Pea oligopeptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of vegetable protein peptide preparation, and particularly relates to pea oligopeptide and a preparation method and application thereof. The pea oligopeptide is obtained by carrying out enzymolysis on pea protein by adopting alkaline protease and neutral protease, and the pea oligopeptide has great lipid-lowering potential and has the characteristics of easiness in absorption and digestion and high bioavailability. By constructing a non-alcoholic fatty liver cell model, it is found that the pea oligopeptide has 3-hydroxy-3-methylglutaryl coenzyme A (HMG-COA) reductase inhibitory activity, has the effects of improving lipid accumulation in liver cells and regulating and controlling lipid metabolism related signal protein expression, and can achieve the effect of effectively preventing and improving the non-alcoholic fatty liver.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protein peptide preparation, and in particular relates to a pea oligopeptide and a preparation method and application thereof. Background Art

[0002] Non-alcoholic fatty liver (NAFLD) refers to a pathological syndrome characterized by excessive lipid deposition in liver cells, caused by factors other than alcohol and other clear liver-damaging factors. It is generally considered the liver component of metabolic syndrome and is often accompanied by obesity, dyslipidemia, and insulin resistance. NAFLD is divided into two major categories: primary and secondary. The former is related to insulin resistance and genetic susceptibility, while the latter is caused by specific reasons such as drugs, viral infection, or autoimmunity. Fatty liver associated with metabolic syndrome, such as rapid weight gain and excessive weight caused by overnutrition, obesity, diabetes, and hyperlipidemia, falls into the category of primary non-alcoholic fatty liver disease, and this type of non-alcoholic fatty liver accounts for a larger proportion of NAFLD. Because people are surrounded by excess nutrition today, obesity associated with ectopic lipid accumulation has become an important risk factor for NAFLD.

[0003] Pea oligopeptides are small-molecule peptides obtained by enzymatic or hydrolytic hydrolysis of pea protein. Some existing studies have shown that pea oligopeptides possess antimicrobial, antioxidant, and angiotensin-converting enzyme (ACE) inhibitory activities. However, the functions of pea oligopeptides obtained by different preparation methods vary significantly. Currently, there are no reports on pea oligopeptides with improved effects on non-alcoholic fatty liver disease and their preparation methods. Summary of the Invention

[0004] The present invention aims to provide a pea oligopeptide and a preparation method and application thereof, wherein the pea oligopeptide has good improvement and prevention effects on non-alcoholic fatty liver.

[0005] The present invention provides a method for preparing pea oligopeptide, comprising the following steps:

[0006] mixing pea protein with water and emulsifying the mixture to obtain a pea protein emulsion;

[0007] mixing the pea protein emulsion with a protease and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis mixture;

[0008] The enzymatic hydrolysis mixture is subjected to enzyme inactivation treatment to obtain an enzyme inactivation-treated enzymatic hydrolysis product;

[0009] The enzymatic hydrolysis product after the enzyme inactivation treatment is subjected to solid-liquid separation, and the obtained supernatant is subjected to ultrafiltration, so that the obtained permeate contains the pea oligopeptide;

[0010] The protease includes alkaline protease and neutral protease; based on the volume of the pea protein emulsion, the added amount of the alkaline protease is 8-10 mg / mL; the added amount of the neutral protease is 7-9 mg / mL.

[0011] Preferably, the enzymatic hydrolysis temperature is 40-55° C., the enzymatic hydrolysis time is 3.5-4.5 h, and the enzymatic hydrolysis system pH value is 7-8.5.

[0012] Preferably, the volume ratio of the pea protein mass to water is (1.8-3.3) g: (10-15) mL.

[0013] Preferably, the ultrafiltration step comprises: sequentially passing the supernatant through ultrafiltration membranes with molecular weights of 10 kDa and 1 kDa, respectively, to obtain the permeate.

[0014] Preferably, the preparation method further comprises post-processing the permeate; the post-processing step comprises:

[0015] The permeate is subjected to nanofiltration to obtain a purified solution;

[0016] The purified solution is concentrated at low temperature, and when the obtained concentrate has a Brix value of 28 to 32, it is decolorized to obtain decolorized pea peptide;

[0017] The decolorized pea peptide is dried to obtain the pea oligopeptide.

[0018] Preferably, the decolorization method is activated carbon treatment, and the time is 25 to 35 minutes;

[0019] The drying method includes spray drying;

[0020] The spray drying pressure is 15-25 MPa, the evaporation rate is 180-220 kg / h, and the time is 5-10 minutes.

[0021] The present invention also provides a pea oligopeptide, which is prepared by the preparation method described in the above technical solution.

[0022] The present invention also provides the use of the pea oligopeptide described in the above technical solution in the preparation of functional foods or health products for preventing and / or improving non-alcoholic fatty liver disease or in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

[0023] The present invention also provides the use of the pea oligopeptide described in the above technical solution in the preparation of functional foods or health products having one or more functions of inhibiting HMG-COA reductase activity, improving lipid accumulation in liver cells, and regulating the expression of lipid metabolism-related signal proteins.

[0024] The present invention also provides a functional food or health product for preventing and / or improving non-alcoholic fatty liver disease, wherein the effective ingredients of the functional food or health product include the pea oligopeptide described in the above technical solution.

[0025] Beneficial effects:

[0026] The present invention provides a method for preparing pea oligopeptides, comprising the following steps: mixing pea protein with water and emulsifying to obtain a pea protein emulsion; mixing the pea protein emulsion with a protease and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis mixture; performing enzyme inactivation treatment on the enzymatic hydrolysis mixture to obtain an enzyme-inactivated enzymatic hydrolysis product; performing solid-liquid separation on the enzyme-inactivated enzymatic hydrolysis product, ultrafiltration of the obtained supernatant, and obtaining a permeate containing the pea oligopeptide; the protease includes alkaline protease and neutral protease; based on the volume of the pea protein emulsion, the amount of the alkaline protease added is 8 to 10 mg / mL; the amount of the neutral protease added is 7 to 9 mg / mL. The present invention uses alkaline protease and neutral protease to enzymatically hydrolyze pea protein to obtain pea oligopeptides, which have great lipid-lowering potential and are easy to absorb and digest and have high bioavailability. The present invention uses a non-alcoholic fatty liver cell model as the research object and studies the effect of the pea oligopeptide prepared above in improving non-alcoholic fatty liver. The results show that the pea oligopeptide has the effects of inhibiting HMG-COA reductase activity, improving lipid accumulation in liver cells and regulating the expression of lipid metabolism-related signal proteins, and can effectively prevent and improve non-alcoholic fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0028] Figure 1 is the HPLC chromatogram of pea oligopeptide in Example 2;

[0029] Figure 2 This is a graph showing the inhibitory activity of pea oligopeptides on HMG-CoA reductase in Example 3;

[0030] Figure 3 The figure shows the effects of pea oligopeptide (A) and atorvastatin (B) on the activity of HepG2 cells in Example 3, wherein different lowercase letters in the figure indicate statistically significant differences (P<0.05);

[0031] Figure 4 This is a graph showing the effect of FFA on the activity of HepG2 cells in Example 3, where different lowercase letters in the graph indicate statistically significant differences (P < 0.05);

[0032] Figure 5 This is the Oil Red O staining result of pea oligopeptide on lipid accumulation in NAFLD cell model in Example 3, where the scale bar in the figure is 50 μm;

[0033] Figure 6 This is a quantitative result of Oil Red O staining of lipid accumulation in the NAFLD cell model by pea oligopeptides in Example 3;

[0034] Figure 7 This is a graph showing the effect of pea oligopeptide intervention on intracellular triglycerides in Example 3;

[0035] Figure 8 This is a graph showing the effect of pea oligopeptide intervention on intracellular cholesterol in Example 3;

[0036] Figure 9 This is a graph showing the effect of pea oligopeptides on the lipid metabolism-related signal AMPK / ACC signaling pathway in the NAFLD cell model in Example 3, where # and ## in the bar graph indicate statistically significant differences (P < 0.05) and significant differences (P < 0.01), respectively; wherein A is a graph showing immunoblotting results, B is a bar graph showing the relative expression levels of p-AMPK / AMPK signaling proteins, C is a bar graph showing the relative expression levels of p-ACC / ACC signaling proteins, and D is a bar graph showing the relative expression levels of CPT1 signaling proteins;

[0037] Figure 10 This is a graph showing the effect of pea oligopeptides on the lipid metabolism-related signal SPEBP2 / HMGCR signaling pathway in the NAFLD cell model in Example 3, where # and ## in the bar graph respectively indicate statistical differences (P < 0.05) and significant differences (P < 0.01); the left figure is an immunoblot result graph, and the right figure is a bar graph of the relative expression levels of SPEBP2 and HMGCR signaling proteins. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing pea oligopeptide, comprising the following steps:

[0039] mixing pea protein with water and emulsifying the mixture to obtain a pea protein emulsion;

[0040] mixing the pea protein emulsion with a protease and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis mixture;

[0041] The enzymatic hydrolysis mixture is subjected to enzyme inactivation treatment to obtain an enzyme inactivation-treated enzymatic hydrolysis product;

[0042] The enzymatic hydrolysis product after the enzyme inactivation treatment is subjected to solid-liquid separation, and the obtained supernatant is subjected to ultrafiltration, so that the obtained permeate contains the pea oligopeptide;

[0043] The protease includes alkaline protease and neutral protease; the protease includes alkaline protease and neutral protease; based on the volume of the pea protein emulsion, the added amount of the alkaline protease is 8-10 mg / mL; the added amount of the neutral protease is 7-9 mg / mL.

[0044] The present invention mixes pea protein with water and then emulsifies to obtain a pea protein emulsion. As one embodiment, the volume ratio of the pea protein mass to water of the present invention is 1.8-3.3 g:10-15 mL; as another embodiment, the volume ratio of the pea protein mass to water is 1 g:5 mL. As one embodiment, the pea protein of the present invention is purchased from Yantai Oriental Protein Technology Co., Ltd. As one embodiment, the emulsification temperature is 45-55°C; as another embodiment, the emulsification temperature is 50°C. The present invention does not specifically limit the specific process of the emulsification, and the conventional emulsification process in this field can be adopted.

[0045] After obtaining the pea protein emulsion, the present invention mixes the pea protein emulsion with a protease and then performs enzymatic hydrolysis to obtain an enzymatic hydrolysis mixture. The protease of the present invention includes alkaline protease and neutral protease; based on the volume of the pea protein emulsion, the alkaline protease is added in an amount of 8-10 mg / mL; the neutral protease is added in an amount of 7-9 mg / mL. In a specific embodiment of the present invention, the alkaline protease used has an activity of 580,000-650,000 DU / g; the neutral protease has an activity of 350,000-390,000 U / g. In one embodiment, the enzymatic hydrolysis temperature can be 40-55°C; in another embodiment, the enzymatic hydrolysis temperature is 50°C. In one embodiment, the enzymatic hydrolysis time can be 3.5-4.5 hours; in another embodiment, the enzymatic hydrolysis temperature is 4 hours. In one embodiment, the enzymatic hydrolysis pH can be 7-8.5; in another embodiment, the pH can be 8.

[0046] After obtaining the enzymatic hydrolysis mixture, the present invention performs an enzyme inactivation treatment on the enzymatic hydrolysis mixture to obtain an enzyme-inactivated enzymatic hydrolysis product. As an embodiment, the enzyme inactivation treatment is a high-temperature enzyme inactivation treatment, and the temperature of the high-temperature enzyme inactivation treatment can be 95° C. and the time is 15 minutes.

[0047] After obtaining the enzyme-inactivated hydrolysis product, the present invention performs solid-liquid separation on the enzyme-inactivated hydrolysis product, and ultrafilters the resulting supernatant to obtain a permeate. In one embodiment, the solid-liquid separation method can be centrifugation; in another embodiment, the centrifugation speed can be 3000 r / min and the time can be 15 minutes. In one embodiment, the ultrafiltration step can be: sequentially passing the supernatant through ultrafiltration membranes with molecular weights of 10 kDa and 1 kDa, respectively, to obtain the permeate. In one embodiment, the ultrafiltration membrane can be an ultrafiltration ceramic membrane.

[0048] After obtaining the permeate, as an embodiment, the present invention performs nanofiltration on the permeate to obtain a purified solution. As an embodiment, the nanofiltration membrane used in the nanofiltration is a nanofiltration membrane of 140 to 200 Da.

[0049] After obtaining the purified solution, in one embodiment, the present invention performs cryogenic concentration on the purified solution. When the resulting concentrate has a Brix value of 28-32, decolorization is performed to obtain decolorized pea peptide. In one embodiment, the concentration temperature can be 60-75°C. In one embodiment, decolorization is performed when the resulting concentrate has a Brix value of 30. In one embodiment, the decolorization method can be activated carbon treatment; the decolorization time can be 25-35 minutes; in another embodiment, the decolorization time can be 30 minutes.

[0050] After obtaining the decolorized pea peptide, as an embodiment, the present invention dries the decolorized pea peptide to obtain the pea oligopeptide. As an embodiment, the drying method of the present invention can be spray drying; the spray drying pressure is 15 to 25 MPa; as another embodiment, the spray drying pressure can be 20 MPa; as an embodiment, the evaporation rate can be 180 to 220 kg / h; as another embodiment, the evaporation rate can be 200 kg / h; as an embodiment, the spray drying time is 5 to 10 minutes; as another embodiment, the spray drying time is 8 minutes.

[0051] The present invention also provides a pea oligopeptide, which is prepared using the preparation method described in the above technical solution. Through physical and chemical property measurements, it was found that the pea oligopeptide prepared using the preparation method of the present invention has high protein and peptide contents, a balanced amino acid composition, and a rich content of essential amino acids; the branched-chain amino acid content is high, thereby confirming that the pea oligopeptide can play an important role in nutrient metabolism and energy homeostasis through multiple signaling pathways, thereby inhibiting fat accumulation and having great lipid-lowering potential. At the same time, it was found that the components with a relative molecular mass of less than 1000u accounted for 87.89% of the pea oligopeptide, the components with a relative molecular mass range of 150-1000u accounted for as high as 82.94%, and the components with a relative molecular mass of more than 2000u accounted for only 12.08%. The weight-average molecular mass of the pea oligopeptide was 559.10u, indicating that the pea oligopeptide is mainly composed of 2-5 peptides. Therefore, it is confirmed that the pea oligopeptide has the characteristics of easy absorption and digestion and high bioavailability.

[0052] Based on the above advantages, the present invention also provides the use of the pea oligopeptide described in the above technical solution in the preparation of functional foods or health products for preventing and / or improving non-alcoholic fatty liver disease, or in the preparation of medicaments for preventing and / or treating non-alcoholic fatty liver disease. The pea oligopeptide described in the present invention has the effects of inhibiting HMG-COA reductase, improving lipid accumulation in liver cells, and regulating the expression of lipid metabolism-related signaling proteins, effectively preventing and improving non-alcoholic fatty liver disease. As an embodiment, the dosage form of the medicament may include, but is not limited to, an injection, tablet, capsule, oral liquid dosage form, or granules.

[0053] The present invention also provides the use of the pea oligopeptide described in the above technical solution in the preparation of functional foods or health products having one or more functions of lowering lipids, inhibiting HMG-COA reductase activity, improving lipid accumulation in liver cells, and regulating the expression of lipid metabolism-related signal proteins. As one embodiment, the functional food or health product can be a functional food or health product with HMG-COA reductase inhibitory activity, improving lipid accumulation in liver cells and regulating the expression of lipid metabolism-related signal proteins, or it can be a lipid-lowering functional food or health product; as another embodiment, the regulation of lipid metabolism-related signal protein expression can upregulate the expression of one or more proteins among p-AMPK, p-ACC and CPT1A, thereby concluding that pea oligopeptides can inhibit ACC activity by activating AMPK phosphorylation and promote the expression of CPT1A (carnitine palmitoyltransferase 1A), thereby enhancing the β-oxidation of fatty acids, weakening the process of fatty acid synthesis, and reducing new fat formation and liver lipid accumulation; as another embodiment, the regulation of lipid metabolism-related signal protein expression can be downregulating the expression of SREBP2 (sterol regulatory element binding protein 2) and / or HMGCR protein (3-hydroxy-3-methylglutaryl coenzyme A reductase) to weaken the cholesterol synthesis pathway and reduce cholesterol accumulation.

[0054] The present invention also provides a functional food or health product for preventing and / or improving non-alcoholic fatty liver disease, wherein the active ingredient of the functional food or health product includes the pea oligopeptide described in the above technical solution. In one embodiment, the effective concentration of the pea oligopeptide in the functional food or health product of the present invention can be 200 to 800 μg / mL; in another embodiment, the effective concentration of the pea oligopeptide in the functional food or health product can be 400 to 800 μg / mL.

[0055] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] A method for preparing pea oligopeptide comprises the following steps:

[0058] 1) 1 g of pea protein was suspended in 5 mL of distilled water, combined and emulsified at 50° C. to obtain a pea protein emulsion;

[0059] 2) adding alkaline protease and neutral protease to the pea protein emulsion, and performing enzymatic hydrolysis at pH 8 and 50° C. for 4 h to obtain an enzymatic hydrolysis product;

[0060] The enzymatic activity of alkaline protease is 600,000 Du / g, and the final concentration in the enzymatic hydrolysis system is 9 mg / mL; the enzymatic activity of neutral protease is 370,000 U / g, and the final concentration in the enzymatic hydrolysis system is 8 mg / mL;

[0061] 3) heating the enzymatic hydrolysis product at 95° C. for 15 minutes to terminate the hydrolysis and inactivate the protease, thereby obtaining an enzyme-inactivated enzymatic hydrolysis product;

[0062] 4) The enzyme-inactivated hydrolyzate was centrifuged at 3000 rpm for 15 min, and the supernatant was passed through ceramic membranes with molecular weights of 10 kDa and 1 kDa in sequence to obtain a permeate containing low molecular weight oligopeptides;

[0063] 5) Using nanofiltration (nanofiltration membrane with a molecular weight cutoff of 200 Da) to remove trace salts and free amino acids in the permeate to obtain a purified solution;

[0064] 6) The purified solution was cryogenically concentrated, and when the concentration was close to 30 Brix, decolorization was performed for 30 minutes. The solution was spray-dried at a pressure of 20 MPa and an evaporation rate of 200 kg / h to remove most of the water and obtain pea oligopeptides.

[0065] Example 2

[0066] Physical and chemical properties of the pea oligopeptide prepared in Example 1

[0067] 1. Detection method

[0068] 1.1 Determination of basic physical and chemical components

[0069] Pea oligopeptides were used as raw materials and the moisture, ash and total protein contents of pea oligopeptides were determined with reference to GB 5009.3-2016 “Determination of Water in Foods”, GB 5009.4-2016 “Determination of Ash in Foods” and GB 5009.5-2016 “Determination of Protein in Foods”. The peptide content was determined based on the difference between the acid-soluble protein content and the free amino acids.

[0070] 1.2 Determination of amino acid composition

[0071] Pea oligopeptides were used as raw materials and the amino acid composition of pea oligopeptides was determined according to GB 5009.124-2016 “Determination of amino acids in foods”.

[0072] 1.3 Molecular weight distribution determination

[0073] The molecular weight distribution of pea oligopeptides was determined using high-performance liquid chromatography (HPLC). A 1 mg / mL solution of pea oligopeptides was prepared in deionized water and filtered through a 0.22 μm tetrafluoroethylene membrane before analysis. The sample was subjected to gel filtration using a HPLC instrument and detected with a UV detector. Chromatographic data were then processed using GPC software. A calibration curve was generated using 0.1% peptide standard solutions. Four peptide standards were used: tyrosine-tyrosine-tyrosine (189 U), tyrosine-tyrosine-tyrosine-arginine (451 U), bacillus enzyme (1450 U), and cytochrome C (12500 U).

[0074] The specific chromatography parameters are: chromatographic column (300mm×7.8mm), mobile phase is a solution of V(acetonitrile):V(water):V(trifluoroacetic acid)=45:55:0.1, injection volume is 10μL, flow rate is 0.5mL / min, column temperature is 30℃, and detection wavelength is 220nm.

[0075] 2. Physical and chemical properties test results

[0076] 2.1 The physical and chemical composition of pea oligopeptides are shown in Table 1.

[0077] Table 1 Physicochemical composition of pea oligopeptides

[0078]

[0079] 2.2 The amino acid composition of pea oligopeptides is shown in Table 2.

[0080] Table 2 Amino acid composition of pea oligopeptides

[0081] Amino acid name Content (g / 100g) Amino acid name Content (g / 100g) Tryptophan (Trp) 0.23 Asparagine (ASP) 10.77 Threonine (Thr) 2.93 Methionine (Met) 0.81 Serine (Ser) 4.42 Isoleucine (Ile) 3.36 Glutamate (Glu) 18.2 Leucine (Leu) 6.29 Glycine (Gly) 3.24 Tyrosine (Tyr) 2.25 Alanine (Ala) 3.96 Phenylalanine (Phe) 3.26 Valine (Val) 4.07 Histidine (His) 1.79 Cystine (Cys)2 0.37 Lysine (Lys) 6.56 Proline (Pro) 3.12 Arginine (Arg) 6.88

[0082] Tables 1 and 2 list the basic physicochemical components and amino acid composition of pea oligopeptides. From Table 1, it can be concluded that pea oligopeptides have high protein and peptide contents, with a protein content of 90.09 g / 100 g and a peptide content of 81.44 g / 100 g. However, its ash content is 5.48 g / 100 g, and the main component of the ash is NaCl salt. Table 2 shows that pea oligopeptides have a balanced amino acid composition, containing large amounts of glutamic acid (18.20g / 100g), aspartic acid (10.77g / 100g), lysine (6.56g / 100g), arginine (6.88g / 100g), leucine (6.29g / 100g), serine (4.42g / 100g), valine (4.07g / 100g), and alanine (3.96g / 100g). It is rich in essential amino acids, with a content of up to 27.51g / 100g. Amino acids are components of protein and play an important role in the intracellular signaling process of cell growth. A rich amino acid composition plays a vital role in regulating human physiological functions. Furthermore, pea oligopeptides contain a high content of branched-chain amino acids, including leucine (6.29g / 100g), isoleucine (3.36g / 100g), and valine (4.07g / 100g). Branched-chain amino acids are amino acids with branches on the side chains of fats. They play an important role in nutrient metabolism and energy homeostasis through various signaling pathways, and can inhibit fat accumulation. Therefore, pea oligopeptides have great lipid-lowering potential and can be used as a natural product to alleviate and intervene in NAFLD.

[0083] 2.3 Molecular weight distribution of pea oligopeptides

[0084] The relative molecular mass distribution of the pea oligopeptide prepared by the enzymatic hydrolysis method in Example 1 was analyzed by high performance liquid chromatography. Figure 1 The HPLC chromatogram of pea oligopeptides is shown, and the relative molecular mass distribution is shown in Table 3.

[0085] Table 3 Molecular weight distribution of pea oligopeptides

[0086]

[0087] Depend on Figure 1From Table 3, it can be concluded that the components with a relative molecular mass of less than 1000u in pea oligopeptides account for 87.89%, the components with a relative molecular mass in the range of 150-1000u account for as high as 82.94%, and the components with a relative molecular mass above 2000u account for only 12.08%. The weight-average molecular mass of pea oligopeptides is 559.10u, which shows that pea oligopeptides are mainly composed of 2-5 peptides. In addition, relevant studies have shown that oligopeptides are more easily absorbed and transported than free amino acids or complete proteins. Therefore, compared with pea protein, pea oligopeptides are easy to absorb and digest and have high bioavailability.

[0088] Example 3

[0089] Verification of the inhibitory effect of pea oligopeptides prepared in Example 1 on non-alcoholic fatty liver disease

[0090] 1. Experimental methods

[0091] 1.13-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition assay

[0092] HMG-CoA reductase (EC 1.1.1.34) is the rate-controlling enzyme of the mevalonate pathway, a metabolic pathway that produces cholesterol from acetyl-CoA. In an NADPH-dependent reaction, HMG-CoA reductase reduces HMG-CoA to mevalonate and CoA. This enzyme is the target of statins, a class of cholesterol-lowering drugs. Inhibition of HMG-CoA reductase induces LDL receptor expression in the liver, thereby lowering plasma cholesterol concentrations.

[0093] The HMG-CoA reductase inhibitory activity assay kit can be used to determine the absorbance decrease (OD 340nm ) to measure the utilization of NADPH, thereby screening for HMG-CoA reductase inhibitors. The inhibition rate is calculated using the following formula:

[0094]

[0095] △A 340nm It is the difference in absorbance at 340nm at two time points (T1 and T2), which is the measurement time interval. The measurement time in this experiment is 10 minutes.

[0096] 1.2 Evaluation of NAFLD cell models

[0097] 1.2.1 Cell culture

[0098] (1) Cell recovery

[0099] Remove the frozen HepG2 cells from the liquid nitrogen tank, quickly place them in a 37°C water bath and shake them for 1 minute to quickly thaw. Then place the cryovial in a centrifuge at 1000 rpm for 3 minutes, discard the supernatant and add 1 mL of DMEM culture medium containing 10% fetal bovine serum and 1% double antibody. Use a pipette to slowly aspirate and mix the cells, then transfer them to a T25 culture flask, add an appropriate amount of culture medium, and culture in an incubator at 37°C and 5% CO2.

[0100] (2) Cell passage

[0101] When the cell confluence in the cell culture flask reaches more than 80%, discard the culture medium in the culture flask, add 1 mL of 1× PBS solution to rinse for 10 seconds, discard the PBS, add 1 mL of 0.25% trypsin cell digestion solution to cover the entire bottom of the T25 culture flask, digest for about 3 to 4 minutes (cell shrinkage and cell gaps become larger), discard the trypsin cell digestion solution, add 2 mL of complete culture medium to terminate digestion, gently blow off the cells, mix well, and divide evenly into two T25 culture flasks, add appropriate amount of culture medium, and continue to culture in the incubator.

[0102] (3) Cell cryopreservation

[0103] When the cell confluence in the cell culture flask reaches more than 80%, discard the culture medium in the culture flask, add 1 mL of 1× PBS solution to rinse for 10 seconds, discard the PBS, add 1 mL of 0.25% trypsin cell digestion solution to cover the entire bottom of the T25 culture flask, and digest for about 3 to 4 minutes (cell shrinkage and cell gaps become larger), discard the trypsin cell digestion solution, add 2 mL of complete culture medium to terminate the digestion, gently blow off the cells, collect the cell suspension, and dispense it into cryopreservation tubes. Centrifuge at 1000g for 3 minutes, discard the supernatant, and add cell freezing solution (92% complete culture medium + 8% DMSO). The final cell concentration in the freezing solution is (5 to 10) × 10 6 pieces / mL.

[0104] (4) Cell counting and plating

[0105] When the cell confluence in the cell culture flask reaches more than 80%, discard the culture medium in the culture flask, add 1 mL of 1× PBS solution to rinse for 10 seconds, discard the PBS, add 1 mL of 0.25% trypsin cell digestion solution to cover the entire bottom of the T25 culture flask, digest for about 3 to 4 minutes, discard the trypsin cell digestion solution, add 2 mL of complete culture medium, gently blow off the cells, mix and dilute appropriately, mix the diluted cell suspension, and draw 100 μL to count using a flow cytometer. After calculating the cell concentration, the required plating concentration (the cell concentration of a 96-well plate is 1×10 4 The cell concentration in a 6-well plate is 1×10 6The cell suspension was diluted and added to the culture plate, mixed and cultured.

[0106] 1.2.2 Detection of cytotoxicity of samples by CCK8 assay

[0107] Cells in the logarithmic growth phase were taken and 1×10 4 Cells were seeded into 96-well cell culture plates, 100 μL of cell suspension was added to each well, complete medium was discarded after culturing for 24 h, 96-well plates were rinsed with 1× PBS, 100 μL of samples of different concentrations diluted with complete medium were added to each well, the concentrations of pea oligopeptides were 50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, 6000, 8000 μg / mL, the concentrations of free fatty acids were 0, 0.25, 0.5, 0.75, 1, 1.25, 2 mM, and the concentrations of atorvastatin were 0, 1, 5, 10, 20, 30, 40 μM. Complete medium was discarded after culturing for 24 h, 96-well plates were rinsed with 1× PBS, 100 μL of new complete medium and 10 μL of CCK8, then place the culture plate in an incubator and incubate for 1 to 3 hours, and measure the absorbance at 450 nm using a microplate reader.

[0108]

[0109] A sample 、A control and A blank are the absorbance of the sample group, control group and blank group at 450 nm, respectively.

[0110] 1.2.3 Oil Red O staining

[0111] (1) Take cells in the logarithmic growth phase and use 1×10 6 Cells were seeded into 6-well cell culture plates at 100 μg / well. 2 mL of cell suspension was added to each well. After culturing for 24 h, the complete medium was discarded and the 6-well plates were rinsed with 1× PBS. Four experimental groups were set up, namely, a blank group (DMEM containing 1% BSA), a free fatty acid group (0.5 mM FFA), a pea oligomer group (0.5 mM FFA + 800 μg / mL pea oligopeptide), and an atorvastatin group (0.5 mM FFA + 10 μM atorvastatin). Culture was continued for 24 h.

[0112] The free fatty acids used in step (1) are prepared by mixing oleic acid and palmitic acid in a ratio of 2:1 (molar concentration ratio). The oleic acid is obtained from Sigma-Aldrich; the palmitic acid is obtained from Maclean. The free fatty acids used in other parts of the present invention are all prepared by this method.

[0113] (2) After culturing for 24 h, the cell culture medium was slowly aspirated, the cells were washed once with PBS, fixed with 4% paraformaldehyde for 20 min, and washed twice with PBS.

[0114] (3) Add an appropriate amount of staining solution (60% isopropanol) to cover the cells for 10 seconds, remove the staining solution, add an appropriate amount of Oil Red O staining solution (1 mL for a 6-well plate), and stain for 30 minutes.

[0115] (4) Remove the Oil Red O staining solution, add an appropriate amount of staining wash solution, let it stand for 30 seconds, then discard the staining wash solution, wash with PBS for 20 seconds, add an appropriate amount of PBS to evenly cover the cells, and observe and photograph under a microscope.

[0116] (5) Add 200 μL of isopropanol to each well and incubate at room temperature for 15 min for extraction. Take 200 μL of the drawer solution and add it to a 96-well plate. Measure the absorbance at a wavelength of 490 nm.

[0117] 1.2.4 Cell triglyceride (TG) and total cholesterol (TC) detection

[0118] (1) Take cells in the logarithmic growth phase and use 1×10 6 Cells were seeded into 6-well cell culture plates, 2 mL of cell suspension was added to each well, and the complete culture medium was discarded after culturing for 24 h. The 6-well plates were rinsed with 1×PBS, and 6 experimental groups were set up, namely, blank group (DMEM containing 1% BSA), free fatty acid group (0.5 mM FFA), low-dose pea oligopeptide group (0.5 mM FFA + 200 μg / mL pea oligopeptide), medium-dose pea oligopeptide group (0.5 mM FFA + 400 μg / mL pea oligopeptide), high-dose pea oligopeptide group (0.5 mM FFA + 800 μg / mL pea oligopeptide) and atorvastatin group (0.5 mM FFA + 10 μM atorvastatin), and cultured for 24 h.

[0119] (2) After culturing for 24 h, the cell culture medium was slowly aspirated, the cells were washed twice with PBS, 200 μL of cell lysis buffer was added, and the cell lysate was collected after lysis.

[0120] (3) The protein concentration of each experimental group was detected using a BCA kit (Biyuntian product number P0012). First, an appropriate amount of working solution was prepared according to the volume ratio (reagent A:reagent B = 50:1) based on the number of samples. Then, 0.5 mg / mL protein standard was added to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL. If the amount was less than 20 μL, PBS was added to make it up to 20 μL, which corresponds to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. After diluting the samples of each experimental group 25 times, 20 μL was taken and added to a 96-well plate. Then, 200 μL of BCA working solution was added to each well and placed at 37°C for 25 minutes. After taking out, the absorbance value was measured at 562 nm using a microplate reader. A standard curve was drawn with the protein standard concentration as the horizontal axis and the absorbance value as the vertical axis. The protein concentration in each group of samples was calculated using the standard curve.

[0121] (4) TG determination

[0122] Triglyceride determination was performed according to the instructions for the triglyceride test kit. Blank wells (2.5 μL distilled water), standard wells (2.5 μL calibrator), and sample wells (2.5 μL sample) were set up. 250 μL of working solution was then added to each well. The plate was shaken to mix thoroughly, and the wells were incubated at 37°C for 10 minutes. After removal, the absorbance was measured at 500 nm using a microplate reader. Calculation was performed according to the formula:

[0123]

[0124] A 样本孔 Indicates the absorbance value of the sample at 500nm, A 标准孔 Indicates the absorbance value of the standard at 500nm, A 空白孔 Indicates the absorbance value of the solvent at 500nm, C 标准 =2.39mM, C pr is the protein concentration of the sample to be tested, gprot / L.

[0125] (5) TC determination

[0126] Total cholesterol was determined by following the instructions for the cholesterol test kit. A blank well (2.5 μL distilled water), a standard well (2.5 μL calibrator), and a sample well (2.5 μL sample) were set up. 250 μL of working solution was then added to each well. The plate was shaken to mix thoroughly, and the wells were incubated at 37°C for 10 minutes. After removal, the absorbance was measured at 500 nm using a microplate reader. Calculation was performed according to the formula:

[0127]

[0128] A 样本孔Indicates the absorbance value of the sample at 500nm, A 标准孔 Indicates the absorbance value of the standard at 500nm, A 空白孔 Indicates the absorbance value of the solvent at 500nm, C 标准 =6.56mM, C pr is the protein concentration of the sample to be tested, gprot / L.

[0129] 1.1.5Western blot

[0130] Western blot was used to detect the effect of pea oligopeptides on the expression of related proteins in NAFLD cell model.

[0131] (1) Cell plating, grouping, and drug administration

[0132] Cells in the logarithmic growth phase were taken and 1×10 6 / well, inoculated into 6-well cell culture plates, added 2 mL of cell suspension to each well, discarded the complete culture medium after culturing for 24 h, rinsed the 6-well plates with 1×PBS, and set up 4 experimental groups, namely blank group (DMEM containing 1% BSA), free fatty acid group (0.5 mM FFA), pea oligopeptide group (0.5 mM FFA + 800 μg / mL pea oligopeptide) and atorvastatin group (0.5 mM FFA + 10 μM atorvastatin), and cultured for 24 h.

[0133] (2) Protein extraction

[0134] After 24 hours of culture, slowly aspirate the cell culture medium, wash twice with PBS, add 200 μL of cell lysis buffer and mix thoroughly by pipetting. PMSF, protease inhibitors, and phosphatase inhibitors need to be added to the lysis buffer. After sufficient lysis, collect the cell lysate, centrifuge at 10,000-14,000 g for 3-5 minutes, and take the supernatant.

[0135] (3) Protein quantification

[0136] Protein concentration in each experimental group was determined using a BCA assay kit (Biyuntian Biotechnology, product number P0012). First, an appropriate amount of working solution was prepared based on the number of samples using a volume ratio (reagent A:reagent B = 50:1). Then, 0.5 mg / mL protein standard was added to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL. Any remaining amount less than 20 μL was made up to 20 μL with PBS, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Samples from each experimental group were diluted 25-fold, and 20 μL was added to a 96-well plate. Then, 200 μL of BCA working solution was added to each well of the plate. The plates were incubated at 37°C for 25 min. After removal, the absorbance at 562 nm was measured using a microplate reader. A standard curve was constructed with the protein standard concentration as the abscissa and the absorbance as the ordinate. Protein concentration in each sample group was calculated using the standard curve. Dilute the sample with lysis buffer to a protein concentration of 5 μg / μL per sample. Then, dilute the 5× loading buffer to 1× with the protein solution. Add 4 volumes of sample to 1 volume of 5× loading buffer, resulting in a final protein concentration of 4 μg / μL per sample. Boil the sample in a boiling water bath for 5 minutes to fully denature the protein. Aliquot the prepared sample and store at -80°C.

[0137] (4) Electrophoresis

[0138] Use precast gel with a separation gel concentration of 10% for electrophoresis. Fix the precast gel in the electrophoresis tank, add an appropriate amount of electrophoresis fluid, evenly pull out the electrophoresis comb, add each group of samples and markers to the electrophoresis wells in turn, connect the positive and negative electrodes, turn on the power supply, set it to constant voltage mode, run at 80V for 30 minutes, then run at 120V for 60 minutes, and wait until the sample electrophoresis reaches the bottom of the electrophoresis tank.

[0139] (5) Wet transfer

[0140] Remove the glass, remove the precast gel, determine the position of the target protein according to the distribution of the marker bands, cut off the excess part, measure the length and width of the target protein gel and record them, cut the transfer filter paper and PVDF membrane according to the size of the gel, and mark them. The size of the PVDF membrane should be slightly larger than the gel. Then place the PVDF membrane in methanol to activate for 5 minutes. Place them in the order of filter paper-gel-PVDF membrane-filter paper, while removing bubbles while placing them. After placing them, fix them and place them in the wet transfer tank. Add transfer solution, cover the electrotransfer cover, pay attention to the positive and negative poles, turn on the power, set it to constant current mode for transfer, and perform the entire membrane wet transfer at 4°C.

[0141] (6) Blocking and incubation with primary antibody

[0142] After transfer, place the PVDF membrane in TBST containing 5% skim milk powder and shake slowly to block for 2 hours. Then use TBST to shake and wash the membrane 3 times at room temperature, 9 minutes each time. Dilute the primary antibody with primary antibody diluent 1:1000 according to the primary antibody configuration ratio. Place the blocked PVDF membrane in the corresponding primary antibody and incubate on a shaker at 4°C overnight.

[0143] (7) Incubation with secondary antibody and ECL color development

[0144] After the primary antibody incubation is completed, the PVDF membrane is placed in TBST and shaken at room temperature for 3 times, each time for 9 minutes. Then, the secondary antibody is diluted with secondary antibody diluent at a ratio of 1:10000 according to the secondary antibody configuration ratio, and the PVDF membrane is placed in the corresponding secondary antibody and incubated on a shaker at room temperature for 2 hours. After the secondary antibody incubation is completed, the PVDF membrane is placed in TBST and shaken at room temperature for 3 times, each time for 9 minutes. During the washing period, ECL developer solution A and solution B are mixed in the dark at a volume of 1:1 to the required volume. After the washing is completed, each band is gently clamped and placed in a gel imaging instrument. ECL developer solution is added dropwise and developed using a chemiluminescence imaging system.

[0145] Antibodies were purchased from Beyotime Biotechnology Co., Ltd.

[0146] (8) Data analysis and processing

[0147] ImageJ software was used to quantify the development results, where the grayscale ratio of phosphorylated proteins to non-phosphorylated proteins was used to represent the relative expression of proteins, and the grayscale ratio of other target proteins to GAPDH was used to represent the relative expression of proteins.

[0148] 2. Experimental results

[0149] 2.1 HMG-CoA reductase inhibitory activity of pea oligopeptides

[0150] Altered cholesterol homeostasis and free cholesterol accumulation in the liver are associated with the pathogenesis of NAFLD. HMG-CoA reductase is the rate-limiting enzyme in the cholesterol biosynthesis pathway, catalyzing the conversion of HMG-CoA to mevalonate. Its regulation is crucial for maintaining cholesterol homeostasis in the liver and circulation. Therefore, regulating HMG-CoA reductase activity is one of the main approaches to preventing and alleviating non-alcoholic fatty liver disease.

[0151] This example measured the inhibitory activity of pea oligopeptides on HMG-CoA reductase, and the results were as follows: Figure 2As shown, pea oligopeptides showed certain inhibitory activity on HMG-CoA reductase. The inhibition rates of 50 mg / mL and 100 mg / mL pea oligopeptides on HMG-COA reductase were 24.45% and 56.62%, respectively. This shows that pea oligopeptides contain components that inhibit HMG-COA reductase activity and have the potential to regulate HMG-CoA reductase. Peptides with HMG-COA reductase inhibitory activity can be screened from pea oligopeptides.

[0152] 2.2 Effects of pea oligopeptides on HepG2 cell viability

[0153] Human liver cancer cell line HepG2 has the physiological characteristics of human liver tissue and is easy to culture. It has been widely used to study liver steatosis, excessive lipid deposition and to construct NAFLD cell models. In this example, HepG2 cells were used to construct a NAFLD cell model. The CCK8 method was used to evaluate the effect of pea oligopeptides on the viability of HepG2 cells. The results are shown in Figure 2. Figure 3 As shown in Figure A, after treating HepG2 cells with pea oligopeptides at different concentrations (50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 4000, 6000 and 8000 μg / mL) for 24 hours, the cell viability showed an obvious concentration-dependent downward trend. When the concentration of pea oligopeptides was 1000-8000 μg / mL, the cell viability decreased significantly, affecting the normal survival of the cells. Therefore, this example selected three pea oligopeptide concentrations (200, 400 and 800 μg / mL) below 1000 μg / mL for subsequent experiments, and set them as pea oligopeptide low-dose group, medium-dose group and high-dose group according to the concentration gradient. In addition, atorvatin was used as a positive control, and its effect on cell viability was also evaluated by CCK8 method. The results are shown in Figure 5. Figure 3 In Figure B, it was found that after HepG2 cells were treated with different concentrations (1, 5, 10, 20, 30 and 40 μM) of atorvastatin for 24 hours, the cell viability showed an obvious concentration-dependent downward trend, among which atorvastatin at a concentration of 20 μM significantly affected the cell viability, and atorvastatin at 10 μM had no effect on the viability of HepG2 cells. Therefore, 10 μM atorvastatin was selected as the concentration of subsequent positive controls.

[0154] 2.3 Effects of pea oligopeptides on lipid accumulation in NAFLD cell models

[0155] 2.3.1 Free fatty acid-induced lipid deposition in HepG2 cells

[0156] Related studies have shown that FFA can be used to induce lipid deposition in HepG2 cells. Within a certain concentration range, FFA induces lipid accumulation and lipotoxicity in HepG2 cells in a dose-dependent manner. FFA has been widely used to induce HepG2 cells to establish NAFLD cell models. In this example, HepG2 cells were mixed with different concentrations of free fatty acids (0, 0.25, 0.50, 0.75, 1, 1.50, and 2 mM) and cultured for 24 hours to observe the effect of free fatty acids on the in vitro viability of hepatocytes. The CCK8 method was used to detect the cytotoxic effect of FFA on HepG2 cells. The results are as follows: Figure 4 As shown, compared with the control group, with the increase of FFA concentration, cell viability showed an obvious concentration-dependent downward trend, especially when the FFA concentration was greater than 1 mM, the cell viability decreased significantly. 0.5 mM FFA did not affect the viability of HepG2 cells. Therefore, in this example, 0.5 mM FFA was selected to induce HepG2 cells to establish a NAFLD cell model.

[0157] 2.3.2 Pea oligopeptides improve lipid deposition in HepG2 cells

[0158] In order to more intuitively evaluate the effect of pea oligopeptides on FFA-induced NAFLD cell model, this example performed Oil Red O staining on normal HepG2 cells, FFA-induced NAFLD cell model, HepG2 cells co-treated with pea oligopeptides (POP) and FFA, and HepG2 cells co-treated with atorvastatin and FFA (positive control). The results are shown in Figure 2. Figure 5 As shown in the figure, compared with normal HepG2 cells, after 24 hours of FFA treatment, the cell morphology became round and a large number of red lipid droplets accumulated in the cytoplasm. However, under the intervention of pea oligopeptide, the red lipid droplets stained with Oil Red O were significantly less than those in the FFA-induced NAFLD cell model group, and a similar phenomenon was also observed in the positive control. The results of Oil Red O staining were quantitatively analyzed at a wavelength of 500nm (as shown in Figure 2). Figure 6 ), the test results were consistent with the photographic results. Compared with the FFA-induced NAFLD cell model, co-treatment of pea oligopeptides with FFA significantly reduced lipid accumulation in HepG2 cells. Here, this example confirms that pea oligopeptides can improve intracellular lipid accumulation. Next, this example further measured the levels of intracellular factors related to lipid metabolism.

[0159] In order to further determine the effect of pea oligopeptides on lipid accumulation in NAFLD cell models, this example measured the total triglyceride (TG) and total cholesterol (TC) contents in normal HepG2 cells, FFA-induced NAFLD cell models, HepG2 cells co-treated with different concentrations of pea oligopeptides and FFA, and HepG2 cells co-treated with atorvastatin and FFA to confirm the results of Oil Red O staining. Figure 7 and Figure 8 As shown in Figure 2, compared with normal HepG2 cells, FFA treatment significantly increased the content of TG and TC in cells ( Figure 7 and Figure 8 ), however, treatment with 200-800 μg / mL pea oligopeptides alleviated this phenomenon. Compared with FFA treatment alone, co-treatment of pea oligopeptides and FFA significantly reduced the content of intracellular TG and TC, but there was no obvious concentration dependence. The positive control (atorvastatin and FFA co-treatment) showed a more significant alleviation phenomenon, which all reflected the results of Oil Red O staining. Changes in liver lipid metabolism in NAFLD promote the development of dyslipidemia, especially the increase in TG and TC levels. Related studies have also observed that FFA induces an increase in the content of TG and TC in HepG2 cells, which is consistent with the results in the example. In summary, the results show that pea oligopeptides can improve and intervene in lipid accumulation in NAFLD cell models.

[0160] 2.4 Effects of pea oligopeptides on the expression levels of lipid metabolism-related signaling proteins in NAFLD cell models

[0161] In order to study the mechanism of pea oligopeptides in alleviating and intervening lipid accumulation in FFA-induced NAFLD cell models, the expression of proteins related to fatty acid metabolism and cholesterol synthesis was detected. In this example, HepG2 cells were treated with 0.5mM FFA and 800μg / mL pea oligopeptides for 24h, and atorvastatin was used as a positive control. Proteins of each group of cells were extracted and analyzed by Western Blot. The results are shown in Figure 2. Figure 9 and Figure 10As shown. AMPK is an important energy detector for intracellular energy metabolism. It regulates the production of cellular triglycerides and cholesterol. The phosphorylation of AMPK weakens the de novo fat formation and liver lipid accumulation mediated by free fatty acids. The phosphorylation of AMPK can effectively increase the phosphorylation level of ACC and promote the inactivation of ACC in cells. The activation of the AMPK / ACC signaling pathway can inhibit the production of TG, because ACC is the rate-limiting enzyme for the synthesis of malonyl-CoA, and malonyl-CoA is a key substrate for fatty acid biosynthesis and an effective inhibitor of fatty acid oxidation. In addition, under the condition of liver lipid overload, the downregulation of CPT1A expression leads to excessive fatty acid β-oxidation, which further leads to liver steatosis and lipid peroxidation. The results of the present example show that the intervention of pea oligopeptides significantly upregulated the expression of p-AMPK, p-ACC and CPT1A ( Figure 9 ), therefore, it can be inferred that pea oligopeptides can inhibit ACC activity by activating AMPK phosphorylation, and promote the expression of CPT1A, thereby enhancing the β-oxidation of fatty acids, weakening the process of fatty acid synthesis, and reducing neoadipogenesis and liver lipid accumulation. SREBP2 is a transcription factor in the liver that regulates genes involved in cholesterol biosynthesis and homeostasis, such as HMG-CoA reductase. SREB P2 is a key transcriptional regulatory factor that regulates cholesterol biosynthesis, which activates the transcription of mevalonate pathway genes and HMGCR. Increased cholesterol synthesis leads to the accumulation of free cholesterol, including increased expression of SREBP2 and HMGCR, which are characteristics of NAFLD. At the same time, this example found that compared with the blank control group, the expression levels of SREBP2 and HMGCR in the NAFLD model group were significantly increased ( Figure 10 ), which indicates that the NAFLD cell model was successfully constructed. Compared with the FFA-induced NAFLD cell model group, the expression levels of SREBP2 and HMGCR in the pea oligopeptide-treated group were significantly downregulated ( Figure 10 ), therefore, pea oligopeptides reduce cholesterol synthesis by intervening in the expression of key factors in the cholesterol synthesis pathway. In addition, SREBP2 is regulated by the upstream AMPK. AMPK phosphorylation inhibits SREBP2 expression, thereby downregulating HMGCR expression, weakening the cholesterol synthesis pathway, and reducing cholesterol accumulation.

[0162] In summary, pea oligopeptides regulate lipid accumulation in NAFLD cell models by activating the AMPK / ACC signaling pathway. Specifically, activating AMPK phosphorylation inhibits ACC activity and SREBP2 expression, further promoting CPT1A expression and downregulating HMGCR expression, thereby enhancing fatty acid β-oxidation, weakening the fatty acid and cholesterol synthesis pathway, and ultimately reducing intracellular lipid accumulation.

[0163] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing pea oligopeptide, characterized in that: The steps include: mixing pea protein with water and emulsifying the mixture to obtain a pea protein emulsion; mixing the pea protein emulsion with a protease and performing enzymatic hydrolysis to obtain an enzymatic hydrolysis mixture; The enzymatic hydrolysis mixture is subjected to enzyme inactivation treatment to obtain an enzyme inactivation-treated enzymatic hydrolysis product; The enzymatic hydrolysis product after the enzyme inactivation treatment is subjected to solid-liquid separation, and the obtained supernatant is subjected to ultrafiltration, so that the obtained permeate contains the pea oligopeptide; The protease includes alkaline protease and neutral protease; based on the volume of the pea protein emulsion, the added amount of the alkaline protease is 8-10 mg / mL; the added amount of the neutral protease is 7-9 mg / mL.

2. The preparation method according to claim 1, characterized in that The enzymatic hydrolysis temperature is 40-55° C., the enzymatic hydrolysis time is 3.5-4.5 hours, and the enzymatic hydrolysis system pH value is 7-8.

5.

3. The preparation method according to claim 1 or 2, characterized in that The volume ratio of the pea protein mass to water is (1.8-3.3) g: (10-15) mL.

4. The preparation method according to claim 1, characterized in that The ultrafiltration step comprises: passing the supernatant through ultrafiltration membranes with molecular weights of 10 kDa and 1 kDa in sequence to obtain the permeate.

5. The preparation method according to claim 1, characterized in that The preparation method further comprises post-processing the permeate; the post-processing step comprises: The permeate is subjected to nanofiltration to obtain a purified solution; The purified solution is concentrated at low temperature, and when the obtained concentrate has a Brix value of 28 to 32, it is decolorized to obtain decolorized pea peptide; The decolorized pea peptide is dried to obtain the pea oligopeptide.

6. The preparation method according to claim 5, characterized in that The decolorization method is activated carbon treatment, and the time is 25 to 35 minutes; The drying method includes spray drying; The spray drying pressure is 15-25 MPa, the evaporation rate is 180-220 kg / h, and the time is 5-10 minutes.

7. A pea oligopeptide, characterized in that: The preparation method according to any one of claims 1 to 6 is used for preparation.

8. Use of the pea oligopeptide according to claim 7 in the preparation of functional foods or health products for preventing and / or improving non-alcoholic fatty liver disease, or in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

9. Use of the pea oligopeptide according to claim 7 in the preparation of functional foods or health products having one or more of the functions of lowering lipids, inhibiting HMG-COA reductase activity, improving lipid accumulation in liver cells, and regulating the expression of lipid metabolism-related signal proteins.

10. A functional food or health product for preventing and / or improving non-alcoholic fatty liver disease, characterized in that: The effective ingredient of the functional food or health product includes the pea oligopeptide according to claim 7.

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