Preparation method of corn oligopeptide and application of corn oligopeptide in dispelling alcohol and protecting liver

Abstract

The application relates to the technical field of corn oligopeptides, in particular to a preparation method of corn oligopeptides and application of the corn oligopeptides in alcohol elimination and liver protection. The corn oligopeptides provided by the application can be taken in three stages of before, during and after drinking, can effectively improve the tolerance of the liver to toxins, can quickly and effectively eliminate alcohol after drinking, can enhance the alcohol elimination and liver protection effects, has the effects of clearing heat and eliminating toxins, warming the middle to stop vomiting, waking up the liver, supplementing the kidney, regulating the intestines and stomach, and overcomes the defects of the prior art.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of corn oligopeptide, and in particular to a preparation method of corn oligopeptide and application of the corn oligopeptide in alcohol elimination and liver protection. BACKGROUND

[0002] Corn peptide is a small molecular polypeptide substance obtained from corn protein through directional enzyme cutting and specific small peptide separation technology, and has the characteristics of good solubility, high stability, safety, rich nutrition and easy absorption, and also has the functions of reducing blood pressure, inhibiting alcohol poisoning, resisting fatigue, protecting liver and improving immunity.

[0003] Corn peptide has great use in food seasoning. After corn protein is enzymatically hydrolyzed, a large amount of flavor peptides can be obtained, which can enhance food quality and flavor, provide flavor precursors or produce other aromatic substances through Maillard reaction, and are important raw materials for natural compound seasonings. The flavor peptides can be widely used in seasoning of instant noodles, frozen foods, seasonings, meat products and pickled vegetables, can reduce the salty taste of salt and the astringency of monosodium glutamate, enhance the umami and thickness of products, and enhance the coordination of various flavors. At the same time, the flavor peptides meet the food development trend of "natural, nutritious and safe", have very good development prospect and high utilization value. SUMMARY

[0004] In view of this, the present application provides a preparation method of corn oligopeptide and application of the corn oligopeptide in alcohol elimination and liver protection. The corn oligopeptide provided by the present application can be taken before, during and after drinking, can effectively improve the tolerance of liver to toxins, has fast and good effects after drinking, can enhance the effects of alcohol elimination and liver protection, has the effects of clearing heat and resolving toxins, warming middle and stopping vomiting, refreshing, tonifying kidney and regulating intestines and stomach, and overcomes the defects of the prior art.

[0005] To achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0006] In a first aspect, the embodiment discloses a preparation method of corn oligopeptide, comprising:

[0007] S1: preparing a corn yellow powder aqueous solution, and homogenizing the solution by using a homogenizer;

[0008] S2: performing alkaline protease enzymolysis on the homogenized solution obtained in step S1;

[0009] S3: continuing to perform neutral protease enzymolysis on the enzymolysis solution obtained in step S2;

[0010] S4: performing enzyme inactivation on the enzymolysis solution obtained in step S3, centrifuging, and taking the supernatant;

[0011] S5: The supernatant obtained in step S4 is subjected to ceramic membrane filtration and ultrafiltration with an ultrafiltration membrane of 1000 U, and the filtrate is subjected to spray drying to obtain a dry peptide powder;

[0012] S6: The dry peptide powder obtained in step S5 is subjected to RP-HPLC separation and purification, and the component peaks are collected to obtain corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ.

[0013] In particular, in step S1, the corn meal is dissolved in distilled water at a mass fraction of 8%, and homogenized for 15 min with a homogenizer.

[0014] In particular, in step S2, the pH is adjusted to 8.5, the temperature is 55℃, and alkaline protease is added at an amount of 3000 units per gram of protein, and the enzyme is hydrolyzed for 3 h.

[0015] In particular, in step S3, the pH is adjusted to 7.0, the temperature is 50℃, and neutral protease is added at an amount of 3000 units per gram of protein, and the enzyme is hydrolyzed for 2 h.

[0016] In particular, in step S4, the temperature is raised to 90℃, the enzyme is inactivated for 15 min, and centrifugation is performed at 6000 x g for 10 min, and the supernatant is taken.

[0017] In particular, in step S5, ceramic membrane filtration is performed with a molecular weight cutoff of 2 x 106 U, the intermediate clear liquid is taken, and ultrafiltration is performed with an ultrafiltration membrane with a molecular weight cutoff of 1000 U to obtain a filtrate with a molecular weight of less than 1000 U, which is spray dried to obtain a dry peptide powder.

[0018] In particular, in step S6, the chromatographic conditions are as follows: sample mass concentration, 10 mg / mL; mobile phase A, V(water):V(trifluoroacetic acid)=100:0.1; mobile phase B, V(acetonitrile):V(water):V(trifluoroacetic acid)=80:20:0.1; detection wavelength, UV 220 nm; flow rate, 0.6 mL / min; column temperature, 40℃; injection volume, 40 μL. The gradient elution program is as follows: 0-10 min, 0-5% mobile phase B; 10-25 min, 5%-9% mobile phase B; 25-35 min, 9%-15% mobile phase B; 35-55 min, 15%-15% mobile phase B; 55-100 min, 15%-40% mobile phase B; 100-120 min, 40%-80% mobile phase B. Twelve main component peaks are collected, the trifluoroacetic acid, acetonitrile and other organic solvents are removed by nitrogen blowing instrument, and then vacuum freeze-dried to obtain dry powder of the 12 component peaks for standby.

[0019] In a second aspect, the embodiments disclose application of the corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared in the first aspect in preparation of a preparation for improving liver fibrosis. The preparation for improving liver fibrosis can be granules, tablets, capsules, pills or powders; the dissolving medium in a liquid preparation can be a buffer, physiological saline or drinking water.

[0020] In a third aspect, the embodiments disclose application of the corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared in the first aspect in preparation of a preparation for protecting liver and relieving alcoholism. The preparation for protecting liver and relieving alcoholism can be granules, tablets, capsules, pills or powders; the dissolving medium in a liquid preparation can be a buffer, physiological saline or drinking water.

[0021] In combination with the third aspect or the fourth aspect, the particle size of the granules is 18-22 mesh.

[0022] The granules or tablets of the corn oligopeptides can be taken in wine or after wine, once a day, and the dosage (calculated by crude drug) is 1 g per 60 Kg of body weight, which can not only prevent drunkenness and relieve alcoholism quickly, but also relieve symptoms of acute and chronic alcoholism, and has the effects of protecting liver and spleen, calming the nerves, stopping vomiting and protecting cardiovascular system.

[0023] It is also found in the application that, after the four corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ are respectively co-cultured with liver cells, AYPQ and LSPY have significant differences relative to a control group, and AYPQ can promote L-O2 cell proliferation to the greatest extent.

[0024] It is also found in the application that the corn oligopeptide AYPQ can reduce CCl4-induced liver injury in mice at a serological level, while the other three corn oligopeptides have poor effects.

[0025] It is also found in the application that the corn oligopeptide AYPQ can improve CCl4-induced liver fibrosis in mice by inhibiting abnormal expression of inflammatory factor levels, while the corn oligopeptides YGPQ, LSPY and AYLQQQ cannot.

[0026] It is also found in the application that the corn oligopeptide AYPQ can improve liver fibrosis by reducing collagen deposition in the liver, while the corn oligopeptides YGPQ, LSPY and AYLQQQ cannot. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The blank group, the model group and the four corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ respectively intervene in the liver tissue HE staining diagram of the liver fibrosis model mice. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from the prior art.

[0029] Example 1: Preparation of corn oligopeptide

[0030] Dissolve corn yellow powder in distilled water with a mass fraction of 8%, and homogenize it for 15 min with a homogenizer; adjust the pH to 8.5 and the temperature to 55℃, and add alkaline protease at an enzyme amount of 3000 units per gram of protein, and hydrolyze for 3 h; adjust the pH to 7.0 and the temperature to 50℃, and add neutral protease at an enzyme amount of 3000 units per gram of protein, and hydrolyze for 2 h. Increase the temperature to 90℃, and inactivate the enzyme for 15 min, and centrifuge at 6000 x g for 10 min, and take the supernatant, and filter it with a ceramic membrane with a molecular weight cut-off of 2 x 106 U, and take the middle clear liquid, and ultrafilter it with an ultrafiltration membrane with a molecular weight cut-off of 1000 U, and obtain a filtrate with a molecular weight of less than 1000 U, and spray dry it to obtain a peptide dry powder.

[0031] RP-HPLC is used to separate and purify the corn oligopeptide. The chromatographic conditions are as follows: sample mass concentration, 10 mg / mL; mobile phase A, V(water):V(trifluoroacetic acid)=100:0.1; mobile phase B, V(acetonitrile):V(water):V(trifluoroacetic acid)=80:20:0.1; detection wavelength, UV 220 nm; flow rate, 0.6 mL / min; column temperature, 40℃; injection volume, 40 μL. The gradient elution program is as follows: 0-10 min, 0-5% mobile phase B; 10-25 min, 5%-9% mobile phase B; 25-35 min, 9%-15% mobile phase B; 35-55 min, 15%-15% mobile phase B; 55-100 min, 15%-40% mobile phase B; 100-120 min, 40%-80% mobile phase B. Twelve main component peaks are selected for collection, the organic solvents such as trifluoroacetic acid and acetonitrile are removed by nitrogen blowing instrument, and then vacuum freeze-dried to obtain dry powder of the 12 component peaks for standby.

[0032] Referring to the method of "Separation and Purification of Antioxidant Peptides in Corn Oligopeptide and Structure Identification", Food Industry, Vol. 40, No. 5, 2019, corn oligopeptide YGPQ, LSPY, AYPQ and AYLQQQ are prepared.

[0033] Example 2: Corn oligopeptide promotes liver cell proliferation

[0034] 1. Grouping experiment

[0035] The peptide powders YGPQ, LSPY, AYPQ, and AYLQQQ were each prepared into solutions with a concentration of 0.4 g / mL using PBS, sterilized by filtration through a 0.22 μm microporous membrane, and stored at -20℃ for later use.

[0036] L-O2 human hepatocytes (STM-CL-5770, Stemer Biotech (Shanghai) Co., Ltd.) in the logarithmic growth phase were divided into a control group and a corn oligopeptide group, respectively, and were injected with 5×10⁻⁶ cells. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates, and incubated for 24 h until complete cell adhesion. Cells were then treated with culture medium containing 5% fetal bovine serum and corn oligopeptides at a final concentration of 0.15 g / mL, with six replicates for each oligopeptide concentration. Control wells without oligopeptides were also included, along with a zeroing well. After incubation at 37°C and 5% CO2 for 5 days, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. The plates were shaken for 10 min to dissolve any crystals, and the absorbance was measured at 570 nm.

[0037] The results are shown in Table 1. In Table 1, compared with the control group, "*" indicates P < 0.05 and "**" indicates P < 0.01. As shown in Table 1, after co-culturing the four maize oligopeptides with hepatocytes, AYPQ and LSPY showed significant differences compared with the control group, and AYPQ was able to promote L-O2 cell proliferation to the greatest extent.

[0038] Table 1. Effects of maize oligopeptides on L-O2 cell proliferation (x±s, n=6)

[0039] Group OD value Control group 0.861±0.042 YGPQ 0.878±0.038 LSPY 0.942±0.066* AYPQ 1.269±0.045** AYLQQQ 0.915±0.035

[0040] Example 3: The effect of corn oligopeptides in improving liver fibrosis

[0041] 1. Grouping and Modeling

[0042] Sixty mice were randomly divided into a control group, a model group, and a corn oligopeptide group (containing corn oligopeptides YGPQ, LSPY, AYPQ, and AYLQQQ solutions administered via gavage at a dose of 4 g / kg), with 10 mice in each group. The mice were cultured for 7 days. From week 2, the control group received olive oil via gavage on Mondays and Thursdays, and saline via gavage for the remaining 5 days. The model group received olive oil solution containing 10% CCl4 (2 mL / kg) via gavage twice a week (Mondays and Thursdays) from week 1 to week 8, and saline via gavage for the remaining 5 days. The corn oligopeptide group received different concentrations of corn oligopeptide solution via gavage 7 times a week from week 1 to week 8, for a total of 8 weeks. After the experiment, the mice were fasted for 12 hours but allowed free access to water. Liver tissue and serum were collected for subsequent assays.

[0043] 2. HE staining

[0044] Liver samples were fixed with 10% neutral buffered formalin for histopathological analysis. The samples were embedded in paraffin and 5-μm-thick sections were cut. HE staining was used to highlight liver injury and collagen deposition, respectively, and analyzed by image analysis software CaseViewer 2.4.

[0045] 3. Detection of serum and collagen markers

[0046] When the liver is damaged, part of the hepatocytes are necrotic, and the necrotic hepatocytes release various enzymes into the blood, such as AST, ALT and AKP, thereby increasing the content of AST, ALT and AKP in the blood. Therefore, the content of AST, ALT and AKP in the serum has become a commonly used indicator for evaluating liver damage.

[0047] Serum AST, ALT and AKP were determined using the corresponding alanine aminotransferase (ALT) test kit, aspartate aminotransferase (AST) test kit and akaline phosphatase (AKP) kit, with the unit being U / L. The expression levels of HA, LN and type IV collagen were detected by enzyme-linked immunosorbent assay. The above-mentioned index detection methods were detected according to the kit instructions.

[0048] Table 2 Effect of corn oligopeptide on serum AST, ALT and AKP of mice (x ± s, n = 10)

[0049] Group AST (U / L) ALT (U / L) AKP (U / L) Control group 8.25±0.52 6.98±0.48 4.31±0.61 Model group 13.68±1.89 24.05±2.35 17.21±3.19 YGPQ 12.99±1.74 22.33±3.28 15.68±2.68* LSPY 11.33±1.68 17.68±1.43* 14.80±2.54* AYPQ 9.83±0.82** 12.35±1.56** 8.35±0.87** AYLQQQ 11.79±1.86 19.86±2.13* 16.79±4.21

[0050] The results of AST, ALT and AKP in the serum of mice are shown in Table 2. In Table 1, “*” indicates P < 0.05, and “**” indicates P < 0.01 compared with the model group.

[0051] As shown in Table 2, compared with the normal group, the levels of AST, ALT and AKP in the model group were significantly increased (P < 0.05), and the intragastric administration of corn oligopeptide could reverse the abnormal increase in the content of AST, ALT and AKP in the serum. The corn oligopeptide AYPQ could simultaneously reduce the abnormal expression of AST, ALT and AKP in the serum of model mice, thereby proving that the corn oligopeptide AYPQ could alleviate CCl4-induced liver damage in mice at the serological level.

[0052] 4. RT-PCR detection of mRNA expression levels of inflammatory factors

[0053] Total RNA was extracted from tissue samples using a total RNA extraction kit for reverse transcription polymerase chain reaction analysis. The concentration and purity were measured using a Nanodrop 2000 spectrophotometer. Reverse transcription was then performed using All-In-One 5X RT Master-Mix, which was reversed into cDNA. Primers were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. RT-PCR was then performed using a Platinum® SYBR® Green SuperMix UDG Reagents Kit, Platinum® Fidelity 2X PCR MasterMix Kit, and a 7900 Real-Time PCR System. Expression levels were normalized according to endogenous GAPDH. TM Fidelity 2X PCR MasterMix Kit and a 7900 Real-Time PCR System. Expression levels were normalized according to endogenous GAPDH.

[0054] GAPDH-F: 5'-AGGTCGGTGTGAACGGATTTG-3', SEQ ID NO: 1

[0055] GAPDH-R: 5'-TGTAGACCATGTAGTTGAGGTCA-3', SEQ ID NO: 2

[0056] IL-6-F: 5'-TAGTCCTTCCTACCCCAATTTCC-3', SEQ ID NO: 3

[0057] IL-6-R: 5'-TTGGTCCTTAGCCACTCCTTC-3', SEQ ID NO: 4

[0058] TNF-a-F: 5'-CCCTCACACTCAGATCATCTTCT-3', SEQ ID NO: 5

[0059] TNF-a-R: 5'-GCTACGACGTGGGCTACAG-3', SEQ ID NO: 6

[0060] Table 3 Effects of corn oligopeptides on mRNA expression levels of inflammatory factors in mice (x ± s, n = 10)

[0061] Group IL-6 mRNA relative expression level TNF-α mRNA relative expression level Control group 0.51±0.08 0.39±0.06 Model group 1.25±0.36 0.72±0.05 YGPQ 1.09±0.24 0.68±0.04 LSPY 1.13±0.18 0.70±0.03 AYPQ 0.66±0.13** 0.43±0.04** AYLQQQ 0.91±0.54 0.69±0.06

[0062] Tumor necrosis factor-a (TNF-a) and interleukin (IL)-6 are inflammatory factors secreted by Kupffer cells and monocyte-derived macrophages, which can stimulate hepatic stellate cell activation to further secrete extracellular matrix.

[0063] The relative expression levels of IL-6 and TNF-a mRNA in mouse tissue samples are shown in Table 3, in which "**" indicates P < 0.01 compared with the model group.

[0064] As shown in Table 3, the model group has significantly higher levels of TNF-α and IL-6 mRNA and protein in the liver tissue (P<0.05), and after the intervention of corn oligopeptide AYPQ, the levels of inflammatory factors in the liver tissue of mice are significantly reduced, thereby improving the inflammatory response related to liver fibrosis. Thus, the present application demonstrates that corn oligopeptide AYPQ can improve CCl4-induced liver fibrosis in mice by inhibiting the abnormal expression of inflammatory factor levels, while corn oligopeptides YGPQ, LSPY and AYLQQQ cannot.

[0065] 5. Detection of laminin, hyaluronic acid and type IV collagen content

[0066] LN and type IV collagen are important components of the basement membrane, and HA is a major component of the extracellular matrix, and their high expression is closely related to liver fibrosis, and is an important indicator reflecting the progression of liver fibrosis and hepatocyte damage.

[0067] The frozen liver tissue was weighed and homogenized in a cold lysis buffer containing protease inhibitors. The liver tissue homogenate was centrifuged at 12000 r / min for 20-30 min at 4°C, and the supernatant was immediately collected for use. The protein concentration was determined by BCA kit. The laminin (LN) test kit, hyaluronic acid (HA) test kit and type IV collagen (type IV collagen, Nanjing Jiancheng Biological Engineering Institute) were used to detect the contents of laminin, hyaluronic acid and type IV collagen in the liver tissue of mice in each group.

[0068] Table 4 Effect of corn oligopeptide on LN, HA and IV-C in the liver of mice (x±s, n=10)

[0069] Group LN (mg / mL) HA (U / L) IV-C (U / L) Control group 125.18±25.25 3.82±0.35 1.87±0.51 Model group 142.52±36.72 8.41±0.61 4.84±0.42 YGPQ 137.92±28.35 8.29±0.48 4.69±0.73 LSPY 131.78±38.09 8.38±0.70 4.57±0.68 AYPQ 128.25±18.35* 4.25±0.25** 2.27±0.24** AYLQQQ 134.34±28.84 7.89±0.33 3.45±0.39*

[0070] As shown in Table 4, the model group has significantly higher levels of LN, HA and IV-C in the liver tissue (P<0.05), and after the intervention of corn oligopeptide AYPQ, the levels of LN, HA and IV-C in the liver tissue of mice are significantly reduced, thereby reducing the collagen deposition in the liver of CCl4-induced liver fibrosis mice to improve liver fibrosis. Thus, the present application demonstrates that corn oligopeptide AYPQ can improve liver fibrosis by reducing collagen deposition in the liver, while corn oligopeptides YGPQ, LSPY and AYLQQQ cannot.

[0071] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which shall be covered within the protection scope of the present application.

Claims

1. Application of corn oligopeptide AYPQ in the preparation of agents to improve liver fibrosis.

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