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

Abstract

The invention relates to the technical field of corn oligopeptides, in particular to a preparation method of corn oligopeptides and application of the corn oligopeptides in hangover alleviating and liver protection. The corn oligopeptide provided by the invention can be taken before drinking, during drinking and after drinking, can effectively improve the tolerance of the liver to toxins, is fast in hangover alleviating and good in effect after drinking, can enhance the hangover alleviating and liver protecting effects, has the effects of clearing away heat and toxic materials, warming middle and arresting vomiting, alleviating hangover and promoting the secretion of saliva or body fluid, tonifying and regulating the kidney and coordinating intestines and stomach, and can be used as a health-care food. The defects in the prior art are overcome.

Description

Technical Field

[0001] The present application relates to the technical field of corn oligopeptides, and in particular to a preparation method of corn oligopeptides and their application in alcohol sobering and liver protection. Background Art

[0002] Corn peptide is a protein extracted from corn. It is a small molecule polypeptide substance obtained through targeted enzymatic cleavage and specific small peptide separation technology. It has the characteristics of good solubility, high stability, safety, rich nutrition and easy absorption. It also has the functions of lowering blood pressure, inhibiting alcohol poisoning, resisting fatigue, protecting the liver and improving the body's immunity.

[0003] Corn peptides are widely used in food seasoning. After enzymatic hydrolysis of corn protein, a large number of flavor peptides can be obtained. These flavor peptides can enhance food quality and flavor, provide flavor precursors or produce other aromatic substances through Maillard reaction. They are important base materials for natural compound seasonings. Flavor peptides can be widely used in the seasoning of instant noodles, quick-frozen foods, seasonings, meat products, pickled vegetables and other convenient foods. They can reduce the saltiness of salt and the dryness of MSG, enhance the umami and thick taste of the product, and enhance the coordination of various flavors. At the same time, flavor peptides are in line with the development trend of "natural, nutritious and safe" food, and have very good development prospects and high utilization value. Summary of the invention

[0004] In view of this, the present invention provides a preparation method of corn oligopeptides and an application of the corn oligopeptides in sobering up and protecting the liver. The corn oligopeptides provided by the present invention can be taken in three stages: before, during and after drinking, and can effectively improve the liver's tolerance to toxins. After drinking, the sobering up is fast and effective, and the sobering up and protecting the liver effect can be enhanced. It also has the effects of clearing away heat and detoxifying, warming the middle and stopping vomiting, sobering up and promoting salivation, nourishing the kidneys and regulating the stomach, thereby overcoming the defects of the prior art.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] In the first aspect, the embodiment discloses a method for preparing corn oligopeptides, comprising:

[0007] S1: prepare an aqueous solution containing corn yellow powder and homogenize it using a homogenizer;

[0008] S2: hydrolyzing the homogenized solution obtained in step S1 with alkaline protease;

[0009] S3: The enzymatic solution obtained in step S2 is further enzymatically hydrolyzed with a neutral protease;

[0010] S4: inactivate the enzyme in the enzymatic hydrolyzate obtained in step S3, centrifuge, and collect the supernatant;

[0011] S5: The supernatant obtained in step S4 is filtered through a ceramic membrane and a 1000U ultrafiltration membrane, and the obtained filtrate is spray-dried to obtain a peptide dry powder;

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

[0013] In the specific step S1, corn yellow powder is dissolved in distilled water with a mass fraction of 8%, and homogenized with a homogenizer for 15 minutes.

[0014] In the specific step S2, the pH is adjusted to 8.5, the temperature is 55°C, and alkaline protease is added at an enzyme amount of 3000 units per gram of protein, and enzymolysis is performed for 3 hours.

[0015] In the specific step S3, the pH is adjusted to 7.0, the temperature is 50°C, and neutral protease is added at an enzyme amount of 3000 units per gram of protein, and enzymolysis is performed for 2 hours.

[0016] In the specific step S4, the temperature is raised to 90° C., the enzyme is inactivated for 15 min, centrifuged at 6000×g for 10 min, and the supernatant is collected.

[0017] In the specific step S5, the solution is filtered with a ceramic membrane having a molecular weight cutoff of 2×106U, the intermediate clear liquid is ultrafiltered with an ultrafiltration membrane having a molecular weight cutoff of 1000U to obtain a filtrate having a molecular weight less than 1000U, and the filtrate is spray-dried to obtain a peptide dry powder.

[0018] Specifically, 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, UV220 nm; flow rate, 0.6 mL / min; column temperature, 40°C; injection volume, 40 μL. Gradient elution program: 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 were selected for collection, and organic solvents such as trifluoroacetic acid and acetonitrile were removed by nitrogen blowdown, followed by vacuum freeze drying to obtain dry powders of the 12 component peaks for later use.

[0019] In the second aspect, the examples disclose the use of corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared in the first aspect in the preparation of a preparation for improving liver fibrosis. The preparation for improving liver fibrosis can be a granule, tablet, capsule, pill or powder; the dissolving medium in the liquid preparation can be a buffer, saline or drinking water.

[0020] In the third aspect, the embodiment discloses the use of the corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared in the first aspect in the preparation of a hangover and liver protection preparation. The hangover and liver protection preparation can be in the form of granules, tablets, capsules, pills or powders; the dissolving medium in the liquid preparation can be a buffer solution, saline or drinking water.

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

[0022] The granules or tablets of the corn oligopeptide can be taken in or after drinking, once a day, with each dosage (calculated as crude drug) being 1 gram for every 60 kg of body weight. It can not only prevent drunkenness and sober up quickly, but also relieve various symptoms of acute and chronic alcohol poisoning, and has the effects of protecting the liver and spleen, calming the nerves and stopping vomiting, and protecting the cardiovascular and cerebrovascular systems.

[0023] The present invention also found that after four corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ were co-cultured with hepatocytes respectively, AYPQ and LSPY had significant differences compared with the control group, and AYPQ could promote the proliferation of L-O2 cells to the greatest extent.

[0024] The present invention also found that corn oligopeptide AYPQ can alleviate CCl4-induced liver damage in mice at the serological level, while the other three corn oligopeptides have poor effects.

[0025] The present invention also found that corn oligopeptide AYPQ may improve CCl4-induced liver fibrosis in mice by inhibiting the abnormal expression of inflammatory factors, while corn oligopeptides YGPQ, LSPY and AYLQQQ cannot.

[0026] The present invention also found that corn oligopeptide AYPQ may improve liver fibrosis by reducing collagen deposition in the liver, while corn oligopeptides YGPQ, LSPY and AYLQQQ cannot. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HE staining images of liver tissues of mice with liver fibrosis model respectively intervened by the blank group, model group, and four corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ provided in the examples. DETAILED DESCRIPTION

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

[0029] Example 1: Preparation of corn oligopeptides

[0030] Dissolve corn yellow powder in distilled water with a mass fraction of 8%, and homogenize with a homogenizer for 15 minutes; adjust the pH to 8.5, the temperature to 55°C, add alkaline protease at 3000 units per gram of protein, and enzymolyze for 3 hours; adjust the pH to 7.0, the temperature to 50°C, add neutral protease at 3000 units per gram of protein, and enzymolyze for 2 hours. Raise the temperature to 90°C, inactivate the enzyme for 15 minutes, centrifuge at 6000×g for 10 minutes, take the supernatant, filter with a ceramic membrane with a molecular weight cutoff of 2×106U, take the intermediate clear liquid, and ultrafilter with an ultrafiltration membrane with a molecular weight cutoff of 1000U to obtain a filtrate with a molecular weight less than 1000U, spray dry, and obtain a peptide dry powder.

[0031] RP-HPLC was used to separate and purify corn oligopeptides. Chromatographic conditions: 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, UV220 nm; flow rate, 0.6 mL / min; column temperature, 40 ° C; injection volume, 40 μL. Gradient elution program: 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 were selected for collection, and organic solvents such as trifluoroacetic acid and acetonitrile were removed by nitrogen blowdown, followed by vacuum freeze drying to obtain dry powders of the 12 component peaks for later use.

[0032] Corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ were prepared by referring to the method of “Isolation, Purification and Structural Identification of Antioxidant Peptides from Corn Oligopeptides, Food Industry, Vol. 40, No. 5, 2019”.

[0033] Example 2: Corn oligopeptides promote hepatocyte proliferation

[0034] 1. Group experiment

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

[0036] L-O2 human hepatocytes (STM-CL-5770, STM Biotechnology Co., Ltd., Shanghai) in the logarithmic phase were divided into a control group and a corn oligopeptide group. 4 / mL was inoculated into a 96-well plate, 100μL of suspension was added to each well, and after incubation for 24h, the cells were treated with culture medium containing 5% fetal bovine serum and a final concentration of 0.15g / mL corn oligopeptide. Six replicate wells were set for each oligopeptide concentration, and a control well without oligopeptide was set. A zeroing well was also set. After incubation in a 37℃, 5% CO2 incubator for 5 days, 10μL of MTT solution (5mg / mL) was added to each well and incubated at 37℃ for 4h. The supernatant was discarded, 100μL / well of DMSO was added, and the crystals were fully dissolved. The light absorption value of each well was measured at 570nm.

[0037] The results are shown in Table 1. In Table 1, "*" indicates P < 0.05 and "**" indicates P < 0.01 compared with the control group. As shown in Table 1, after the four corn oligopeptides were co-cultured with hepatocytes, AYPQ and LSPY had significant differences compared with the control group, and AYPQ could promote the proliferation of L-O2 cells to the greatest extent.

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

[0039] Grouping OD value Control group 0.861±0.042 YQ 0.878±0.038 LSPY 0.942±0.066* QUR 1.269±0.045** AYLQQQ 0.915±0.035

[0040] Example 3: Effect of corn oligopeptides on improving liver fibrosis

[0041] 1. Grouping and modeling

[0042] 60 mice were randomly divided into control group, model group and corn oligopeptide group (gavage dose of 4g / kg corn oligopeptide YGPQ, LSPY, AYPQ and AYLQQQ solution), 10 mice in each group, and adaptive culture for 7 days. From the 2nd week, the control group was gavaged with olive oil on Monday and Thursday, and normal saline for the remaining 5 days; the model group was gavaged with olive oil solution (2mL / kg) containing 10% CCl4, twice a week (Monday and Thursday) from the 1st to the 8th week, and normal saline for the remaining 5 days. The corn oligopeptide group was gavaged with corn oligopeptide solution of different concentrations 7 times a week, 1w to 8w, for a total of 8w. After the experiment, fasting but not water deprivation for 12h, anesthetized mice, and liver tissue and serum were collected for subsequent index detection.

[0043] 2. HE staining

[0044] Liver specimens were fixed with 10% neutral formalin buffer for histopathological analysis. The samples were embedded in paraffin and 5 μm thick sections were cut. HE staining was used to highlight liver damage and collagen deposition, respectively, and HE staining was analyzed by image analysis software CaseViewer2.4.

[0045] 3. Detection of serum indexes and collagen indexes

[0046] When the liver is damaged, partial necrosis of liver cells occurs. Necrotic liver cells release various enzymes into the blood, such as AST, ALT and AKP, which increases the levels of AST, ALT and AKP in the blood. Therefore, the levels of AST, ALT and AKP in serum have become a commonly used indicator for evaluating liver damage.

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

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

[0049] Grouping 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 YQ 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* QUR 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 mouse serum are shown in Table 2. In Table 1, compared with the model group, "*" indicates P < 0.05 and "**" indicates P < 0.01.

[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 oral administration of corn oligopeptides could reverse the abnormal increase of AST, ALT and AKP in serum. Corn oligopeptide AYPQ could simultaneously reduce the abnormal expression of AST, ALT and AKP in the serum of model mice, thus proving that corn oligopeptide AYPQ could alleviate CCl4-induced liver injury in mice at the serological level.

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

[0053] Total RNA was extracted from tissue samples using a total RNA extraction kit for reverse transcription polymerase chain reaction analysis. Concentration and purity were measured using a Nanodrop 2000 spectrophotometer. It was then reverse transcribed using All-In-One 5X RT Master-Mix and converted into cDNA. Primers were synthesized by Shanghai Sanggong Bioengineering Co., Ltd. and then PCR was performed using MegaFi TM RT-PCR was performed using the Fidelity 2X PCR MasterMix Kit and the 7900 Real-Time PCR System. Expression levels were normalized 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-α-F: 5'-CCCTCACACTCAGATCATCTTCT-3', SEQ ID NO: 5

[0059] TNF-α-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] Grouping Relative expression level of IL-6 mRNA Relative expression level of TNF-α mRNA Control group 0.51±0.08 0.39±0.06 Model Group 1.25±0.36 0.72±0.05 YQ 1.09±0.24 0.68±0.04 LSPY 1.13±0.18 0.70±0.03 QUR 0.66±0.13** 0.43±0.04** AYLQQQ 0.91±0.54 0.69±0.06

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

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

[0064] As shown in Table 3, the mRNA and protein levels of TNF-α and IL-6 in the liver tissue of the model group were significantly increased (P < 0.05). After intervention with corn oligopeptide AYPQ, the levels of inflammatory factors in the liver tissue of mice were significantly reduced, thereby improving the inflammatory response associated with liver fibrosis. This shows that the present invention confirms that corn oligopeptide AYPQ may improve CCl4-induced mouse liver fibrosis 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 the main component of the extracellular matrix. Their high expression is closely related to liver fibrosis and is an important indicator reflecting the progress of liver fibrosis and liver cell damage.

[0067] The frozen liver tissue was weighed and homogenized in cold lysis buffer containing protease inhibitors. The liver tissue homogenate was centrifuged at 12000r / min for 20min-30min 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 (Ⅳ-C, Nanjing Jiancheng Bioengineering Institute) were used to detect the content of laminin, hyaluronic acid, and type Ⅳ collagen in the liver tissue of each group of mice.

[0068] Table 4 Effects of corn oligopeptides on LN, HA, and IV-C in mouse liver (x±s, n=10)

[0069] Grouping 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 YQ 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 QUR 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 levels of LN, HA, and IV-C in the liver tissue of the model group were significantly increased (P < 0.05). After intervention with corn oligopeptide AYPQ, the levels of LN, HA, and IV-C in the liver tissue of mice were significantly reduced, thereby reducing the collagen deposition in the liver of CCl4-induced liver fibrosis mice to improve liver fibrosis. This shows that the present invention confirms that corn oligopeptide AYPQ may improve liver fibrosis by reducing collagen deposition in the liver, while corn oligopeptides YGPQ, LSPY, and AYLQQQ cannot.

[0071] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing corn oligopeptide, characterized in that: include: S1: prepare an aqueous solution containing corn yellow powder and homogenize it using a homogenizer; S2: hydrolyzing the homogenized solution obtained in step S1 with alkaline protease; S3: The enzymatic solution obtained in step S2 is further enzymatically hydrolyzed with a neutral protease; S4: inactivate the enzyme in the enzymatic hydrolyzate obtained in step S3, centrifuge, and collect the supernatant; S5: The supernatant obtained in step S4 is filtered through a ceramic membrane and a 1000U ultrafiltration membrane, and the obtained filtrate is spray-dried to obtain a peptide dry powder; S6: The peptide dry powder obtained in step S5 is separated and purified by RP-HPLC, and the component peaks are collected to obtain corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ.

2. The preparation method according to claim 1, characterized in that: In step S1, corn yellow powder is dissolved in distilled water with a mass fraction of 8%, and homogenized with a homogenizer for 15 minutes.

3. The preparation method according to claim 1, characterized in that: In step S2, the pH is adjusted to 8.5, the temperature is 55°C, and alkaline protease is added at an enzyme amount of 3000 units per gram of protein, and enzymolysis is carried out for 3 hours.

4. The preparation method according to claim 1, characterized in that: In step S3, the pH is adjusted to 7.0, the temperature is 50°C, and neutral protease is added at an enzyme amount of 3000 units per gram of protein, and enzymolysis is carried out for 2 hours.

5. The preparation method according to claim 1, characterized in that: In step S4, the temperature was raised to 90°C, the enzyme was inactivated for 15 min, centrifuged at 6000×g for 10 min, and the supernatant was collected.

6. The preparation method according to claim 1, characterized in that: In step S5, the solution is filtered with a ceramic membrane having a molecular weight cutoff of 2×106U, the intermediate clear liquid is taken, and ultrafiltration is performed with an ultrafiltration membrane having a molecular weight cutoff of 1000U to obtain a filtrate having a molecular weight less than 1000U, which is spray-dried to obtain a peptide dry powder.

7. The preparation method according to claim 1, characterized in that: In step S6, the chromatographic conditions are: 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 220nm; flow rate, 0.6mL / min; column temperature, 40°C; injection volume, 40μL; gradient elution program: 0-10min, 0-5% mobile phase B; 10-25min, 5%-9% mobile phase B; 25-35min, 9%-15% mobile phase B; 35-55min, 15%-15% mobile phase B; 55-100min, 15%-40% mobile phase B; 100-120min, 40%-80% mobile phase B; select 12 main component peaks for collection, remove organic solvents such as trifluoroacetic acid and acetonitrile through a nitrogen blowdown instrument, and then vacuum freeze-dry to obtain dry powders of the 12 component peaks for use.

8. Use of the corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared according to claim 1 in the preparation of preparations for improving liver fibrosis.

9. Use of the corn oligopeptides YGPQ, LSPY, AYPQ and AYLQQQ prepared according to claim 1 in the preparation of alcohol sobering and liver protecting preparations.

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