Yellow wine vinasse active component capable of reducing blood fat as well as extraction method and application of yellow wine vinasse active component

Abstract

The invention belongs to the technical field of vinasse functional development, and particularly relates to a yellow wine vinasse active component capable of reducing blood fat and an extraction method and application thereof. Polypeptides and / or proteins in the active ingredients of the yellow wine vinasse can play a role in reducing blood fat through four biological pathways, functional application capable of reducing blood fat and preventing or treating hyperlipemia is developed by utilizing the yellow wine vinasse, the economic value of the yellow wine vinasse is improved, and the application prospect is wide. The yellow wine vinasse is subjected to high-value functional development, and the defects that the existing yellow wine vinasse is mainly used for preparing feed and the functional development degree is low are overcome; the technical problem that in the prior art, the rice wine vinasse functional development degree is low is solved.

Description

Technical Field

[0001] The present application belongs to the technical field of functional development of lees, and in particular relates to an active ingredient of yellow wine lees capable of lowering blood lipids, an extraction method and an application thereof. Background Art

[0002] Hyperlipidemia is a systemic metabolic chronic disease caused by abnormal fat transport or metabolism. The levels of total cholesterol, triglycerides and other blood lipid components in the patient's plasma are too high, and the blood lipid concentration exceeds the normal range. Hyperlipidemia will directly damage the health of blood vessels. When the blood lipid concentration reaches a certain high level, it may induce atherosclerosis and coronary heart disease. At the same time, hyperlipidemia will significantly increase the burden on the heart. If the burden on the heart is too heavy for a long time, it is easy to induce coronary heart disease. Therefore, hyperlipidemia may lead to the occurrence of many diseases such as atherosclerosis, coronary heart disease, obesity, and hypertension.

[0003] At present, chemical synthetic drugs such as statins, fibric acids, and niacin are used in medicine to lower lipids to treat hyperlipidemia. However, these chemical synthetic drugs may cause a series of side effects such as skin allergies, liver toxicity, and impaired renal function after use. In addition to obtaining bioactive ingredients through chemical synthesis, bioactive ingredients can also be obtained through microbial fermentation, natural extraction, etc. Huangjiu lees are the solids remaining after Huangjiu is fermented and matured, and the wine is pressed to separate the wine from the solids. It is the main by-product of Huangjiu production enterprises and has a huge output. As a winemaking waste, Huangjiu lees are rich in nutrients and active ingredients such as starch, protein, fat and cellulose. The rice protein content in Huangjiu lees is as high as 29.58%~41.29% (dry basis), which has the characteristics of high biological value and low allergenicity. It has extremely high nutritional value and development value and is an important protein resource. However, Huangjiu lees are currently mainly used to prepare feed, which has poor economic value and low degree of functional development of Huangjiu lees. Summary of the invention

[0004] In view of this, the present application provides an active ingredient of rice wine lees that can lower blood lipids, as well as an extraction method and application, to solve the technical problem of the low level of functional development of rice wine lees in the prior art.

[0005] In a first aspect, the present application provides a yellow wine lees active ingredient capable of lowering blood lipids, wherein the yellow wine lees active ingredient is selected from polypeptides and / or proteins;

[0006] The rice wine lees active ingredients are selected from rice wine lees active ingredients that inhibit pancreatic lipase and / or cholesterol esterase activity, rice wine lees active ingredients that inhibit cholesterol micelles, or rice wine lees active ingredients that enhance bile salt activity.

[0007] Preferably, the inhibiting of pancreatic lipase and / or cholesterol esterase activity is specifically inhibiting the pancreatic lipase and / or cholesterol esterase activity by binding to at least one of pancreatic lipase and cholesterol esterase via a reversible chemical bond.

[0008] Preferably, the rice wine lees active ingredients are selected from polypeptides with a molecular weight not greater than 3 kDa.

[0009] Preferably, among the polypeptides not greater than 3 kDa, polypeptides having an amino acid sequence of YQVQY, YIIPY, TPLQY, VRSPF, IVQGR, SLHLE, KPHER, WFYND, RIHEY, FFHCY, KYLAL, YYPGL, IHQFR, WNELW, IHRFR, LTPHW, IHFQF, FRHW, KEAFR, WFYN, YKHGL, WSLDY, IYVYD, SFFY, KQFR, HWCY, WQSSR, LLQPY, YYPGF, FNLPR, YVFR, WLSY, KWLQ, TLSPY, FRGCR, IHHTY, IFHR, WLYN, FTFPR, KPTSW, LGYPR, LSPFW, RPVR or YPLPR are screened out, and these polypeptides can inhibit at least one of pancreatic lipase or cholesterol esterase.

[0010] The second aspect of the present application provides a method for extracting the active ingredients of rice wine lees capable of lowering blood lipids as described in the first aspect, the extraction method comprising: adding rice wine lees into boric acid-potassium chloride buffer for extraction, filtering and centrifuging to obtain the active ingredients of rice wine lees capable of lowering blood lipids.

[0011] Preferably, the extraction process is: at pH 9.0, constant temperature extraction at 57°C for 2 hours, and ultrasonic assisted extraction at 250w for 30 minutes;

[0012] The filtering and centrifuging process includes: filtering the rice wine lees extraction residue, adjusting the pH of the filtrate to 4.0, and centrifuging at 8000 r / min for 15 minutes.

[0013] The third aspect of the present application provides the use of the active ingredient of rice wine lees capable of lowering blood lipids described in the first aspect in the preparation of a composition for preventing or treating hyperlipidemia.

[0014] Preferably, the application is specifically: use of a polypeptide with an amino acid sequence of YQVQY, YIIPY, TPLQY, VRSPF, WFYND, RIHEY, FFHCY, IHQFR or WNELW in the preparation of a composition for inhibiting the activity of pancreatic lipase and cholesterol esterase to prevent or treat hyperlipidemia.

[0015] Preferably, the application is specifically: use of a polypeptide with an amino acid sequence of IVQGR, SLHLE, KPHER, KYLAL, YYPGL, IHRFR, LTPHW, IHFQF, FRHW, KEAFR or WFYN in the preparation of a composition for inhibiting pancreatic lipase activity to prevent or treat hyperlipidemia.

[0016] Preferably, the application specifically includes: use of a polypeptide having an amino acid sequence of YKHGL, WSLDY, IYVYD, SFFY, KQFR, HWCY, WQSSR, LLQPY, YYPGF, FNLPR, YVFR, WLSY, KWLQ, TLSPY, FRGCR, IHHTY, IFHR, WLYN, FTFPR, KPTSW, LGYPR, LSPFW, RPVR or YPLPR in the preparation of a composition for inhibiting cholesterol esterase activity to prevent or treat hyperlipidemia.

[0017] The fourth aspect of the present application provides a composition for preventing or treating hyperlipidemia, comprising the yellow wine lees active ingredient capable of lowering blood lipids as described in the first aspect and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The electrophoresis spectrum of the active components of rice wine lees; Figure 2 This is a graph showing the inhibition results of active ingredients of rice wine lees and pancreatic lipase and cholesterol esterase; Figure 3 This is the result diagram of the interaction between the active ingredients of rice wine lees and bile salt; Figure 4 This is the result diagram of the interaction between the active ingredients of rice wine lees and cholesterol micelles; Figure 5 This is the result diagram of the inhibition type of the active ingredients of rice wine lees and pancreatic lipase and cholesterol esterase; Figure 6 This is a diagram showing the results of a cell proliferation toxicity experiment on the active ingredients of rice wine lees; Figure 7 This is the result of the Cell Oil Red O experiment of the active ingredient of rice wine lees; Figure 8 This is a graph showing the results of the biochemical index experiment of the active components of rice wine lees; Fig. 9 The results of inhibition of active peptides with different molecular weights and pancreatic lipase and cholesterol esterase are shown in the figure; Fig.10 This is a diagram showing the results of isolating and purifying the active polypeptide MU1 (<3 kDa). DETAILED DESCRIPTION

[0020] The present application provides an active ingredient of rice wine lees capable of lowering blood lipids, as well as an extraction method and application thereof, which are used to solve the technical problem of low degree of functional development of rice wine lees in the prior art.

[0021] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0022] In view of the fact that rice wine lees are currently mainly used to prepare feed, the economic value of feed is poor, and the functional development level of rice wine lees is low, the present application provides a rice wine lees active ingredient capable of lowering blood lipids, as well as an extraction method and application; the rice wine lees active ingredient provided in the present application is selected from polypeptides and / or proteins in the rice wine lees active ingredient, and these polypeptides and / or proteins can inhibit the activity of pancreatic lipase and / or cholesterol esterase, inhibit cholesterol micelles or enhance the activity of bile salts.

[0023] Yellow wine lees include multiple components such as water, ash, protein, fat, crude fiber, starch, etc. Past studies have shown that proteins (peptides) have multiple biological activities such as information transmission and biological metabolism regulation. Therefore, proteins (peptides) in yellow wine lees may have functional development potential and are expected to be used as active ingredients for lowering blood lipids. Therefore, the present application obtains proteins in the active ingredients of yellow wine lees through ultrasound-assisted alkaline extraction. At the same time, part of the protein will be degraded to generate polypeptides during the ultrasound-assisted alkaline extraction process, so that the obtained yellow wine lees active ingredients include proteins and / or polypeptides. Regarding the specific role of proteins and / or polypeptides in the active ingredients of yellow wine lees, pancreatic lipase is an important enzyme for hydrolyzing dietary fat. Inhibiting the activity of pancreatic lipase can effectively reduce the absorption efficiency of fat in the small intestine, and cholesterol esterase can convert cholesterol esters into free cholesterol. Inhibiting the activity of cholesterol esterase can slow down the process of converting cholesterol esters into free cholesterol, reduce the rate of absorption by the human body, and help reduce the cholesterol content in plasma. The polypeptides and / or proteins in the active ingredients of yellow wine lees provided in the present application can be combined with pancreatic lipase and / or cholesterol esterase Inhibit its activity and play a lipid-lowering effect; in addition to the two biological pathways of combining with pancreatic lipase and / or cholesterol esterase to inhibit their activity, bile salt is a key medium for cholesterol absorption in the intestine, and the strength of its binding ability directly affects the absorption efficiency of cholesterol. The polypeptides and / or proteins in the active ingredients of rice wine lees provided in the present application enhance the activity of bile salt through the third biological pathway, thereby enhancing its binding ability with cholesterol and effectively reducing the cholesterol level in the blood; and the polypeptides and / or proteins in the active ingredients of rice wine lees provided in the present application can also effectively reduce the absorption efficiency of cholesterol in the intestine through the fourth biological pathway by inhibiting the assembly process of cholesterol micelles or reducing their solubility; therefore, the polypeptides and / or proteins in the active ingredients of rice wine lees provided in the present application can play a lipid-lowering effect through four biological pathways, and the functional use of rice wine lees that can lower blood lipids and prevent or treat hyperlipidemia is developed using rice wine lees, thereby improving the economic value of rice wine lees, and carrying out high-value functional development of rice wine lees, overcoming the defect that the current rice wine lees are mainly used for preparing feed and have a low degree of functional development.

[0024] Preferably, for the combination type of polypeptides and / or proteins in the rice wine lees active ingredients provided in the present application and pancreatic lipase and / or cholesterol esterase, the polypeptides and / or proteins and pancreatic lipase and / or cholesterol esterase are combined by reversible chemical bonds such as hydrogen bonds and electrostatic interactions, thereby not causing permanent inactivation of pancreatic lipase and / or cholesterol esterase, and the inhibition of pancreatic lipase and / or cholesterol esterase activity is reversible.

[0025] Preferably, the polypeptides and / or proteins in the rice wine lees active ingredients provided in the present application are all active ingredients formed by amino acids connected by peptide bonds, but the lipid-lowering effects of the polypeptides and / or proteins may vary; polypeptides or proteins with higher molecular weight will be spatially hindered from binding with pancreatic lipase and cholesterol esterase at the active sites, and polypeptides with smaller molecular weight will have better lipid-lowering effects; therefore, the rice wine lees active ingredients provided in the present application are preferably polypeptides with a molecular weight not greater than 3 kDa.

[0026] Preferably, the polypeptides in the rice wine lees active ingredients provided in the present application have different binding abilities to pancreatic lipase and cholesterol esterase; the preferred amino acid sequences of the present application are YQVQY, YIIPY, TPLQY, VRSPF, IVQGR, SLHLE, KPHER, WFYND, RIHEY, FFHCY, KYLAL, YYPGL, IHQFR, WNELW, IHRFR, LTPHW, IHFQF, FRHW, KEAFR, WFYN, YKHGL, WSLDY, Polypeptides such as IYVYD, SFFY, KQFR, HWCY, WQSSR, LLQPY, YYPGF, FNLPR, YVFR, WLSY, KWLQ, TLSPY, FRGCR, IHHTY, IFHR, WLYN, FTFPR, KPTSW, LGYPR, LSPFW, RPVR or YPLPR have similar or stronger binding abilities to pancreatic lipase or cholesterol esterase than the commercially available drug orlistat, and are the most biologically active polypeptide components among the active ingredients of rice wine lees.

[0027] At the same time, the present application provides a corresponding extraction method for polypeptides and / or proteins in the active ingredients of rice wine lees. The extraction adopts ultrasound-assisted alkaline extraction method. The dried rice wine lees powder is added to boric acid-potassium chloride buffer (pH 9.0), and then ultrasonic-assisted extraction is performed for a period of time at a constant temperature. The filtrate is then filtered to obtain the filtrate, and the filtrate is then adjusted to acidic and centrifuged to obtain the active ingredients of rice wine lees. The active ingredients of rice wine lees were analyzed by SDS-PAGE gel electrophoresis, and it was found that the molecular weight of the protein was mainly below 25 kDa, and the electrophoresis spectrum showed that there were obvious bands below 15 kDa, and the molecular weight was relatively small, indicating that during the ultrasound-assisted alkaline extraction process, part of the protein was hydrolyzed into smaller polypeptides. The active ingredients obtained from rice wine lees using ultrasound-assisted alkaline extraction method are proteins and polypeptides.

[0028] Preferably, the present application also provides the use of polypeptides and / or proteins in the active ingredients of rice wine lees for preparing a composition for preventing or treating hyperlipidemia; the composition described in the present application includes food, health products and medicines.

[0029] Preferably, for the target of preparing a composition for preventing or treating hyperlipidemia, when polypeptides with amino acid sequences such as YQVQY, YIIPY, TPLQY, VRSPF, WFYND, RIHEY, FFHCY, IHQFR, WNELW are selected, these polypeptides have a strong binding ability with pancreatic lipase and cholesterol esterase, and can use pancreatic lipase and cholesterol esterase as targets to inhibit the activity of pancreatic lipase and cholesterol esterase.

[0030] Preferably, for the target of preparing a composition for preventing or treating hyperlipidemia, when the amino acid sequences are selected as polypeptides such as IVQGR, SLHLE, KPHER, KYLAL, YYPGL, IHRFR, LTPHW, IHFQF, FRHW, KEAFR, WFYN, etc., these polypeptides have a strong binding ability with pancreatic lipase and can use pancreatic lipase as a target to inhibit the activity of pancreatic lipase.

[0031] Preferably, for the target of preparing a composition for preventing or treating hyperlipidemia, when the amino acid sequence is selected as a polypeptide such as YKHGL, WSLDY, IYVYD, SFFY, KQFR, HWCY, WQSSR, LLQPY, YYPGF, FNLPR, YVFR, WLSY, KWLQ, TLSPY, FRGCR, IHHTY, IFHR, WLYN, FTFPR, KPTSW, LGYPR, LSPFW, RPVR, YPLPR, etc., these polypeptides have a strong binding ability with cholesterol esterase and can use cholesterol esterase as a target to inhibit the activity of cholesterol esterase.

[0032] At the same time, the present application also provides a composition for preventing or treating hyperlipidemia, which may be a medicine, a health product or a food; the composition includes the protein and / or polypeptide in the above-mentioned rice wine lees active ingredient and a pharmaceutically acceptable carrier or excipient; for the dosage form of the composition for preventing or treating hyperlipidemia, granular powder, tablets, capsules, liquid preparations and other dosage forms can be selected; the administration method may be oral, subcutaneous injection, intravenous injection, intramuscular injection and other administration methods; the carrier or excipient may be an excipient such as starch, an adhesive such as polyvinyl pyrrolidone, a lubricant such as magnesium stearate, a diluent such as physiological saline.

[0033] The active ingredients of rice wine lees capable of lowering blood lipids provided by the present application will be specifically described below with reference to the examples.

[0034] Example 1

[0035] Example 1 of the present application provides a method for extracting active ingredients from rice wine lees, which includes the steps of analyzing rice wine lees samples, extracting active ingredients from rice wine lees using an ultrasound-assisted alkaline extraction method, and filtering active ingredients from rice wine lees.

[0036] Among them, the steps of analyzing the rice wine lees sample include: analyzing the moisture, ash, protein, fat, crude fiber and starch contents in the rice wine lees sample according to the test methods recommended in the national standards GB / T5009.3-2016, GB / T5009.4-2016, GB / T5009.5-2016, GB / T5009.6-2016, GB / T5009.10-2003 and GB / T5009.9-2016; the analysis results show that the rice wine lees sample includes 93.1g / 100g moisture, 0.22g / 100g ash, 3.52g / 100g protein, 0.3g / 100g fat, 0.4g / 100g crude fiber and 2.38g / 100g starch; in addition to water, the protein content in the rice wine lees is relatively high, and it is expected to be used as an active ingredient for lowering blood lipids for functional development.

[0037] The steps of extracting the active components of rice wine lees by ultrasound-assisted alkaline extraction method include: weighing 200g of dry rice wine lees powder, adding 2L of boric acid-potassium chloride buffer solution with pH 9.0, and extracting at a constant temperature of 57°C for 2 hours; then using 250w ultrasound-assisted extraction for 30 minutes, filtering the rice wine lees extraction residue and centrifuging the filtrate, adjusting the pH to 4.0, and centrifuging at 8000r / min for 15 minutes to obtain protein precipitate, which is the active components of rice wine lees; using SDS-PAGE gel to perform electrophoresis analysis on the active components of rice wine lees, and the analysis results are as follows: Figure 1 The electrophoresis pattern shown in the figure shows that the molecular weight of the protein in the active ingredients of rice wine lees is mainly concentrated below 25 kDa, and there are obvious bands below 15 kDa, indicating that this part of the protein is hydrolyzed into smaller polypeptides during the extraction process of the ultrasonic-assisted alkaline extraction method. Therefore, the active ingredients of the rice wine lees in this example include proteins and polypeptides.

[0038] In order to further explore the active ingredients of rice wine lees, the active ingredients of rice wine lees were screened in this example. The step of filtering the active ingredients of rice wine lees included: dissolving the active ingredients of rice wine lees in Tris-HCl buffer to an appropriate concentration, filtering with a 0.22 mm water filter membrane, and then using filter membranes with pore sizes of 3kDa, 10kDa and 30kDa to separate the sample into four fractions through a tubular membrane ultrafiltration device, which are four groups of active polypeptide components MU1 (<3kDa), MU2 (3kDa~10kDa), MU3 (10~30kDa), and MU4 (>30kDa).

[0039] Experimental Example 1

[0040] This experimental example analyzes the interactions between the active ingredients of rice wine lees and four groups of active polypeptides provided in Example 1 and pancreatic lipase and cholesterol esterase, respectively, to analyze the lipid-lowering activity of the active ingredients of rice wine lees.

[0041] The principle of the analytical experiment on the interaction between the active ingredients of rice wine lees, four groups of active peptides and pancreatic lipase and cholesterol esterase is as follows: pancreatic lipase is an enzyme responsible for hydrolyzing dietary fat, and 4-nitrophenyl laurate (PNPL) is its substrate. After hydrolysis, 4-nitrophenol is generated, which has a characteristic absorbance at a wavelength of 405nm. By measuring the change in absorbance before and after the reaction, the activity of pancreatic lipase can be indirectly reflected; and cholesterol esterase can hydrolyze 4-nitrophenyl butyrate (PNPB) to generate 4-nitrophenol with a characteristic absorbance, and its absorbance can be detected at 405nm. By measuring the change in absorbance before and after the reaction, the activity of pancreatic lipase can be indirectly reflected; if the active ingredients of rice wine lees combine with pancreatic lipase and cholesterol esterase to inhibit the activity of pancreatic lipase and cholesterol esterase, the hydrolysis of PNPL and PNPB will be reduced, thereby reducing the absorbance of the reaction solution.

[0042] The raw materials for the analytical experiment on the interaction between the active ingredients and active polypeptides of rice wine lees and pancreatic lipase and cholesterol esterase are: pancreatic lipase, 4-nitrophenyl laurate (PNPL), sodium acetate, Triton, orlistat, sodium taurocholate, 4-nitrophenyl butyrate (PNPB), and cholesterol esterase; the instruments are: multifunctional microplate reader, water bath, electronic balance, micropipette, and constant temperature incubator.

[0043] The steps of the analytical experiment on the interaction between the active ingredients of rice wine lees and pancreatic lipase and cholesterol esterase are as follows: pancreatic lipase or cholesterol esterase (1 mg / mL) was dissolved in 100 mM Tris-HCl buffer at pH = 8; 2.0 mM 4-nitrophenyl laurate (PNPL) or 4-nitrophenyl butyrate (PNPB) was prepared using 5 mmol / L sodium acetate solution (containing 1% Triton X-100), 50 μL of rice wine lees active ingredients of different concentrations were mixed with 50 μL of 2.0 mg / mg pancreatic lipase or cholesterol esterase solution, and 100 μL of 2.0 mg / mg pancreatic lipase or cholesterol esterase solution was added after incubation at 37°C for 10 min. PNPL or PNPB, after incubation at 37°C for 30min, the absorbance was measured at 405nm. Buffer and orlistat were used as blank control and positive control, respectively. Three lines were set for each experimental system. The inhibition rate of activity was calculated as follows: inhibition rate (%) = (1-(A3-A4) / (A1-A2)) × 100, where A1, A2, A3 and A4 represent the absorbance values ​​of pancreatic lipase or cholesterol esterase + substrate, buffer + substrate, pancreatic lipase or cholesterol esterase + sample + substrate, and sample + substrate in the reaction system, respectively. The steps of the analytical experiment on the interaction between the four groups of active peptides and pancreatic lipase and cholesterol esterase are the same as those for the active ingredients of rice wine lees, except that the active ingredients of rice wine lees are replaced by four groups of active peptides.

[0044] The results of the analysis of the interaction between the active components of rice wine lees and pancreatic lipase and cholesterol esterase are as follows Figure 2 As shown, from Figure 2 As shown in Figures A and B, the active ingredients of rice wine lees have a good inhibitory effect on pancreatic lipase, and the inhibition rate increases with the increase of concentration. When it reaches 2.0 mg / mL, the inhibition rate of rice wine lees protein is 64.32±1.06%; the IC 50 Equivalent to 0.39 mg / mL, approximately the IC of the drug orlistat for pancreatic lipase inhibition 50 (0.002mg / mL) 200 times; at the same time from Figure 2 As can be seen from Figures C and D, the inhibitory effect of the active ingredients of rice wine lees on cholesterol esterase is better than that on pancreatic lipase. At a protein concentration of 2.0 mg / mL, the inhibition rate of cholesterol esterase reached 95.53±0.39%, and the IC50 was 0.27 mg / mL.

[0045] The experimental results show that the active ingredients of rice wine lees provided in the present application can inhibit the activity of pancreatic lipase and cholesterol esterase. Pancreatic lipase is an important enzyme for hydrolyzing dietary fat. Inhibiting the activity of pancreatic lipase can effectively reduce the absorption efficiency of fat in the small intestine, thereby achieving the purpose of lowering blood lipids. At the same time, cholesterol esterase plays an important role in the conversion of cholesterol esters to free cholesterol. By inhibiting the activity of cholesterol esterase, the process of converting cholesterol esters to free cholesterol can be slowed down, thereby reducing its absorption rate by the human body, helping to reduce the cholesterol content in plasma and exerting a lipid-lowering effect. Therefore, the active ingredients of rice wine lees provided in the present application are active ingredients that can lower blood lipids.

[0046] The results of the analysis of the interaction between the four groups of active peptides and pancreatic lipase and cholesterol esterase are as follows Fig. 9 As shown, from Fig. 9 Figures A and B show that there are differences in the inhibitory activities of pancreatic lipase and cholesterol esterase between peptides of different molecular weights. When the active peptide components are less than 3 kDa at the same concentration, the inhibitory effects on pancreatic lipase and cholesterol esterase are the best. This is because the higher molecular weight peptides are spatially hindered from binding to the active sites of pancreatic lipase and cholesterol esterase, resulting in their inhibitory activity being short peptides with fewer amino acids and smaller molecular weight peptides, while low molecular weight peptides can act as hydrogen donors to exert biological activity.

[0047] Experimental Example 2

[0048] This experimental example analyzes the interaction between the active ingredients of rice wine lees provided in Example 1 and bile salts, so as to analyze the lipid-lowering activity of the active ingredients of rice wine lees.

[0049] The principle of the analytical experiment on the interaction between the active ingredients of rice wine lees and bile salts is that bile salts help the absorption of lipids in the digestive system. Certain proteins or other components can bind to them, reducing the effectiveness of bile salts, thereby affecting the digestion and absorption of fat. By measuring the binding of samples with sodium taurocholate, the bile salt binding ability of the sample in a simulated digestive environment can be understood.

[0050] The raw materials for the analytical experiment on the interaction between the active components of rice wine lees and bile salts are: pepsin, hydrochloric acid, trypsin, sodium taurocholate, sulfuric acid, sodium cholate hydrate; the instruments are: centrifuge, 37°C constant temperature oscillator, multifunctional microplate reader, pH meter, electronic balance, micropipette.

[0051] The steps of the analytical experiment on the interaction between the active ingredients of rice wine lees and bile salts are as follows: prepare sodium taurocholate standard solutions with concentrations of 0.05, 0.10, 0.15, 0.20, 0.25, and 0.30 mmol using Tris-Hcl buffer; and sodium cholate hydrate standard solutions with concentrations of 0.03, 0.06, 0.12, 0.18, 0.24, and 0.30 mmol, for later use. Take 2 mL of sodium taurocholate standard solutions and sodium cholate hydrate standard solutions of different concentrations in test tubes, add 5 mL of 60% sulfuric acid solution, shake in a water bath at 70°C for 30 min, take out and ice bath for 5 min, measure the absorbance at 387 nm, and obtain the sodium taurocholate content standard curve and sodium cholate hydrate content standard curve respectively;

[0052] Place 3 mL of sample solution of different concentrations in a centrifuge tube, add 3 mL of 10 mg / mL trypsin solution and 1 mL of 10 mM hydrochloric acid solution, and shake at 37°C for 1 hour; take out and adjust the pH value to 6.3, add 4 mL of 10 mg / mL trypsin solution, and shake at 37°C for 1 hour. Take out and add 4 mL of 0.5 mmol / L sodium taurocholate solution, place at 37°C for 1 hour, and centrifuge at 4000 r / min for 15 minutes. Take the supernatant and put it in another test tube, add 5 mL of 60% sulfuric acid solution, shake in a water bath at 70°C for 30 minutes, take out and put in an ice bath for 5 minutes, and measure the absorbance at 387 nm. The steps for determining the binding rate of hydrated sodium cholate were the same as above, with a concentration of 0.4 mM; no bile salt was added, and the determination steps were the same as above as the background group; the binding rate was calculated according to the formula: binding rate = (1-A1 / A2) × 100%, where A1 is the remaining amount of sodium cholate and A2 is the amount of sodium cholate added.

[0053] The results of the analysis of the interaction between the active ingredients of rice wine lees and bile salts are as follows Figure 3 As shown, from Figure 3It can be seen that the binding ability of rice wine lees active ingredients to sodium cholate (SC) and sodium taurocholate (STC) at lower concentrations tends to increase with increasing concentrations. When the concentration of rice wine lees active ingredients is 0.0625 mg / mL, the binding energy to SC is 12.95±1.19%, and the binding energy to STC is 29.74±1.36%. When the concentration of rice wine lees protein doubles to 0.125 mg / mL, the binding energy to SC is 18.54±0.72%, and the binding energy to STC is 34.63±2.69%. Between these two concentrations, rice wine lees protein has a more obvious binding energy increase for the two bile salts. Then it can be observed that the increase in the concentration of rice wine lees active ingredients does not significantly promote the increase in bile salt binding energy, and there is only a weak positive correlation between the two. At the same time, we can use Figure 3 It is obvious that rice wine lees protein has a stronger binding energy to STC. This is because STC contains taurine groups, and its molecular structure may be easier to interact with specific components in the sample, making sodium taurocholate show higher affinity when binding to the sample. In addition, the solubility and bioavailability of STC may also be higher than those of sodium cholate, further enhancing its binding ability to the sample, resulting in stronger binding energy.

[0054] The experimental results show that the active ingredients of rice wine lees provided in the present application can enhance the binding of bile salts, and the importance of bile salts in the study of lowering blood lipids is reflected in their regulatory effects on cholesterol absorption and metabolism. Cholesterol is an important component of blood lipids in the human body, and its abnormal increase in levels is closely related to the risk of cardiovascular disease. Bile salts are the key medium for cholesterol absorption in the intestine, and the strength of their binding ability directly affects the absorption efficiency of cholesterol. Studies have shown that by enhancing the binding ability of bile salts, the cholesterol level in the blood can be effectively reduced, thereby playing a role in lowering blood lipids. Therefore, the active ingredients of rice wine lees provided in the present application are active ingredients that can lower blood lipids.

[0055] Experimental Example 3

[0056] This experimental example analyzes the interaction between the active ingredients of rice wine lees provided in Example 1 and cholesterol micelles, so as to analyze the hypolipidemic activity of the active ingredients of rice wine lees.

[0057] The principle of the analytical experiment on the interaction between rice wine lees active ingredients and cholesterol micelles is that cholesterol and fatty acids (such as oleic acid) can form micelles under the action of bile salts (such as sodium taurocholate); the rice wine lees active ingredients may affect the cholesterol content by interacting with cholesterol or bile salts. A uniform micellar solution is formed by ultrasonic homogenization, and different concentrations of rice wine lees active ingredients are added to it to evaluate their binding ability to cholesterol. After the reaction is completed, the unbound components are removed by centrifugation, and the cholesterol concentration in the supernatant is measured.

[0058] The raw materials for the analytical experiment on the interaction between the active components of rice wine lees and cholesterol micelles are: sodium taurocholate, cholesterol, oleic acid, NaCl, and a total cholesterol kit; the instruments are: an ultrasonic homogenizer, a constant temperature oscillator (37°C), a high-speed centrifuge, a multifunctional microplate reader, an electronic balance, and a micropipette gun.

[0059] The steps of the analytical experiment on the interaction between the active ingredients of rice wine lees and cholesterol micelles are as follows: 15mM pH7.4 PBS buffer (containing 10mM sodium taurocholate, 2mM cholesterol, 5mM oleic acid, 132mM NaCl) was homogenized by ultrasound, incubated at 37°C for 12h, 3mL was taken in a 15mL centrifuge tube, and samples with a concentration range of (0.1875, 0.375, 0.75, 1.5, 3, 6mg) were dissolved in 3mL micelle solution. No rice wine lees protein was added to the blank group, and the culture was carried out at 37°C with constant temperature shaking for 2h. The supernatant was collected and the cholesterol content of the supernatant was determined using a total cholesterol kit according to the formula: Cholesterol micelle dissolution inhibition rate (%) = (1-A1 / A2) × 100, A1 is the absorbance value of the blank group; A2 is the absorbance value of the sample group.

[0060] The results of the analysis of the interaction between the active components of rice wine lees and cholesterol micelles are as follows: Figure 4 As shown, from Figure 4 It can be seen that the inhibition rate of the active ingredients of rice wine lees on cholesterol micelles is relatively good. The inhibition rate of the active ingredients of rice wine lees increases steadily with the increase of concentration at a concentration of 0.0625-2.0 mg / mL. This may be because after the active ingredients of rice wine lees are added to the micelles, they react with one or more components in the micelles to form an insoluble complex, thereby inhibiting the formation of cholesterol micelles; the IC50 of the inhibition rate of cholesterol micelles by the active ingredients of rice wine lees is 0.41 mg / mL, and when the concentration of the active ingredients of rice wine lees reaches 2.0 mg / mL, the cholesterol micelle inhibition rate reaches 69.20±0.23%.

[0061] The experimental results show that the active ingredients of rice wine lees provided in the present application can inhibit cholesterol micelles, which play a vital role in the intestine. Cholesterol micelles are composed of components such as cholesterol, bile salts and lecithin. This combination can increase the solubility of cholesterol in the small intestine and promote its absorption. If the assembly process of cholesterol micelles can be hindered or its solubility can be reduced, this will effectively reduce the absorption efficiency of cholesterol in the intestine and help control blood lipid levels. Therefore, the active ingredients of rice wine lees provided in the present application are active ingredients that can lower blood lipids.

[0062] Experimental Example 4

[0063] This experimental example analyzes the inhibition types of the active ingredients of rice wine lees provided in Example 1 and pancreatic lipase and cholesterol esterase.

[0064] The principle of the analysis experiment of the inhibition type of rice wine lees active ingredients and pancreatic lipase and cholesterol esterase is as follows: different concentrations of rice wine lees active ingredients react with enzymes, and specific substrates (PNPL and PNPB) are used for catalytic reaction. Substrates are hydrolyzed to generate products with absorbance, and the optical density (OD) value per minute is measured at a wavelength of 405 nm. The linear slope of "time-OD value" represents the reaction rate of the enzyme. By drawing a reaction rate graph of different concentrations of rice wine lees active ingredients and enzymes, the inhibition type is analyzed.

[0065] The raw materials for the analysis experiment of the active ingredients of rice wine lees and the inhibition types of pancreatic lipase and cholesterol esterase are: pancreatic lipase, 4-nitrophenyl laurate (PNPL), sodium acetate, Triton, orlistat, sodium taurocholate, 4-nitrophenyl butyrate (PNPB), and cholesterol esterase; the instruments are: multifunctional microplate reader, water bath, electronic balance, micropipette, and constant temperature incubator.

[0066] The steps of the analysis experiment of the active components of rice wine lees and the inhibition types of pancreatic lipase and cholesterol esterase are as follows:

[0067] Referring to the method for determining the inhibitory activity of pancreatic lipase, the active ingredients of rice wine lees were diluted to different concentrations with Tris-HCl buffer, and the reaction rates of pancreatic lipase of different concentrations in the presence of the inhibitor were determined; 50 μL of sample solutions of different concentrations, 50 μL of pancreatic lipase of different concentrations and 100 μL of PNPL were added to each well of a 96-well plate, and the OD value was determined every 1 min within 10 min at a wavelength of 405 nm. Each group of experiments was repeated 3 times; the slope of the "time-OD value" linear graph represented the reaction rate of pancreatic lipase in the presence of the sample, and a fitted linear graph of the enzyme reaction rate OD / min and the enzyme concentration at different sample concentrations was drawn in the same graph to analyze the inhibition type of pancreatic lipase by the active ingredients of rice wine lees. The results are shown in Figure 2. Figure 5 As shown in Figure A.

[0068] Referring to the cholesterol esterase inhibitory activity determination method, the active ingredients of rice wine lees were diluted to different concentrations with Tris-HCl buffer, and the reaction rates of cholesterol esterases of different concentrations in the presence of inhibitors were determined; 50 μL of sample solutions of different concentrations, 50 μL of cholesterol esterases of different concentrations and 100 μL of PNPB were added to each well of a 96-well plate, and the OD value per minute within 10 minutes was determined at a wavelength of 405 nm. Each group of experiments was repeated 3 times; the slope of the "time-OD value" linear graph represented the reaction rate of cholesterol esterase in the presence of the sample, and a fitted linear graph of the enzyme reaction rate OD / min and the enzyme concentration at different sample concentrations was drawn in the same graph to analyze the inhibition type of the active ingredients of rice wine lees on cholesterol esterase. The results are shown in FIG. Figure 5 As shown in Figure B.

[0069] The results of the analysis of the active components of rice wine lees and the inhibitory types of pancreatic lipase and cholesterol esterase are as follows Figure 5 As shown, from Figure 5 As can be seen from Figure A, the relationship between pancreatic lipase concentration and reaction rate is a straight line, and when the concentration of rice wine lees active ingredients is changed, the graph passes through the origin, and the slope of the fitting equation decreases with the increase of rice wine lees protein concentration. These results show that the rice wine lees active ingredients do not completely inactivate pancreatic lipase, but only reduce the catalytic rate of the enzyme, and the rice wine lees active ingredients bind to pancreatic lipase through non-covalent bonds, reversibly inhibiting pancreatic lipase; similarly, Figure 5 As can be seen from Figure B, the concentration of cholesterol esterase and the reaction rate are fitted into a straight line. Even if the concentration of cholesterol esterase is changed, all straight lines pass through the origin, and the slope of the fitting equation decreases with the increase of the concentration of active ingredients in rice wine lees, indicating that the active ingredients in rice wine lees bind to cholesterol esterase through non-covalent bonds and reversibly inhibit the activity of cholesterol esterase.

[0070] The experimental results show that the active ingredients of rice wine lees and pancreatic lipase and cholesterol esterase are of reversible chemical bond inhibition type; reversible binding inhibition will not lead to permanent inactivation of pancreatic lipase and / or cholesterol esterase, and under certain conditions they can dissociate to restore the activity of the enzyme. The active ingredients of rice wine lees and pancreatic lipase and cholesterol esterase form a temporary complex.

[0071] Experimental Example 5

[0072] In this experimental example, the active ingredients of rice wine lees provided in Example 1 were subjected to an experiment on the effects on cells, and the experiment included a cell proliferation toxicity experiment, a cell oil red O experiment, and a biochemical index experiment.

[0073] Among them, the steps of the cell proliferation toxicity experiment include: digesting HepG2 cells in the logarithmic growth phase with trypsin, preparing a cell suspension, inoculating 6000 cells / well in a 96-well plate, setting 3 replicates for each group, and continuing to culture for 24 hours; adding drugs to each group of cells, and continuing to culture for 24 hours. Remove the original culture medium, prepare a culture medium with a content of 10% CCK8, add 100μL of 10% CCK8 culture medium to each well, and incubate in an incubator for 2 hours; measure the absorbance of each well at a wavelength of 450nm, and calculate the cell survival rate according to the formula: cell survival rate (%) = (1-(A1-A0) / (A2-A0))×100; where A0 is the absorbance value of the empty well, A1 is the absorbance value of the experimental group, and A2 is the absorbance value of the control group without a sample.

[0074] The results of cell proliferation toxicity experiments were as follows Figure 6 As shown, from Figure 6 It can be seen that the active ingredients of rice wine lees can reduce cell viability. At a concentration of 500μg / mL and below, the cell viability is greater than 90%, which is not considered cytotoxic; when the concentration of the active ingredients of rice wine lees is 2000μg / mL, the cell survival rate is only 55.75%; therefore, in subsequent experiments, it is mainly necessary to focus on using no more than 500μg / mL of active ingredients of rice wine lees for cell experiments; at the same time, when adding rice wine lees active ingredients to food, health products and medicines, it is also necessary to control the amount of active ingredients added.

[0075] The steps of the cell oil red O experiment include: after taking out the HepG2 cells after climbing and processing, aspirate the culture medium, wash them three times with pre-cooled PBS buffer, add cell fixative (4% paraformaldehyde solution) to fix the cells for 15 minutes. After fixation, stain the cells with oil red O solution for 1-2 hours, and rinse them with 60% isopropanol solution for 10-20 seconds. Finally, seal the slides with glycerol gelatin and collect and analyze images under a microscope.

[0076] The results of the cell oil red O experiment are as follows Figure 7 As shown, from Figure 7 Figure A shows the effect of Oil Red O staining on HepG2 cells. No obvious red-stained lipid droplets were observed in the blank group under the microscope. The proportion of red lipid droplets in the model group was significantly higher than that in the blank group, indicating that the HepG2 cell fat accumulation model was successfully established. Compared with the model group, the number of stained lipid droplets in the cells with added rice wine lees protein after differentiation was significantly reduced, indicating that the sample played a certain role in the cell Oil Red O staining. The specific Oil Red O staining results are shown in the figure. Figure 7 As shown in Figure B, Figure 7As shown in Figure B, different concentrations of rice wine lees protein have a down-regulating effect on the lipid content in the HepG2 cell model. The concentration of rice wine lees protein with a more obvious change in lipid content in the down-regulation model is between 125 and 500 μg / mL, but the downward trend of the red proportion at 250 μg / mL and 500 μg / mL is not significant; while high concentrations of rice wine lees protein increase the red proportion in cell oil red O, which is consistent with the results of biochemical index determination in the previous experiment. After incubating cells with rice wine lees protein of different mass concentrations for 24 h, the proportion of red lipid droplets was significantly reduced compared with the model group, indicating that rice wine lees protein has a certain scavenging effect on lipids. It can be seen that rice wine lees protein can reduce the lipid content in the high-lipid cell model and regulate cellular lipid metabolism.

[0077] Biochemical index experiments include TC, TG, HDL-C, AST, ALT and other biochemical indexes. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in biochemical indexes are important biomarkers for assessing the health status of the liver. When liver cells are damaged, the integrity of the cell membrane will be destroyed, causing the AST and ALT in the cells to leak into the blood, which increases the concentration of these two enzymes in the blood. Therefore, by measuring the levels of AST and ALT in the blood, it is possible to assess whether the liver is damaged and the extent of the damage; and total cholesterol (TC) and triglycerides (TG) in biochemical indexes are two key indicators of blood lipid levels. Their abnormal increase is usually associated with an increased risk of cardiovascular disease. Therefore, maintaining the balance of TC and TG content in cells is of great significance for the prevention and treatment of hyperlipidemia and its related cardiovascular diseases; and the main role of high-density lipoprotein (HDL) in the body is to help transport cholesterol from the blood vessel wall and peripheral tissues to the liver for metabolism and excretion. This process is called reverse cholesterol transport. HDL helps reduce the deposition of cholesterol in blood vessel walls, thereby reducing the risk of atherosclerosis and cardiovascular disease.

[0078] The steps of the biochemical index experiment include: setting up a normal group, a model group, a positive control group and a sample treatment group according to the experimental cell oil red O steps, and culturing cells in the same way. After incubation with yellow wine lees protein for 24 hours, the cells were digested with trypsin for 3-5 minutes, centrifuged (1200g) to collect the cell precipitate, and ultrasonically disrupted (300 W, 3-5s each time, 30s interval, repeated 3-5 times) in an ice water bath until the cells were completely disrupted to obtain the cell contents. According to the instructions, commercial kits were used to detect the content of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Nanjing Jiancheng).

[0079] The results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in biochemical indicators are as follows Figure 8 As shown in Figures A and B, it can be seen from Figures A and B that the content of intracellular ALT and AST in the model group is significantly higher than that in the blank control group, indicating that liver cells are damaged; with the increase of sample dose at 62.5-500μg / ml, the content of intracellular ALT and intracellular AST gradually decreases, but in subsequent experiments, when the concentration of rice wine lees is 1000μg / ml and 2000μg / ml, the content of intracellular ALT and intracellular AST shows an upward trend, which may be due to the influence of excessive concentration of rice wine lees protein on cells. This shows that the concentration of rice wine lees protein is suitable for 0-500μg / ml.

[0080] The results of the total cholesterol (TC) and triglyceride (TG) tests in the biochemical indexes are as follows: Figure 8 As shown in Figures C and D, Figure 8 As can be seen from Figure C, the TC content in the model group obtained in the experiment was significantly increased compared with the blank cells. The TC content in the 62.5μg / mL rice wine lees protein was significantly reduced compared with the model group; the TC content decreased with the increase of the rice wine lees protein concentration at 62.5-250μg / mL; when the sample concentration was greater than 500μg / mL, the TC content in the cells increased, but there was no significant change in the intracellular TC content at 500μg / mL and 250μg / mL, which should also be caused by the effect of excessive concentration on the cells; when the rice wine lees protein concentration was 250μg / mL, the TC content was 0.1683±0.0015mmol / L, which was close to 0.1617±0.0021mmol / L in the positive control group, indicating that rice wine lees protein can effectively reduce the TC level in the cells at a lower concentration; and from Figure 8 As can be seen from Figure D, the influence trend of the blank group, model group, positive control group and sample group on the intracellular TG content is basically the same as the influence on the TC content. When the concentration of rice wine lees protein is 250μg / mL, the intracellular TG content is the lowest, which is 0.1103±0.0025mmol / L, and compared with 0.269±0.03mmol / L in the model group, the intracellular TG content is reduced by 59.0%, indicating that the effect of rice wine lees protein on the TG content in cells is relatively obvious. Therefore, it is shown that rice wine lees protein has the effect of reducing the intracellular TG content in high-lipid HepG2 cells.

[0081] The results of the high-density lipoprotein (HDL) test in biochemical indicators are as follows: Figure 8As shown in Figure E, it can be seen from Figure E that compared with the 62.5-1000μg / mL rice wine lees protein sample group, the blank control group and the positive control group, the HDL content of the model group decreased significantly, but when the sample was 2000μg / ml, the HDL concentration was equivalent to the model group. As the dose of the sample group increased, the intracellular HDL content first increased continuously. When the sample concentration reached 125μg / mL, the intracellular HDL concentration reached the highest, which was better than the positive control group. Then the intracellular HDL content decreased with the increase in concentration; it may be related to the effect of the sample on the cell growth rate. The concentration is too high and it is not suitable for cell experiments. The experiment shows that low concentrations of rice wine lees protein can effectively increase the intracellular HDL content, thereby helping to maintain healthy blood lipid levels and reduce the risk of atherosclerosis and cardiovascular diseases.

[0082] Example 2

[0083] In this experimental example, the active polypeptide MU1 (<3 kDa) provided in Example 1 was further separated and purified to screen out a batch of polypeptide components with the highest biological activity.

[0084] The ultrafiltration performed in Example 1 can achieve the preliminary fractionation and separation of the active components of rice wine lees, and four groups of active polypeptide components MU1 (<3 kDa), MU2 (3 kDa~10 kDa), MU3 (10~30 kDa), and MU4 (>30 kDa) are obtained. The experimental results of Experimental Example 1 show that the component with a molecular weight less than 3 kDa has the highest inhibitory activity on pancreatic lipase and cholesterol esterase. Next, the component is separated and purified to screen a batch of polypeptide components with the highest biological activity.

[0085] The separation and purification of active peptide MU1 (<3 kDa) was performed using LC-MS / MS analysis instrument, and the steps included: using a 100μm id × 180mm analytical column filled with Reprosil-Pur 120 C18 AQ 3μm; mobile phase A was 0.1% formic acid in water, mobile phase B was 80% acetonitrile solution containing 0.1% formic acid, the flow rate was 600nL / min, the analysis time for each component was 66 minutes, 0-55 min mobile phase B gradient elution from 0% to 40%, 56-66min mobile phase B 95% isocratic elution. The full spectrum scan range was 100-1500m / z, the primary mass spectrometry resolution was set to 70000, the automatic gain control (AGC) was 3e6, and the maximum injection time (MaximumIT) was set to 100ms. The parent ion with an ion intensity of 20 in the full scan was selected, and the secondary mass spectrometry detection was performed using the high energy collision fragmentation (HCD) method. The secondary mass spectrometry resolution was set to 17500, AGC was 1e5, MaximumIT was 50ms, and the peptide fragmentation collision energy was set to 28. The mass spectrometry MS / MS data were analyzed using software to verify the matching degree between the collected data and the theoretical fragments of the peptide. The search parameters included fixed modification (Carbamidomethyl on C), variable modification (Oxidation on M and Acetyl on Peptide N-term), non-specific enzyme, Oryza_sativa and Saccharomyces cerevisiae databases, primary mass spectrometry deviation of 20ppm, and secondary mass spectrometry deviation of 0.02Da. The samples were separated by ultrafiltration-affinity chromatography, and DB search -10lgP > 20 were screened out for peptide sequences for subsequent inhibitory peptide screening.

[0086] The results of separation and purification of active polypeptide MU1 (<3 kDa) are as follows Fig.10 As shown, from Fig.10 As shown in Figure A, a total of 3880 peptides less than 3 kDa were identified by LC-MS / MS, of which 1983 peptides with a molecular weight less than 1 kDa accounted for the largest proportion, reaching 51.11%. Fig.10 Figure B shows the number of amino acids in the peptide segment. Among them, the polypeptide with a polypeptide chain length of 4 amino acids is the largest, and then the number of amino acids gradually decreases as the chain length increases. Fig.10Figure C shows the amino acid composition ratio of the polypeptide. The sequence and amino acid properties of the polypeptide are closely related to the inhibitory activity of pancreatic lipase and cholesterol esterase. Studies have reported various polypeptides with blood-lowering potential, whose N-terminal and C-terminal amino acids contain polar amino acids; among the rice wine lees polypeptides less than 3kDa, glutamic acid has the highest amino acid content, accounting for 7.98%, and tryptophan has the lowest content, accounting for 1.23%.

[0087] The physicochemical properties of the isolated and purified active polypeptide MU1 (<3 kDa) are as follows Fig.10 As shown in Figure D, the isoelectric point (pI) and protein hydrophobicity analysis (GRAVY) of the peptide can be seen from Figure D, and its data reflects the folding degree and stability of the detected peptide; the isoelectric point (pI) of the peptide refers to the net charge of the polypeptide molecule is zero in a solution with a specific pH value, that is, the positive and negative charges are equal, and the peptide will not migrate in the electric field; and in the hydrophobicity analysis, greater than 0 usually indicates hydrophobicity, which means that the amino acid residues tend to repel water molecules and aggregate with other hydrophobic molecules, while less than 0 usually indicates hydrophilicity, which means that the amino acid residues tend to attract water molecules and dissolve in water. It can be seen from Figure D that the isoelectric point of the polypeptide is mainly in the acidic and alkaline ranges, and the content is comparable, so at a neutral pH value, the polypeptide may have a net charge close to zero, which can help maintain the solubility and stability of the polypeptide; and in the hydrophobic effect, the content of polypeptide peptides greater than 0 and less than 0 is comparable, indicating that the ratio of hydrophobic and hydrophilic amino acids in the polypeptide is relatively balanced, which is beneficial to the biological activity and structural stability of the polypeptide.

[0088] The secondary mass spectra of the three polypeptides YQVQY, YIIPY, and TPLQY in the isolated and purified active polypeptide MU1 (<3 kDa) are shown in Figures E~G.

[0089] Experimental Example 6

[0090] This experimental example analyzes the interaction between the three polypeptides YQVQY, YIIPY, TPLQY provided in Example 2 and pancreatic lipase and cholesterol esterase, and the analysis is performed by molecular docking of the three polypeptides YQVQY, YIIPY, TPLQY and pancreatic lipase and cholesterol esterase.

[0091] Among them, the molecular docking process of the three polypeptides YQVQY, YIIPY, TPLQY and pancreatic lipase included: molecular docking using Discovery Studio 2019 and AutoDock Tools-1.5.6 software. Firstly, the three-dimensional structure of the peptide was constructed according to the amino acid sequence of the peptide. The three-dimensional structure of pancreatic lipase (PDB number: 1LPB) obtained from the RCSB: PDB protein database (http: / / www.rcsb.org) was imported into the software for structural modification. Phe77, Ser152, Asp176, and His263 on the pancreatic lipase protein crystal were selected as key amino acids and defined as active pockets for pancreatic lipase molecular docking. Then, LibDock semi-flexible molecular docking was used. Semi-flexible docking took into account both data accuracy and calculation speed. The top 20 peptides with the highest LibDock Score were screened out, and Flexibledocking was used for full flexible docking. Autodock molecular docking was used to assist in the screening of lipid-lowering vinasse peptides. Finally, Discovery Studio 2019 was used to visualize the screening results in 2D and 3D to analyze the impact of docking and bond position.

[0092] The difference in the molecular docking process with pancreatic lipase is that the docking process of the three polypeptides YQVQY, YIIPY, TPLQY and cholesterol esterase molecules replaces the key amino acids for docking from Phe77, Ser152, Asp176, His263 to Ser194, Asp320 and His435.

[0093] The screening process of molecular docking of three peptides YQVQY, YIIPY, TPLQY and pancreatic lipase is as follows: computational molecular docking can predict the interaction and direction between ligands and macromolecular receptors, and analyze the interaction mechanism between receptors and ligands by directly observing the binding conformation and binding site of receptors and ligands; the 2-5 amino acid peptides identified from peptides less than 3 kDa were docked using Discovery Studio 2019 software and AutoDock Tools software. A total of 962 peptides were docked by Libdock in Discovery Studio 2019, and 957 peptides were successfully docked, of which 98 peptides had a libdock score>170, accounting for 10.24%; there were 879 peptides with a libdock score>120, accounting for 91.85% of the successfully docked peptides; The higher the value, the better the Libdock docking result. Therefore, it can be explained that the peptides with 2-5 amino acids have a relatively good binding ability with pancreatic lipase, which can well confirm that the yellow wine lees peptides and yellow wine lees proteins with a molecular weight of less than 3kDa in previous experiments showed good binding energy. The flexible docking function in Discovery Studio 2019 was used to further dock the 20 peptides with libdock score>180 and the orlistat molecule, and the docking results of these 21 small molecules were analyzed by Binding Energies. Among them, 7 peptides with -CDOCKER ENERGY greater than orlistat and Binding Energy less than orlistat were screened out; Autodock molecular docking was used to assist in the screening of yellow wine lees peptides for lowering blood lipids, and these 7 small molecules were docked, and 3 peptides with docking binding energy greater than orlistat or similar to orlistat were screened out, namely YQVQY, YIIPY and TPLQY. The docking results of the 3 selected peptides are shown in Table 1. DiscoveryStudio 2019 client software was used to further analyze the interaction patterns and action sites of YQVQY, YIIPY and TPLQY on pancreatic lipase.

[0094] Virtual screening of pancreatic lipase has been shown to be a valuable method for finding new lead compounds. The active site of pancreatic lipase (PDB ID: 1LPB) consists of a highly restricted catalytic triad of Ser 152, Asp 176, and His 263; the peptides mainly bind to the active site of pancreatic lipase through hydrogen bonds, alkyl, Pi-alkyl, and Pi-cations. YQVQY can form four hydrogen bonds with amino acid residues PHE77, SER152, ARG256, and HIS263, and has hydrophobic and electrostatic interactions with PHE77, ILE78, TYR114, PHE215, ARG256, and ALA259. YIIPY formed five hydrogen bonds with amino acid residues GLY76, PHE77, THR115 and SER152, and had hydrophobic and electrostatic interactions with ILE78, TYR114, ALA178, PRO180, PHE215, ARG256, ALA259 and HIS263. TPLQY formed six hydrogen bonds with amino acid residues GLY76, PHE77, SER152, PHE215 and ALA259, and had hydrophobic and electrostatic interactions with ILE78, TYR114, ALA178, PRO180, ARG256 and HIS263. Among them, all three peptides formed hydrogen bonds with amino acid residues PHE77 and SER152 in pancreatic lipase, and had hydrophobic and electrostatic interactions with TYR114 and ARG256. Phe 77 is a hydrogen bond donor and electrostatic stabilizer that can form an oxyanion pore for substrate hydrolysis. All three peptides formed hydrogen bonds or carbon-hydrogen bonds with Phe 77, which may be one of the reasons why they have good pancreatic lipase inhibitory activity.

[0095] Table 1: Molecular docking results of YQVQY, YIIPY, TPLQY, orlistat and pancreatic lipase

[0096]

[0097] After the molecular docking of three peptides YQVQY, YIIPY, TPLQY and pancreatic lipase, the top three peptides with the best binding energy with pancreatic lipase were successfully screened out: YQVQY, YIIPY and TPLQY. In order to further explore the lipid-lowering potential of these three peptides, molecular docking experiments will be further used to analyze their interactions with cholesterol esterase in detail. The three peptides were docked using Discovery Studio 2019 software and Autodock software, and the docking results are shown in Table 2. The docking results are shown in Table 2. The LibDock Score, -CDOCKER ENERGY and BindingEnergy of the three peptides are better than orlistat, but the results of Autodock docking show that the binding energy of YQVQY is only -2.74 kJ / mol, which is not better than orlistat; the interaction mode and action site of YQVQY, YIIPY and TPLQY on cholesterol esterase were further analyzed using Discovery Studio 2019 client software. The docking results showed that YQVYQ could form six hydrogen bonds with amino acid residues ALA108, SER194, TYR427, HIS435 and ASP437, and had hydrophobic and electrostatic interactions with LEU258, PHE324 and ALA436. YIIPY could form six hydrogen bonds with amino acid residues ALA108, SER194, TYR427, HIS435 and ASP437, and had hydrophobic and electrostatic interactions with ALA108, LEU124, TRP227, LEU323, PHE324, HIS435, ALA436 and ASP437. TPLQY can form 9 hydrogen bonds with amino acid residues GLY106, GLY107, ALA108, TYR125, GLU193, SER194 and LEU439, and has hydrophobic and electrostatic interactions with LEU69, MET11, GLU193, TRP227, PHE324 and HIS435. Among them, these three polypeptides all form hydrogen bonds with amino acid residues ALA108 and SER194 in cholesterol esterase, and have hydrophobic and electrostatic interactions with PHE324. The main active site of cholesterol esterase is the catalytic triad at the bottom of the canyon, which is composed of Ser194-His435-Asp320. All three polypeptides form hydrogen bonds with SER194. Therefore, these three polypeptides have a highly effective inhibitory effect on cholesterol esterase.

[0098] Table 2: Molecular docking results of YQVQY, YIIPY, TPLQY, orlistat and cholesterol esterase

[0099]

[0100] Experimental Example 7

[0101] In this experimental example, the remaining polypeptides in the rice wine lees active ingredients provided in Example 1 were docked with pancreatic lipase and cholesterol esterase molecules; the molecular docking process was the same as that in Example 7; wherein, the molecular docking results with pancreatic lipase are shown in Table 3, and the molecular docking results with cholesterol esterase are shown in Table 4.

[0102] Table 3: Molecular docking results of peptides and pancreatic lipase

[0103]

[0104] Table 4: Docking results of peptide and cholesterol esterase molecules

[0105]

[0106] It can be seen from Tables 3 and 4 that in addition to the three peptides QVQY, YIIPY and TPLQY, there are many other peptides that have inhibitory effects on pancreatic lipase and cholesterol esterase; for example, VRSPF, IVQGR, SLHLE, KPHER, WFYND, RIHEY, FFHCY, KYLAL, YYPGL, IHQFR, WNELW, IHRFR, LTPHW, IHFQF, FRHW, KEAFR, WFYN have strong binding ability with pancreatic lipase; while YKHGL , WSLDY, WNELW, IHQFR, IYVYD, FFHCY, SFFY, VRSPF, KQFR, HWCY, WQSSR, RIHEY, LLQPY, YYPGF, FNLPR, YVFR, WLSY, KWLQ, TLSPY, FRGCR, IHHTY, IFHR, WLYN, FTFPR, KPTSW, WFYND, LGYPR, LSPFW, RPVR, YPLPR have strong binding ability with cholesterol esterase. In addition, VRSPF, WFYND, RIHEY, FFHCY, IHQFR, WNELW and other polypeptides have strong binding ability with pancreatic lipase and cholesterol esterase, and have similar effects to QVQY, YIIPY and TPLQY, and can inhibit pancreatic lipase and cholesterol esterase at the same time.

Claims

1. A yellow wine lees active ingredient capable of lowering blood lipids, characterized in that: The rice wine lees active ingredients are selected from polypeptides and / or proteins; The rice wine lees active ingredients are selected from rice wine lees active ingredients that inhibit pancreatic lipase and / or cholesterol esterase activity, rice wine lees active ingredients that inhibit cholesterol micelles, or rice wine lees active ingredients that enhance bile salt activity.

2. The active ingredient of rice wine lees capable of lowering blood lipids according to claim 1, characterized in that: The active ingredients of rice wine lees are selected from polypeptides with a molecular weight not greater than 3 kDa.

3. The active ingredient of rice wine lees capable of lowering blood lipids according to claim 1, characterized in that: The amino acid sequence of the polypeptide is as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No.20, SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25, SEQ ID No.26, SEQ ID No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43 or SEQ ID No.

44.

4. The active ingredient of rice wine lees capable of lowering blood lipids according to claim 1, characterized in that: The inhibition of pancreatic lipase and / or cholesterol esterase activity specifically involves inhibiting the pancreatic lipase and / or cholesterol esterase activity by combining with at least one of pancreatic lipase and cholesterol esterase via a reversible chemical bond.

5. The method for extracting the active component of rice wine lees capable of lowering blood lipids according to any one of claims 1 to 4, characterized in that: The extraction method comprises: adding rice wine lees into boric acid-potassium chloride buffer for extraction, filtering and centrifuging to obtain the active ingredients of rice wine lees capable of lowering blood lipids.

6. Use of an active ingredient from rice wine lees capable of lowering blood lipids obtained by the extraction method of claim 5 in the preparation of a composition for preventing or treating hyperlipidemia.

7. The use according to claim 6, characterized in that: The application specifically includes: use of at least one polypeptide in an amino acid sequence such as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.13 or SEQ ID No.14 in preparing a composition for inhibiting the activity of pancreatic lipase and cholesterol esterase to prevent or treat hyperlipidemia.

8. The use according to claim 6, characterized in that: The specific application is: the use of at least one polypeptide in the amino acid sequence such as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.11, SEQ ID No.12, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 or SEQ ID No.20 in the preparation of a composition for inhibiting pancreatic lipase activity to prevent or treat hyperlipidemia.

9. The use according to claim 6, characterized in that: The application specifically includes: use of at least one polypeptide in the amino acid sequence such as SEQ ID No.21, SEQ ID No.22, SEQ ID No.23, SEQ ID No.24, SEQ ID No.25, SEQ ID No.26, SEQ ID No.27, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No.32, SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37, SEQ ID No.38, SEQ ID No.39, SEQ ID No.40, SEQ ID No.41, SEQ ID No.42, SEQ ID No.43 or SEQ ID No.44 in the preparation of a composition for inhibiting cholesterol esterase activity to prevent or treat hyperlipidemia.

10. A composition for preventing or treating hyperlipidemia, characterized in that: The invention comprises the yellow wine lees active ingredient capable of lowering blood lipids as described in any one of claims 1 to 4 and a pharmaceutically acceptable carrier or auxiliary material.

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