, then homogenize with a homogenizer under ice bath conditions, and then centrifuge to take the supernatant to obtain dry-cured ham polypeptide extract;
步骤2：对干腌火腿多肽提取物进行超滤，再用凝胶柱分离，洗脱条件为：洗脱液为0.01mol/L的HCl，恒温分离，流速0.8mL/min，用280nm紫外检测器测定馏分，用自动馏分收集器收集每管馏分；馏分在真空冷冻干燥机中干燥；Step 2: Perform ultrafiltration on the dry-cured ham polypeptide extract, and then separate it with a gel column. The elution conditions are: the eluent is 0.01mol/L HCl, separated at a constant temperature, the flow rate is 0.8mL/min, and detected by 280nm ultraviolet light Fractions were measured with an automatic fraction collector, and each tube fraction was collected with an automatic fraction collector; the fractions were dried in a vacuum freeze dryer;
步骤3：采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离，获得最具缓解酒精性肝损伤活性的多肽；Step 3: Using liquid phase separation to further separate the most effective component for alleviating alcoholic liver injury obtained in step 2, to obtain the most active polypeptide for alleviating alcoholic liver injury;
步骤4：采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析，鉴定活性肽的结构，得到一种保护肝活性的肽序列为：Lys-Arg-Gln-Lys-Tyr-Asp。Step 4: Using mass spectrometry to analyze the peptide with the most active alcohol-induced liver injury obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: Lys-Arg-Gln-Lys-Tyr -Asp.
优选的技术方案为：步骤3包括将冻干后的样品溶于1mL蒸馏水，注入HPLC系统，采用BEH C18色谱柱进行梯度洗脱，流速为0.3mL/min，洗脱液A为0.1％甲酸，洗脱液B为100％乙腈；流速梯度为：0-10min，100％A，10-22min，30-80％B；22-23min，100％A；肽峰在280nm紫外波长下检测；将多肽对应的峰分为七个部分，冷冻干燥；收集七个干腌火腿小肽分离组分，干燥后测定各个小肽的缓解酒精性肝损伤的活性。The preferred technical scheme is: Step 3 includes dissolving the freeze-dried sample in 1 mL of distilled water, injecting it into the HPLC system, and adopting BEH C18 chromatographic column to carry out gradient elution, the flow rate is 0.3 mL/min, and the eluent A is 0.1% formic acid, The eluent B is 100% acetonitrile; the flow rate gradient is: 0-10min, 100%A, 10-22min, 30-80%B; 22-23min, 100%A; the peptide peak is detected at 280nm ultraviolet wavelength; the peptide The corresponding peaks were divided into seven fractions, which were freeze-dried; seven fractions of dry-cured ham small peptides were collected, and the activity of each small peptide in alleviating alcoholic liver injury was determined after drying.
优选的技术方案为：鉴定方法包括：采用Acquity高效液相色谱系统，采用反相BEH C18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离；梯度洗脱：流速0.3mL/min，洗脱液a为0.1％甲酸，洗脱液b为100％CAN；流动梯度为:0-10min，100％a，10-22min，30-80％b；22-23min，100％a；柱温保持在25℃；流量直接进入MS/MS系统进行多反应测量；前体离子记录的质量范围为m/z＝200-4000；使用Mass Lynx V4.1操作仪器，分析质谱图信息。The preferred technical solution is: the identification method includes: adopting Acquity high-performance liquid chromatography system, adopting reversed-phase BEH C18 chromatographic column to separate the polypeptide peak with the most activity of alleviating alcoholic liver injury; gradient elution: flow rate 0.3mL/min, Eluent a is 0.1% formic acid, eluent b is 100% CAN; flow gradient is: 0-10min, 100% a, 10-22min, 30-80% b; 22-23min, 100% a; column temperature Keep at 25°C; the flow directly enters the MS/MS system for multiple reaction measurements; the mass range of the precursor ion records is m/z=200-4000; use Mass Lynx V4.1 to operate the instrument and analyze the mass spectrum information.
由于上述技术方案运用，本发明与现有技术相比具有的优点是：Owing to above-mentioned technical scheme uses, the advantage that the present invention has compared with prior art is:
1、本发明的干腌火腿源的缓解酒精性肝损伤肽具有潜在的医学价值。1. The dry-cured ham-derived peptide of the present invention has potential medical value for alleviating alcoholic liver injury.
2、本发明所涉及的活性肽同时兼具改善肠道菌群和减少肝脏脂质堆积，缓解肝压力功能，具有结构简单、安全、活性强等特点，发挥出营养和保健的作用，有望为开发无毒副作用的缓解酒精性肝损伤新食品提供有效成分，具有广泛的应用前景。2. The active peptides involved in the present invention have the functions of improving intestinal flora, reducing liver lipid accumulation, and relieving liver pressure. They have the characteristics of simple structure, safety, and strong activity, and play the role of nutrition and health care. The development of new food for relieving alcoholic liver injury without toxic and side effects provides active ingredients and has broad application prospects.
图1为排阻色谱法获得的肽对雄性小鼠酒精性肝损伤(ALD)的影响。Figure 1 shows the effect of peptides obtained by size-exclusion chromatography on alcohol-induced liver damage (ALD) in male mice.
图2为空白组(CTRL)、酒精组(EtOH)、酒精+联苯双酯组(EtOH)、酒精+肽组(EtOH+JHP)喂食35天对小鼠肠道内酶表达的影响。Figure 2 shows the effects of feeding for 35 days on the expression of intestinal enzymes in mice in the blank group (CTRL), alcohol group (EtOH), alcohol+bifendate group (EtOH), and alcohol+peptide group (EtOH+JHP).
图3A为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的HPLC图。Fig. 3A is the HPLC chart of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
图3B为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的一级质谱(MS)图。Fig. 3B is a primary mass spectrum (MS) diagram of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
图3C为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的二级质谱(MS/MS)图。Figure 3C is a MS/MS spectrum of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD).
图4为喂食对照饮食或含乙醇饮食(含或不含JHP)对小鼠肠道菌群的影响。Figure 4 shows the effect of feeding a control diet or an ethanol-containing diet (with or without JHP) on the intestinal flora of mice.
图5为KRQKYD对小鼠肠道紧密连接的影响。Figure 5 shows the effect of KRQKYD on intestinal tight junctions in mice.
图6为KRQKYD对小鼠肝脏氧化应激反应的影响。Figure 6 shows the effect of KRQKYD on the oxidative stress response of the mouse liver.
图7为本发明的技术路线图。Fig. 7 is a technical roadmap of the present invention.
以下由特定的具体实施例说明本发明的实施方式，熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。The implementation of the present invention will be illustrated by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.
请参阅图1-7。须知，本说明书所附图式所绘示的结构、比例、大小等，均仅用以配合说明书所揭示的内容，以供熟悉此技术的人士了解与阅读，并非用以限定本发明可实施的限定条件，故不具技术上的实质意义，任何结构的修饰、比例关系的改变或大小的调整。提供以下实施例以便更好地理解本发明，而非限制本发明。以下实施例中的实验方法如无特殊说明，均为常规方法。下述实施例中所用的实验材料如无特殊说明，均为常规生化试剂商店购买所得。See Figure 1-7. It should be noted that the structures, proportions, sizes, etc. shown in the drawings attached to this specification are only used to match the content disclosed in the specification, for those who are familiar with this technology to understand and read, and are not used to limit the implementation of the present invention. Restricted conditions, so there is no technical substantive significance, any modification of structure, change of proportional relationship or adjustment of size. The following examples are provided for a better understanding of the invention, but not to limit the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.
实施例1：一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法Example 1: A dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and its preparation method
本发明的可缓解酒精性肝损伤的干腌火腿源多肽，其氨基酸序列为下：The dry-cured ham-derived polypeptide of the present invention that can relieve alcoholic liver damage has the following amino acid sequence:
六肽：Lys-Arg-Gln-Lys-Tyr-Asp；下文中，该六肽也可以简写为：KRQKYD。Hexapeptide: Lys-Arg-Gln-Lys-Tyr-Asp; hereinafter, the hexapeptide can also be abbreviated as: KRQKYD.
本发明的火腿源缓解酒精性肝损伤六肽序列，包括所述活性六肽序列为核心。The ham-derived hexapeptide sequence for alleviating alcoholic liver damage includes the active hexapeptide sequence as the core.
本发明的干腌火腿源缓解酒精性肝损伤肽的制备方法，包括以下步骤：The preparation method of the dry-cured ham source of the present invention for alleviating alcoholic liver damage peptide comprises the following steps:
(1)以金华火腿/宣威火腿/如皋火腿的股二头肌为原料进行均质(1) Homogenize the biceps femoris of Jinhua ham/Xuanwei ham/Rugao ham
将20g股二头肌与80mL磷酸盐缓冲液(0.2mmol/L，pH7.2)混合，于冰浴下使用数显匀浆机均质；均质4次；每次10s；22000rpm。Mix 20 g of biceps femoris with 80 mL of phosphate buffer (0.2 mmol/L, pH 7.2), and homogenize using a digital display homogenizer under an ice bath; homogenize 4 times; each time for 10 seconds; 22000 rpm.
(2)离心(2) centrifugal
对步骤(1)得到的浆料进行离心，取上清液。离心时离心机设置转速12000g；时间20min；温度4℃。The slurry obtained in step (1) is centrifuged, and the supernatant is taken. During centrifugation, the centrifuge was set at a rotational speed of 12000g; the time was 20min; and the temperature was 4°C.
(3)对多肽提取物进行超滤和凝胶过滤(3) Ultrafiltration and gel filtration of peptide extracts
对干腌火腿多肽提取物进行超滤，然后用凝胶柱分离，洗脱条件为：洗脱液为0.01mol/L的HCl，恒温分离，流速0.8mL/min。用280nm紫外检测器(Amersham Biosciences)测定馏分，用自动馏分收集器收集每管馏分。馏分在真空冷冻干燥机中干燥以供进一步分析。The dry-cured ham polypeptide extract was subjected to ultrafiltration, and then separated by a gel column. The elution conditions were: the eluent was 0.01mol/L HCl, separated at a constant temperature, and the flow rate was 0.8mL/min. Fractions were measured with a 280 nm UV detector (Amersham Biosciences), and fractions from each tube were collected with an automatic fraction collector. Fractions were dried in a vacuum freeze dryer for further analysis.
超滤的工艺参数为：P/N:S02-E003-05-N，介质/额定值：mPES/3KDa，表面积：790cm
2。
The process parameters of ultrafiltration are: P/N: S02-E003-05-N, medium/rated value: mPES/3KDa, surface area: 790cm 2 .
真空冷冻干燥的工艺条件为：-40℃～-50℃，24h。The technological conditions of vacuum freeze-drying are: -40°C～-50°C, 24h.
(4)干腌火腿粗肽提取物的反相高效液相色谱(RP-HPLC)分离纯化(4) Separation and purification of dry-cured ham crude peptide extract by reversed-phase high-performance liquid chromatography (RP-HPLC)
将步骤(3)得到的缓解酒精性肝损伤最有效的组分进一步分离。步骤(4)具体为：将冻干后的样品溶于1mL蒸馏水，注入HPLC系统，采用BEH C18色谱柱(1.7μm，2.1×100mm，Waters Inc.，Milford，MA，USA)进行梯度洗脱，流速为0.3mL/min，洗脱液A为0.1％甲酸，洗脱液B为100％乙腈。流速梯度为：0-10min，100％A；10-22min，30-80％B；22-23min，100％A。肽峰在280nm紫外波长下检测。将多肽对应的峰分为七个部分，冷冻干燥；收集七个干腌火腿小肽分离组分，干燥测定各个小肽缓解酒精性肝损伤的活性。The most effective component for alleviating alcoholic liver injury obtained in step (3) is further separated. Step (4) is specifically: dissolve the freeze-dried sample in 1 mL of distilled water, inject it into the HPLC system, and use BEH C18 chromatographic column (1.7 μm, 2.1×100 mm, Waters Inc., Milford, MA, USA) to carry out gradient elution, The flow rate was 0.3 mL/min, the eluent A was 0.1% formic acid, and the eluent B was 100% acetonitrile. The flow rate gradient is: 0-10min, 100%A; 10-22min, 30-80%B; 22-23min, 100%A. Peptide peaks were detected at a UV wavelength of 280 nm. The peaks corresponding to the peptides were divided into seven parts and freeze-dried; seven dry-cured ham small peptide fractions were collected and dried to determine the activity of each small peptide in alleviating alcoholic liver injury.
(5)缓解酒精性肝损伤活性肽结构鉴定(5) Structural identification of active peptides for alleviating alcoholic liver injury
采用质谱对液相分离得到的最具缓解酒精性肝损伤的活性峰进行分析，鉴定活性肽的结构，得到一种保护肝活性的肽序列：KRQKYD；步骤(5)具体技术方案为：采用Acquity(Waters Inc.)高效液相色谱系统，采用反相BEH C18色谱柱(1.7μm，2.1×100mm，Waters Inc.)对具有缓解酒精性肝损伤最有效的峰进行分离。梯度洗脱：流速0.3mL/min，洗脱液a为0.1％甲酸，洗脱液b为100％ACN。流动梯度为:0-10min，100％a；10-22min，30-80％b；22-23min，100％a。柱温保持在25℃。流量直接进入MS/MS系统进行多反应测量。前体离子记录的质量范围为m/z＝200-4000。使用Mass Lynx V4.1操作仪器，分析质谱图信息。Using mass spectrometry to analyze the most active peaks for alleviating alcoholic liver damage obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: KRQKYD; the specific technical scheme of step (5) is: using Acquity (Waters Inc.) high-performance liquid chromatography system, using a reversed-phase BEH C18 column (1.7μm, 2.1×100mm, Waters Inc.) to separate the most effective peaks for alleviating alcoholic liver injury. Gradient elution: flow rate 0.3mL/min, eluent a is 0.1% formic acid, and eluent b is 100% ACN. The flow gradient is: 0-10min, 100%a; 10-22min, 30-80%b; 22-23min, 100%a. The column temperature was maintained at 25°C. The flow goes directly to the MS/MS system for multiple reaction measurements. Precursor ions were recorded in the mass range m/z = 200-4000. Use Mass Lynx V4.1 to operate the instrument and analyze the mass spectrogram information.
(6)人工合成KRQKYD并验证其抗炎活性。(6) Synthesize KRQKYD and verify its anti-inflammatory activity.
步骤(6)中人工合成KRQKYD方法如下：In the step (6), the synthetic KRQKYD method is as follows:
1、准确称量0.2mmol树脂(2-氯三酰氯树脂)，分别加入3ml二甲基甲酰胺(DMF)、二氯甲烷(DCM)溶胀树脂30min；1. Accurately weigh 0.2mmol resin (2-chlorotriacyl chloride resin), add 3ml dimethylformamide (DMF) and dichloromethane (DCM) respectively to swell the resin for 30min;
2、按缩合剂(HCTU)：氨基酸：碱(DIEA)：树脂＝4:4:8:1置入10ml EP管中备用；2. According to condensing agent (HCTU): amino acid: base (DIEA): resin = 4:4:8:1, put it into a 10ml EP tube for later use;
3、连接第一氨基酸(不加HCTU)，将氨基酸溶于3mL DMF中，加入132μl DIEA，摇匀3min，倒入合成管，将合成管置于45℃空气浴摇床中8h；3. Connect the first amino acid (without adding HCTU), dissolve the amino acid in 3mL DMF, add 132μl DIEA, shake well for 3min, pour into the synthesis tube, and place the synthesis tube in an air-bath shaker at 45°C for 8h;
4、树脂清洗：先用DMF洗3次，再用DCM洗3次，最后用DMF洗3次；4. Resin cleaning: first wash 3 times with DMF, then wash 3 times with DCM, and finally wash 3 times with DMF;
5、使用5％甲醇溶液封口:合成管中放入5ml 1％甲醇，摇匀30min；5. Use 5% methanol solution to seal: put 5ml 1% methanol into the synthesis tube, shake well for 30min;
6、清洗树脂:重复步骤4的内容；6, cleaning resin: repeat the content of step 4;
7、去除氨基保护基团(Fmoc):加入20％哌啶试剂摇匀5min；7. Remove amino protecting group (Fmoc): add 20% piperidine reagent and shake well for 5 minutes;
8、连接氨基酸:将称好的氨基酸与缩合后的混合物溶解在3mL DMF中，加入132μL DIEA，摇匀3min，倒入合成管，摇匀30min，重复步骤6、7、8依次插入所需氨基酸，使肽链延伸；8. Connecting amino acids: Dissolve the weighed amino acid and the condensed mixture in 3 mL DMF, add 132 μL DIEA, shake well for 3 minutes, pour into the synthesis tube, shake well for 30 minutes, repeat steps 6, 7, and 8 to insert the required amino acids in sequence , to extend the peptide chain;
9、清洗树脂:重复步骤4、的内容；9, cleaning resin: repeat step 4, content;
10、沥干树脂，用准备好的切割液(TFA:苯酚:水:TIPS＝88:5:5:2)切割肽段；10. Drain the resin and cut the peptide with the prepared cutting solution (TFA: phenol: water: TIPS = 88:5:5:2);
11、将溶液压出，用氮气浓缩至5ml；11. Press out the solution and concentrate to 5ml with nitrogen;
12、加入35mL冰乙醚沉淀肽，3500g离心15min。沉淀的肽为目标肽。采用BEH C18色谱柱(1.7μm，2.1×100mm，Waters Inc.，Milford，MA，USA)，采用反相高效液相色谱(RP-HPLC)对合成的多肽进行纯化。流动相为0.1％甲酸(溶剂A)和100％乙腈(溶剂B)，流动相为:0-10min，100％溶剂A；10-22min，30-80％溶剂B；22-23min，100％溶剂A，流速0.3ml/min。分离过程在280nm紫外波长下进行检测。用液相色谱-质谱联用法对合成的多肽进行鉴定。12. Add 35 mL ice ether to precipitate the peptide, and centrifuge at 3500 g for 15 min. The precipitated peptide is the target peptide. The synthesized peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a BEH C18 column (1.7 μm, 2.1×100 mm, Waters Inc., Milford, MA, USA). Mobile phase is 0.1% formic acid (solvent A) and 100% acetonitrile (solvent B), and mobile phase is: 0-10min, 100% solvent A; 10-22min, 30-80% solvent B; 22-23min, 100% solvent A, flow rate 0.3ml/min. The separation process was detected at a UV wavelength of 280nm. The synthesized peptides were identified by liquid chromatography-mass spectrometry.
本发明的干腌火腿源的缓解酒精性肝损伤的肽同时具有改善肠道菌群和降低肝细胞氧化应激损伤的功能，非常适合用于制备缓解肠道和肝脏不适的食品以及修复酒精性肝损伤的药品。The dry-cured ham-derived peptide for alleviating alcoholic liver injury of the present invention has the functions of improving intestinal flora and reducing liver cell oxidative stress damage, and is very suitable for preparing food for relieving intestinal and liver discomfort and repairing alcoholic liver injury. Medicines for liver damage.
干腌火腿源缓解酒精性肝损伤肽(JHP)对雄性小鼠酒精性肝损伤(ALD)的影响；JHP是一种分子量范围广泛的多肽的复杂混合物。确定36个月的干腌火腿中的功能性成分对ALD预防作用。Dry-cured ham sources attenuate the effects of alcoholic liver damage peptide (JHP); JHP is a complex mixture of peptides with a wide range of molecular weights in male mice. Determining the ALD preventive effect of functional ingredients in 36-month dry-cured ham.
组分A由于分子量最大首先被洗脱出来，而组分I分子量最小，最后被洗脱出来。收集所有馏分，冷冻干燥。用36个月的干腌火腿中分离的不同组分喂养194只小鼠，测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和丙二醛(MDA)(图1的B-D)。G组ALT(128.89U/L)、AST(294.39U/L)、MDA(80.47mmol/mL)水平均低于其他各组。因此，JHP-G具有最大的缓解酒精性肝损伤特性。为了进一步分析，JHP-G在真空冷冻干燥机中干燥。Component A was eluted first due to its largest molecular weight, while component I was eluted last because of its smallest molecular weight. All fractions were collected and freeze-dried. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA) were measured in 194 mice fed different fractions isolated from dry-cured ham for 36 months (Fig. 1B-D). The levels of ALT (128.89U/L), AST (294.39U/L) and MDA (80.47mmol/mL) in group G were lower than those in other groups. Therefore, JHP-G has the greatest alleviating property of alcoholic liver injury. For further analysis, JHP-G was dried in a vacuum freeze dryer.
雄性小鼠中分别喂食对照饮食或含乙醇饮食(含或不含JHP)35天血清中谷丙转氨酶ALT(B)、谷草转氨酶AST(C)和丙二醛MDA(D)浓度的影响；Effects of serum alanine aminotransferase ALT (B), aspartate aminotransferase AST (C) and malondialdehyde MDA (D) concentrations in male mice fed a control diet or an alcohol-containing diet (with or without JHP) for 35 days;
采用反相高效液相色谱法进一步纯化G组分，该组分存在分子间极性差异。从G-1到G-7共分离出7个馏分(图2的A)。将分离的馏分冻干，测定其对ALD的活性(图2的B-D)。G-6部位具有最大的缓解损伤作用。Component G was further purified by reversed-phase high-performance liquid chromatography, which had intermolecular polarity differences. A total of 7 fractions were separated from G-1 to G-7 (A of Fig. 2). The isolated fractions were lyophilized and assayed for their activity on ALD (Figure 2, panels B-D). The G-6 site has the greatest damage relief.
采用反相高效液相色谱质谱联用(LC-MS/MS)对G-6进行氨基酸测序。发现是来自 于Lys-Arg-Gln-Lys-Tyr-Asp(KRQKYD)。Amino acid sequencing of G-6 was carried out by reversed-phase high-performance liquid chromatography-mass spectrometry (LC-MS/MS). Found to be from Lys-Arg-Gln-Lys-Tyr-Asp (KRQKYD).
对合成的干腌火腿源缓解酒精性肝损伤肽进行结构鉴定(图3)；Structural identification of synthetic dry-cured ham-derived peptides for alleviating alcoholic liver injury (Figure 3);
采用Acquity(Waters Inc.)高效液相色谱系统，采用反相BEH C18色谱柱(1.7μm，2.1×100mm，Waters Inc.)对具有缓解酒精性肝损伤最有效的峰进行分离。The Acquity (Waters Inc.) high performance liquid chromatography system was used to separate the most effective peaks for alleviating alcoholic liver injury with a reversed-phase BEH C18 column (1.7μm, 2.1×100mm, Waters Inc.).
梯度洗脱：流速0.3mL/min，洗脱液a为0.1％甲酸，洗脱液b为100％ACN。流动梯度为：0-10min，100％a；10-22min，30-80％b；22-23min，100％a。柱温保持在25℃。流量直接进入MS/MS系统进行多反应测量。Gradient elution: flow rate 0.3mL/min, eluent a is 0.1% formic acid, and eluent b is 100% ACN. The flow gradient is: 0-10min, 100%a; 10-22min, 30-80%b; 22-23min, 100%a. The column temperature was maintained at 25°C. The flow goes directly to the MS/MS system for multiple reaction measurements.
前体离子记录的质量范围为m/z＝200-4000。使用Mass Lynx V4.1操作仪器，分析质谱图信息。Precursor ions were recorded in the mass range m/z = 200-4000. Use Mass Lynx V4.1 to operate the instrument and analyze the mass spectrogram information.
KRQKYD通过抑制氧化应激对小鼠酒精性肝损伤的影响；Effect of KRQKYD on alcoholic liver injury in mice by inhibiting oxidative stress;
为了确定KRQKYD是否影响酒精处理小鼠肝脏氧化应激损伤，需要检测肝脏中活性氧(ROS)、超氧化物歧化酶(SOD)和谷胱甘肽氧化酶(GSH-Px)的水平以及CYP2E1、Nrf2和HO-1基因的表达。In order to determine whether KRQKYD affects liver oxidative stress injury in alcohol-treated mice, it is necessary to detect the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione oxidase (GSH-Px) in the liver as well as the levels of CYP2E1, Expression of Nrf2 and HO-1 genes.
如图4的A所示，酒精处理(EtOH)组的活性氧水平(161.21U/mg prot)高于空白组(94.66U/mg prot)，说明酒精处理小鼠产生了更多的活性氧。KRQKYD处理组活性氧水平为71.19U/mg prot，低于对照组。这表明KRQKYD预处理显著降低了EtOH组活性氧水平。饮酒可显著降低肝脏中GSH-Px和SOD的水平，如图4的B所示。As shown in A of Figure 4, the level of reactive oxygen species (161.21 U/mg prot) in the alcohol-treated (EtOH) group was higher than that in the blank group (94.66 U/mg prot), indicating that alcohol-treated mice produced more reactive oxygen species. The reactive oxygen species level in the KRQKYD treatment group was 71.19 U/mg prot, which was lower than that in the control group. This indicated that KRQKYD pretreatment significantly reduced the ROS level in the EtOH group. Alcohol consumption significantly decreased the levels of GSH-Px and SOD in the liver, as shown in B of Fig. 4 .
KRQKYD预处理显著改善了EtOH组GSH301Px和SOD活性的下降。结果表明，KRQKYD可以通过抑制活性氧的产生，提高GSH-Px和SOD活性来减少酒精诱导的肝脏氧化应激损伤；KRQKYD对ALD的保护作用可能是通过下调CYP2E1的表达降低氧化应激，并通过激活Nrf2/HO319通路增强氧化防御系统。KRQKYD pretreatment significantly improved the decline of GSH301Px and SOD activities in the EtOH group. The results showed that KRQKYD can reduce alcohol-induced liver oxidative stress damage by inhibiting the production of reactive oxygen species and increasing the activity of GSH-Px and SOD; the protective effect of KRQKYD on ALD may be through reducing the expression of CYP2E1 to reduce oxidative stress, and through Activation of the Nrf2/HO319 pathway enhances the oxidative defense system.
KRQKYD对酒精处理小鼠(EtOH组)GM谱的影响；Effect of KRQKYD on GM profile of alcohol-treated mice (EtOH group);
KRQKYD对酒精处理小鼠GM谱的影响通过16S rRNA高通量测序分析结肠微生物区系组成。共在32个样本(每组n＝8)中检测到4201828个细菌16S rRNA，每个样本有131307个，共检测到749个不同的操作分类单元。The effect of KRQKYD on the GM profile of alcohol-treated mice was analyzed by 16S rRNA high-throughput sequencing to analyze colonic microflora composition. A total of 4,201,828 bacterial 16S rRNAs were detected in 32 samples (n = 8 per group), 131,307 in each sample, and a total of 749 different operational taxa were detected.
EtOH组的Chao1和Shannon指数明显高于空白组，KRQKYD组，和EtOH+KRQKYD组，这些结果表明酒精摄入显著提高了变形杆菌的比例，而KRQKYD预处理显著降低了酒精处理小鼠肠道中变形杆菌的比例。The Chao1 and Shannon indices of the EtOH group were significantly higher than those of the blank group, KRQKYD group, and EtOH+KRQKYD group, these results indicated that alcohol intake significantly increased the proportion of Proteus, and KRQKYD pretreatment significantly reduced Proteus in the gut of alcohol-treated mice The proportion of bacilli.
在门水平上，GM主要由疣微菌门、拟杆菌门、放线菌门、厚壁菌门和变形菌门组成(图5的A)。EtOH组疣微菌门、放线菌门和脱硫弧菌门的比例明显减少，而KRQKYD预 处理后的呈进行性增加。酒精摄入显著提高了变形杆菌的比例，而KRQKYD预处理显著降低了酒精处理小鼠的变形杆菌比例。At the phylum level, GM was mainly composed of Verrucobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Proteobacteria (Fig. 5A). The proportions of Verrucomicrobia, Actinomycetes and Desulfovibrio were significantly decreased in the EtOH group, but increased progressively after KRQKYD pretreatment. Alcohol intake significantly increased the proportion of Proteus bacteria, whereas KRQKYD pretreatment significantly decreased the proportion of Proteobacteria in alcohol-treated mice.
在属水平上，EtOH处理组比空白组大肠杆菌和肠球菌的丰度高，而艾克曼菌、杜博氏菌和脱硫弧菌的丰度低(图5的B)。KRQKYD预处理后，艾克曼菌、杜博氏菌和脱硫弧菌的相对丰度增加，大肠杆菌、志贺氏菌和肠球菌的相对丰度降低。这表明KRQKYD对肝脏的保护作用可能与调节酒精诱导小鼠的细菌组成有关。At the genus level, the abundance of E. coli and Enterococcus was higher in the EtOH treatment group than in the blank group, while the abundance of Ekkermansia, Duboella and Desulfovibrio was lower (Fig. 5B). After pretreatment with KRQKYD, the relative abundances of Ekmansia, Duboella, and Desulfovibrio increased, while those of Escherichia coli, Shigella, and Enterococcus decreased. This suggests that the protective effect of KRQKYD on the liver may be related to the regulation of bacterial composition in alcohol-induced mice.
KRQKYD可提高对紧密蛋白的表达继而影响肠道稳态；KRQKYD can increase the expression of compact protein and then affect the intestinal homeostasis;
首先进行阿利新蓝染色以确定KRQKYD是否影响酒精处理小鼠的肠道内稳态根据结肠的组织病理学分析(图6的A)。用Western blot法检测Reg3g和Reg3b的表达，进一步探讨KRQKYD对肠道平衡的保护作用。Alcian blue staining was first performed to determine whether KRQKYD affects intestinal homeostasis in alcohol-treated mice based on histopathological analysis of the colon (Fig. 6A). Western blot was used to detect the expression of Reg3g and Reg3b, and further explore the protective effect of KRQKYD on intestinal balance.
如图6的B-D所示，与空白组相比，酒精处理(Etoh处理)小鼠Reg3b和Reg3g蛋白表达水平略低。然而，用KRQKYD预处理后，在酒精处理过的小鼠中，这些蛋白的含量越来越高。采用RT-qPCR检测结肠中密封蛋白、封闭蛋白-1和紧密连接-1的mRNA表达水平。图6的A显示，酒精处理后小鼠密封蛋白、封闭蛋白-1和紧密连接-1的mRNA表达水平明显降低，而经KRQKYD预处理的小鼠则相反，mRNA表达水平明显升高。证实出调控机制包括了封闭蛋白-1、密封蛋白和紧密连接-1，使用免疫组织化学和Western-blot技术评估了上游信号通路蛋白的变化。如图6的B-Etoh处理小鼠的密封蛋白、封闭蛋白-1和紧密连接-1蛋白表达水平明显降低。As shown in Figure 6B-D, compared with the blank group, the protein expression levels of Reg3b and Reg3g in the alcohol-treated (Etoh-treated) mice were slightly lower. However, after pretreatment with KRQKYD, these proteins were increasingly abundant in alcohol-treated mice. The mRNA expression levels of claudin, claudin-1 and tight junction-1 in the colon were detected by RT-qPCR. Figure 6A shows that the mRNA expression levels of claudin, claudin-1 and tight junction-1 were significantly decreased in mice treated with alcohol, whereas the mRNA expression levels were significantly increased in KRQKYD pretreated mice on the contrary. It was confirmed that the regulatory mechanism included occludin-1, claudin and tight junction-1, and the changes of upstream signaling pathway proteins were evaluated using immunohistochemistry and Western-blot techniques. As shown in Figure 6, the expression levels of claudin, claudin-1 and tight junction-1 proteins in mice treated with B-Etoh were significantly reduced.
KRQKYD预处理可逐步提高酒精处理小鼠中这些蛋白的水平。KRQKYD促进紧密连接的成分，增强了屏障功能，促进了肠道完整性。结果表明，KRQKYD提高了抗菌肽和紧密蛋白的表达，改善了小鼠的肠道平衡。KRQKYD pretreatment progressively increases the levels of these proteins in alcohol-treated mice. KRQKYD promotes components of tight junctions, enhances barrier function, and promotes intestinal integrity. The results showed that KRQKYD increased the expression of antimicrobial peptides and claudin and improved intestinal balance in mice.
图1凝胶排阻色谱法获得的肽对雄性小鼠ALD的影响。(A)干腌火腿中多肽的SephadexTM 10/300 GL尺寸排斥色谱分析；(B-D)：JHP-(A-I)对雄性小鼠血清中谷丙转氨酶ALT；(B)、谷草转氨酶AST；(C)和丙二醛MDA(D)浓度的影响。Figure 1 Effect of peptides obtained by gel exclusion chromatography on ALD in male mice. (A) SephadexTM 10/300 GL size exclusion chromatographic analysis of peptides in dry-cured ham; (B-D): JHP-(A-I) on alanine aminotransferase ALT in serum of male mice; (B), aspartate aminotransferase AST; (C) and Effect of malondialdehyde MDA(D) concentration.
图2空白组(CTRL)、酒精组(EtOH)、酒精+联苯双酯组(EtOH)、酒精+肽组(EtOH+JHP)喂食35天对小鼠肠道内酶表达的影响(A)反相高效液相色谱法分离G组分；(B-D):G-(1-7)对雄性小鼠血清中谷丙转氨酶ALT(B)、谷草转氨酶AST；(C)和丙二醛MDA(D)浓度的影响。Figure 2 Effects of blank group (CTRL), alcohol group (EtOH), alcohol+bifendate group (EtOH), alcohol+peptide group (EtOH+JHP) on the expression of intestinal enzymes in mice fed for 35 days (A) Separation of G components by high-performance liquid chromatography; (B-D): G-(1-7) on alanine aminotransferase ALT (B), aspartate aminotransferase AST in male mouse serum; (C) and malondialdehyde MDA (D) concentration effect.
图3A-图3C分别为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的MS结构鉴定图。图3AMS/MS谱图中G-6组分的总颗粒；图3B峰在9.74min时的质谱(G-6)；图 3C用MS/MS谱鉴定纯化的多肽的分子量和氨基酸序列。Figure 3A-Figure 3C are the MS structure identification diagrams of the hexapeptide Leu-Pro-Gly-Val-Leu-Pro-Val-Ala (KRQKYD), respectively. The total particles of the G-6 component in the figure 3AMS/MS spectrum; the mass spectrum (G-6) of the peak in Figure 3B at 9.74min; Figure 3C using MS/MS spectrum to identify the molecular weight and amino acid sequence of the purified polypeptide.
图4KRQKYD对小鼠酒精性肝脏氧化应激的影响。(A)肝脏活性氧水平；(B)肝脏抗氧化酶活性水平；(C)HO-1、Nrf2和CYP2E1基因在肝脏中的表达水平；(D-E)肝组织HO-1、Nrf2、CYP2E1蛋白表达水平。Fig. 4 Effect of KRQKYD on oxidative stress in alcoholic liver of mice. (A) liver reactive oxygen species level; (B) liver antioxidant enzyme activity level; (C) HO-1, Nrf2 and CYP2E1 gene expression levels in liver; (D-E) liver tissue HO-1, Nrf2, CYP2E1 protein expression level.
图5喂食对照饮食或含乙醇饮食(含或不含JHP)35天对小鼠肠道菌群的影响。(A)肠道细菌门水平的相对丰度；(B)肠道细菌属水平的相对丰度。Figure 5 Effects of 35 days on the gut microbiota of mice fed a control diet or an ethanol-containing diet (with or without JHP). (A) Relative abundance at the level of gut bacterial phylum; (B) Relative abundance at the level of gut bacterial genus.
图6KRQKYD对小鼠肠道屏障稳态的影响。(A)定量RT-PCR检测紧密连接-1(ZO-1)、封闭蛋白-1(Claudin-1)、密封蛋白(Occludin)mRNA表达水平；(B-C)western blot检测ZO-1、Claudin-1、Occludin表达水平。Figure 6 Effect of KRQKYD on intestinal barrier homeostasis in mice. (A) Quantitative RT-PCR detection of tight junction-1 (ZO-1), claudin-1 (Claudin-1), claudin (Occludin) mRNA expression levels; (B-C) western blot detection of ZO-1, Claudin-1 , Occludin expression level.
实施例2：一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法Example 2: A dry-cured ham-derived polypeptide capable of alleviating alcoholic liver injury and its preparation method
一种可缓解酒精性肝损伤的干腌火腿源多肽，氨基酸序列为：Lys-Arg-Gln-Lys-Tyr-Asp。A dry-cured ham-derived polypeptide that can relieve alcoholic liver injury, the amino acid sequence is: Lys-Arg-Gln-Lys-Tyr-Asp.
为实现上述目的及其他相关目的，本发明提供的技术方案是：一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法，包括下列步骤：In order to achieve the above purpose and other related purposes, the technical solution provided by the present invention is: a method for preparing a dry-cured ham-derived polypeptide that can relieve alcoholic liver damage, comprising the following steps:
步骤1：将干腌火腿的股二头肌与磷酸盐缓冲液混合，然后于冰浴条件下用匀浆机进行均质，接着离心取上清，得到干腌火腿多肽提取物；Step 1: Mix the biceps femoris of dry-cured ham with phosphate buffer, then homogenize with a homogenizer under ice bath conditions, and then centrifuge to take the supernatant to obtain dry-cured ham polypeptide extract;
步骤2：对干腌火腿多肽提取物进行超滤，再用凝胶柱分离，洗脱条件为：洗脱液为0.01mol/L的HCl，恒温分离，流速0.8mL/min，用280nm紫外检测器测定馏分，用自动馏分收集器收集每管馏分；馏分在真空冷冻干燥机中干燥；Step 2: Perform ultrafiltration on the dry-cured ham polypeptide extract, and then separate it with a gel column. The elution conditions are: the eluent is 0.01mol/L HCl, separated at a constant temperature, the flow rate is 0.8mL/min, and detected by 280nm ultraviolet light Fractions were measured with an automatic fraction collector, and each tube fraction was collected with an automatic fraction collector; the fractions were dried in a vacuum freeze dryer;
步骤3：采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离，获得最具缓解酒精性肝损伤活性的多肽；Step 3: Using liquid phase separation to further separate the most effective component for alleviating alcoholic liver injury obtained in step 2, to obtain the most active polypeptide for alleviating alcoholic liver injury;
步骤4：采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析，鉴定活性肽的结构，得到一种保护肝活性的肽序列为：Lys-Arg-Gln-Lys-Tyr-Asp。Step 4: Using mass spectrometry to analyze the peptide with the most active alcohol-induced liver injury obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: Lys-Arg-Gln-Lys-Tyr -Asp.
步骤3包括将冻干后的样品溶于1mL蒸馏水，注入HPLC系统，采用BEH C18色谱柱进行梯度洗脱，流速为0.3mL/min，洗脱液A为0.1％甲酸，洗脱液B为100％乙腈；流速梯度为：0-10min，100％A，10-22min，30-80％B；22-23min，100％A；肽峰在280nm紫外波长下检测；将多肽对应的峰分为七个部分，冷冻干燥；收集七个干腌火腿小肽分离组分，干燥后测定各个小肽的缓解酒精性肝损伤的活性。 Step 3 includes dissolving the lyophilized sample in 1 mL of distilled water, injecting it into the HPLC system, and performing gradient elution with a BEH C18 chromatographic column at a flow rate of 0.3 mL/min, eluent A is 0.1% formic acid, and eluent B is 100 % acetonitrile; the flow rate gradient is: 0-10min, 100%A, 10-22min, 30-80%B; 22-23min, 100%A; the peptide peak is detected at 280nm ultraviolet wavelength; the peak corresponding to the peptide is divided into seven The seven parts were freeze-dried; seven dry-cured ham small peptide fractions were collected, and the activity of each small peptide in alleviating alcoholic liver injury was determined after drying.
鉴定方法包括：采用Acquity高效液相色谱系统，采用反相BEH C18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离；梯度洗脱：流速0.3mL/min，洗脱液a为0.1％甲酸，洗脱液b为100％CAN；流动梯度为:0-10min，100％a，10-22min，30-80％b；22-23 min，100％a；柱温保持在25℃；流量直接进入MS/MS系统进行多反应测量；前体离子记录的质量范围为m/z＝200-4000；使用Mass Lynx V4.1操作仪器，分析质谱图信息。The identification methods include: using Acquity high-performance liquid chromatography system, using reversed-phase BEH C18 chromatographic column to separate the peptide peak with the most active activity in alleviating alcoholic liver injury; gradient elution: flow rate 0.3mL/min, eluent a 0.1 % formic acid, the eluent b is 100% CAN; the flow gradient is: 0-10min, 100%a, 10-22min, 30-80%b; 22-23min, 100%a; the column temperature is maintained at 25°C; The flow directly enters the MS/MS system for multiple reaction measurements; the mass range of the precursor ion records is m/z=200-4000; use Mass Lynx V4.1 to operate the instrument and analyze the mass spectrum information.
该六肽同时具有增加肠道中脱硫菌门相对丰度(由5.2±0.04％升至7.1±0.02％)和降低后壁菌门相对丰度(由66.5±0.02％降至57.3±0.01％)等功能，相较于酒精受损肠道，可以增加肠上皮细胞的紧密连接，提高密封蛋白(上调18.2±0.2％)、封闭蛋白-1(上调15.8±0.3％)和紧密连接-1(上调22.5±0.3％)蛋白的表达，降低肝脏炎症级联反应并可用于功能性食品或药品的制备。本发明中干腌火腿源的缓解酒精性肝损伤肽上调了Nrf2/HO-1抗氧化防御系统的表达，降低了肝细胞氧化应激损伤，具有抑制活性氧的生成的功能，从而非常适合作为主要功效配料用于制备缓解酒精性肝损伤的食品或药品。The hexapeptide can also increase the relative abundance of Desulfobacteria in the intestine (from 5.2±0.04% to 7.1±0.02%) and reduce the relative abundance of Deuterobacteria (from 66.5±0.02% to 57.3±0.01%), etc. function, compared with alcohol-impaired gut, can increase the tight junction of intestinal epithelial cells, improve claudin (18.2±0.2% up-regulation), claudin-1 (15.8±0.3% up-regulation) and tight junction-1 (22.5% up-regulation ±0.3%) protein expression, reduce liver inflammation cascade reaction and can be used for the preparation of functional food or medicine. In the present invention, the peptide derived from dry-cured ham for alleviating alcoholic liver injury up-regulates the expression of Nrf2/HO-1 antioxidant defense system, reduces the oxidative stress damage of liver cells, and has the function of inhibiting the generation of active oxygen, so it is very suitable as a The main functional ingredients are used to prepare food or medicine for alleviating alcoholic liver damage.
以上所述者仅为用以解释本发明之较佳实施例，并非企图具以对本发明做任何形式上之限制，是以，凡有在相同之发明精神下所作有关本发明之任何修饰或变更，皆仍应包括在本发明意图保护之范畴。The above are only preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change of the present invention made under the same spirit of the invention , should still be included in the scope of protection intended by the present invention.
Claims (4)
- 一种可缓解酒精性肝损伤的干腌火腿源多肽，其特征在于：氨基酸序列为：Lys-Arg-Gln-Lys-Tyr-Asp。A dry-cured ham-derived polypeptide capable of alleviating alcoholic liver damage, characterized in that the amino acid sequence is: Lys-Arg-Gln-Lys-Tyr-Asp.
- 一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法，其特征在于：包括下列步骤：A method for preparing a dry-cured ham-derived polypeptide capable of alleviating alcoholic liver damage, characterized in that it comprises the following steps:步骤1：将干腌火腿的股二头肌与磷酸盐缓冲液混合，然后于冰浴条件下用匀浆机进行均质，接着离心取上清，得到干腌火腿多肽提取物；Step 1: Mix the biceps femoris of dry-cured ham with phosphate buffer, then homogenize with a homogenizer under ice bath conditions, and then centrifuge to take the supernatant to obtain dry-cured ham polypeptide extract;步骤2：对干腌火腿多肽提取物进行超滤，再用凝胶柱分离，洗脱条件为：洗脱液为0.01mol/L的HCl，恒温分离，流速0.8mL/min，用280nm紫外检测器测定馏分，用自动馏分收集器收集每管馏分；馏分在真空冷冻干燥机中干燥；Step 2: Perform ultrafiltration on the dry-cured ham polypeptide extract, and then separate it with a gel column. The elution conditions are: the eluent is 0.01mol/L HCl, separated at a constant temperature, the flow rate is 0.8mL/min, and detected by 280nm ultraviolet light Fractions were measured with an automatic fraction collector, and each tube fraction was collected with an automatic fraction collector; the fractions were dried in a vacuum freeze dryer;步骤3：采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离，获得最具缓解酒精性肝损伤活性的多肽；Step 3: Using liquid phase separation to further separate the most effective component for alleviating alcoholic liver injury obtained in step 2, to obtain the most active polypeptide for alleviating alcoholic liver injury;步骤4：采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析，鉴定活性肽的结构，得到一种保护肝活性的肽序列为：Lys-Arg-Gln-Lys-Tyr-Asp。Step 4: Using mass spectrometry to analyze the peptide with the most active alcohol-induced liver injury obtained by liquid phase separation, identify the structure of the active peptide, and obtain a peptide sequence with liver protection activity: Lys-Arg-Gln-Lys-Tyr -Asp.
- 根据权利要求2所述的可缓解酒精性肝损伤的干腌火腿源多肽的制备方法，其特征在于：步骤3包括将冻干后的样品溶于1mL蒸馏水，注入HPLC系统，采用BEH C18色谱柱进行梯度洗脱，流速为0.3mL/min，洗脱液A为0.1％甲酸，洗脱液B为100％乙腈；流速梯度为：0-10min，100％A，10-22min，30-80％B；22-23min，100％A；肽峰在280nm紫外波长下检测；将多肽对应的峰分为七个部分，冷冻干燥；收集七个干腌火腿小肽分离组分，干燥后测定各个小肽的缓解酒精性肝损伤的活性。The preparation method of the dry-cured ham-derived polypeptide capable of relieving alcoholic liver injury according to claim 2, characterized in that: Step 3 comprises dissolving the freeze-dried sample in 1 mL of distilled water, injecting it into the HPLC system, and using a BEH C18 chromatographic column Carry out gradient elution, the flow rate is 0.3mL/min, the eluent A is 0.1% formic acid, and the eluent B is 100% acetonitrile; the flow rate gradient is: 0-10min, 100%A, 10-22min, 30-80% B; 22-23min, 100% A; peptide peaks were detected at 280nm ultraviolet wavelength; the peaks corresponding to the peptides were divided into seven parts and freeze-dried; seven dry-cured ham small peptide fractions were collected, and each small peptide fraction was determined after drying The activity of peptides in alleviating alcohol-induced liver injury.
- 根据权利要求2所述的可缓解酒精性肝损伤的干腌火腿源多肽的制备方法，其特征在于：鉴定方法包括：采用Acquity高效液相色谱系统，采用反相BEH C18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离；梯度洗脱：流速0.3mL/min，洗脱液a为0.1％甲酸，洗脱液b为100％CAN；流动梯度为:0-10min，100％a，10-22min，30-80％b；22-23min，100％a；柱温保持在25℃；流量直接进入MS/MS系统进行多反应测量；前体离子记录的质量范围为m/z＝200-4000；使用Mass Lynx V4.1操作仪器，分析质谱图信息。According to claim 2, the preparation method of the dry-cured ham-derived polypeptide that can relieve alcoholic liver damage is characterized in that: the identification method includes: using an Acquity high-performance liquid chromatography system, using a reversed-phase BEH C18 chromatographic column for the most relieving Separation of peptide peaks of alcoholic liver injury activity; gradient elution: flow rate 0.3mL/min, eluent a is 0.1% formic acid, eluent b is 100% CAN; flow gradient: 0-10min, 100% a , 10-22min, 30-80%b; 22-23min, 100%a; the column temperature is maintained at 25°C; the flow directly enters the MS/MS system for multiple reaction measurements; the mass range of the precursor ion record is m/z= 200-4000; use Mass Lynx V4.1 to operate the instrument and analyze the mass spectrum information.
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