Preparation method and application of corn oligopeptide with functions of resisting drunkenness and protecting liver

Abstract

The invention provides a method for preparing corn peptide, which comprises the following steps of: 1) performing enzymolysis on corn protein powder by using protease alcalase, centrifuging and collecting precipitate; 2) suspending the precipitate with water, performing enzymolysis with papain and trypsin, centrifuging, and taking supernate; and 3) concentrating, decolorizing and drying the supernate to obtain faint yellow powder. The invention also provides the corn peptide prepared by the method, and the corn peptide has the functions of resisting drunkenness and protecting the liver.

Description

Technical Field

[0001] The present invention relates to corn peptides, especially corn oligopeptides with anti-hangover and liver protection functions. The present invention also relates to a preparation method of the corn peptides. Background Art

[0002] Corn is a traditional crop in China, and its output accounts for about one-fifth of the grain output, ranking second in the world. For a long time, the structure of corn processing products in China has been single, the utilization rate of raw materials is low, and the processing depth is insufficient, resulting in a vicious cycle in the entire industry. With the rapid development of the starch industry, there are more than 100 processing products using corn starch as the raw material, which are widely used in industries such as food, medicine, chemical industry, textile and papermaking. Producing starch from corn has low cost and high quality, and it is one of the starches with the best chemical composition, with a purity of up to 99.5%. According to the research results, 67 kg of starch can be obtained from every 100 kg of corn. At present, wet corn starch production is adopted in China. After starch is extracted by wet processing, there are many utilizable nutrients in the waste residue, such as corn protein, corn yellow pigment, etc. In China, these by-products are mainly discharged or used as feed, which not only causes environmental pollution but also wastes protein resources.

[0003] Corn protein powder is the main by-product of wet corn starch production, containing about 60% protein, mainly prolamin, glutelin and globulin. Due to its complex composition, rough taste, extremely poor water solubility, etc., its application in the food industry is seriously affected. Bioactive peptides are a class of peptide compounds with a molecular weight less than 6000 Da and beneficial or physiological effects on the life activities of organisms. According to research findings, when the protein ingested by the human body is hydrolyzed by various enzymes in the digestive tract, it is not only absorbed in the form of amino acids, but more is directly absorbed in the form of small-molecule active peptides of proteins. At the same time, the protease in the digestive enzymes of people with low immunity and people prone to fatigue will be reduced a lot, and they cannot decompose macromolecular proteins in food, so they cannot absorb proteins, resulting in a lack of human proteins. As a small-molecule protein, protein peptides can be directly absorbed by the human body. Therefore, through certain technologies, corn protein can be decomposed into small-molecule polypeptides that are easily digested and absorbed by the human body. Corn oligopeptide is a new resource food approved by the Ministry of Health. It is a small-molecule polypeptide extracted from corn protein powder using bioengineering directional enzymatic hydrolysis technology, which can improve the economic value of corn and promote the healthy development of corn in the food processing industry.

[0004] Chinese Patent Application CN102174626A discloses a method for preparing corn peptides. The preparation process includes adding ethanol to corn protein powder, performing enzymatic hydrolysis in the alcohol phase, then evaporating part of the ethanol and adding an equal amount of water, adding a composite enzyme to continue enzymatic hydrolysis in the aqueous phase, then centrifuging the hydrolyzate, performing ultrafiltration fractionation and anion-cation exchange desalting, and finally concentrating and drying it to obtain corn peptide dry powder. Using ethanol as a solvent for extraction has certain limitations in actual production, and ethanol needs to be evaporated later, resulting in a complex preparation process.

[0005] Chinese Patent Application CN107365819A discloses a corn hangover peptide and a method for preparing the same. The method includes the following steps: taking corn protein powder, adding water and starch, then inoculating Aspergillus oryzae and placing it in an incubator for cultivation; after taking it out, shearing for 2 - 5 minutes, and stirring at 30 - 40 °C for 1 - 2 hours to extract crude enzyme; taking corn protein powder, adding water, alkaline protease and crude enzyme, adjusting to the required pH value, performing enzymatic hydrolysis for 4 - 6 hours, and then centrifuging. The supernatant is the corn hangover peptide solution. It uses corn protein powder as the nitrogen source of the basal medium to induce Aspergillus oryzae to secrete protease, and adds starch to induce Aspergillus oryzae to secrete amylase. The produced crude enzyme has high protease activity and starch hydrolase activity, and can efficiently hydrolyze corn protein powder. This method requires self-fermentation to prepare the enzyme, which takes a long time and has low efficiency.

[0006] Therefore, there is still a need in the art for a simple and low-cost method for preparing corn peptides. Summary of the Invention

[0007] In one aspect, the present invention provides a method for preparing corn peptides, including:

[0008] 1) enzymatically hydrolyzing corn protein powder with protease alcalase, and collecting the precipitate after centrifugation; and

[0009] 2) suspending the precipitate in water and then enzymatically hydrolyzing it with papain and trypsin, and taking the supernatant after centrifugation.

[0010] In some embodiments, the enzymatic hydrolysis in step 1) is carried out at 60 - 70 °C, preferably 65 °C; the enzymatic hydrolysis time does not exceed 1 hour, preferably 40 minutes.

[0011] In some embodiments, the enzymatic hydrolysis in step 2) is carried out at 35 - 45 °C, preferably 40 °C; the enzymatic hydrolysis time is 4 - 5 hours, preferably 4.5 hours.

[0012] In some embodiments, the method further includes: 3) concentrating, decolorizing and drying the supernatant to obtain a light yellow powder.

[0013] In some embodiments, the dosage of protease alcalase is 2.5% of the mass of the corn protein powder.

[0014] In some embodiments, the dosage of papain is 1% of the mass of the corn protein powder.

[0015] In some embodiments, the dosage of trypsin is 0.5% of the mass of the corn protein powder.

[0016] In some embodiments, the method further includes inactivating the enzyme of the supernatant before step 3).

[0017] In some embodiments, the method further includes filtering the supernatant with a filter membrane with a pore size of 20 nm before step 3), and collecting the filtrate.

[0018] On the other hand, the present invention provides corn peptides prepared by the above method.

[0019] On the other hand, the present invention provides the use of the above corn peptides in the preparation of foods, beverages or drugs for anti-hangover and / or hangover-relieving and / or liver-protecting.

[0020] The method for preparing corn peptides provided by the present invention is simple in operation and low in cost, and the prepared corn peptides have anti-hangover, hangover-relieving and / or liver-protecting functions. Description of the Drawings

[0021] Figure 1 It is a roadmap for the anti-hangover experiment of corn peptides.

[0022] Figure 2 It shows the results of the ethanol content in the mouse serum. ***, compared with the model group, p < 0.001.

[0023] Figure 3 It shows the results of the detection of ALT and AST activities. ***, compared with the blank group, p < 0.001. Compared with the model group: #, p < 0.05; ##, p < 0.01; , p < 0.001.

[0024] Figure 4 It shows the results of the detection of ADH and ALDH activities. ***, compared with the blank group, p < 0.001. Compared with the model group: #, p < 0.05; ##, p < 0.01; , p < 0.001.

[0025] Figure 5 It shows the results of the detection of MDA content. ***, compared with the blank group, p < 0.001. Compared with the model group: ##, p < 0.01; , p < 0.001.

[0026] Figure 6Showed the results of SOD activity detection. ***, compared with the blank group, p < 0.001. Compared with the model group: #, p < 0.05; ##, p < 0.01; , p < 0.001.

[0027] Figure 7 Showed the results of GSH activity detection. ***, compared with the blank group, p < 0.001. Compared with the model group: ##, p < 0.01; , p < 0.001.

[0028] Figure 8 Showed the representative results of H&E staining of mouse liver tissue sections.

[0029] Figure 9 Showed the representative results of H&E staining of mouse stomach tissue sections.

[0030] Figure 10 Showed the representative results of H&E staining of mouse kidney tissue sections.

[0031] Figure 11 Showed the representative results of AB-PAS staining of mouse colon tissue sections. Detailed implementation methods

[0032] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0033] In this article, the term "and / or" should be understood to mean either any one of the alternatives or both alternatives.

[0034] In this article, the terms "comprising" and "including" generally mean including the specifically designated elements, but do not exclude other elements, and also include the cases consisting of these elements..

[0035] In this article, the term "about" generally means varying within the range of 0.5% - 10% above or below the specified value, for example, varying within the range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0036] Unless otherwise specified, the percentages and ratios herein are all percentages by weight (or mass).

[0037] In this study, corn protein was enzymatically hydrolyzed by a complex enzyme, which changed some functional properties of corn protein. The developed corn oligopeptides have the effect of preventing and reducing ethanol toxicity, can improve the anti-alcohol effect by enhancing the activities of the anti-alcohol enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and can also enhance the antioxidant capacity and reduce alcoholic liver injury. The corn oligopeptides of the present invention have the effects of reducing the degree of drunkenness, shortening the drunkenness time, and protecting the liver. In particular, the inventors found that before treatment with other enzymes (complex enzyme), corn protein was treated with protease alcalase for a short time (not exceeding 1 h), the precipitate was collected after centrifugation, and then degraded with other enzymes, and the obtained corn peptides had better anti-alcohol and liver-protecting functions. In this study, the anti-alcohol and liver-protecting effects of the isolated and purified corn peptides were studied through the corn protein hydrolysis process and animal experiments to realize the optimization and high-value utilization of corn resources.

[0038] The following specific examples are used to further illustrate the present invention.

[0039] Example 1 Preparation of Corn Oligopeptides

[0040] The preparation process of the corn oligopeptides of the present invention is as follows.

[0041] Enzymatic hydrolysis: Take edible corn protein powder, add purified water according to the ratio of solid to liquid of 1:10, and stir evenly at 85 °C; adjust the pH to 8.5 with sodium hydroxide, wait until the temperature drops to 60 °C, add protease alcalase according to 2.5% of the mass of corn protein powder, mix evenly and react at 65 °C for 40 min. Centrifuge at 5000 g for 10 min and collect the precipitate. Add purified water to the obtained precipitate according to the ratio of solid to liquid of 1:10, stir evenly and adjust the pH to 8.0, add papain and trypsin according to 1% and 0.5% of the mass of corn protein powder respectively, and carry out enzymatic hydrolysis reaction at 40 °C for 4.5 h with stirring, and take the supernatant after centrifuging at 10,000 g for 30 min. Heat the supernatant to 100 °C and inactivate the enzyme for 20 s.

[0042] Membrane separation and concentration: After the supernatant is filtered through a ceramic membrane (pore size 20 nm), it is concentrated at 70 °C until the solid content in the feed liquid is about 25%.

[0043] Decolorization and drying: Add activated carbon according to 2% of the solid content, keep the temperature at 80 °C, centrifuge to take the supernatant after decolorizing for 30 min, and carry out spray drying on the supernatant, with the inlet air temperature of 180 °C and the outlet air temperature of 80 °C, and collect the powder to obtain corn oligopeptide powder (light yellow).

[0044] After detection, the proportion of protein hydrolysates with a relative molecular mass less than 1000 Da in the corn oligopeptide powder is 86.4%. Example 2 Preparation of Corn Oligopeptides (comparative example)

[0045] In this example, the preparation of corn oligopeptide powder is basically carried out in the same way as in Example 1, except that the centrifugation step at 5000g is omitted. The specific enzymatic hydrolysis process is as follows.

[0046] Enzymatic hydrolysis: Take edible corn protein powder, add purified water according to the ratio of solid-liquid ratio of 1:10, and stir evenly at 85°C; adjust the pH to 8.5 with sodium hydroxide. After cooling to 60°C, add protease alcalase according to 2.5% of the mass of corn protein powder, mix evenly and react at 65°C for 40 min. Adjust the pH to 8.0, and add papain and trypsin according to 1% and 0.5% of the mass of corn protein powder respectively. Enzymatic hydrolysis reaction is carried out at 40°C for 4.5 h with stirring, and the supernatant is taken after centrifugation at 10,000g for 30 min. Heat the supernatant to 100°C and inactivate the enzyme for 20 s.

[0047] After detection, the proportion of protein hydrolysates with a relative molecular mass less than 1000 Da in the prepared corn oligopeptide powder is 87.5%.

[0048] Preparation of corn oligopeptide in Example 3 (comparative example)

[0049] In this example, the preparation of corn oligopeptide powder is basically carried out in the same way as in Example 1, except that the treatment time of protease alcalase is extended to 2 h. The specific enzymatic hydrolysis process is as follows.

[0050] Enzymatic hydrolysis: Take edible corn protein powder, add purified water according to the ratio of solid-liquid ratio of 1:10, and stir evenly at 85°C; adjust the pH to 8.5 with sodium hydroxide. After cooling to 60°C, add protease alcalase according to 2.5% of the mass of corn protein powder, mix evenly and react at 65°C for 2 h. Centrifuge at 5000g for 10 min to collect the precipitate. Add purified water to the obtained precipitate according to the ratio of solid-liquid ratio of 1:10, stir evenly and then adjust the pH to 8.0. Add papain and trypsin according to 1% and 0.5% of the mass of corn protein powder respectively. Enzymatic hydrolysis reaction is carried out at 40°C for 4.5 h with stirring, and the supernatant is taken after centrifugation at 10,000g for 30 min. Heat the supernatant to 100°C and inactivate the enzyme for 20 s.

[0051] After detection, the proportion of protein hydrolysates with a relative molecular mass less than 1000 Da in the corn oligopeptide powder is 85.3%.

[0052] Example 4 Animal experiment

[0053] In this experiment, an acute drunken mouse model was established, and different doses of corn oligopeptides were intragastrically administered. The effects on the behavioral indices (drunkenness and sobering-up time) of acute drunken mice and the pathological conditions of tissue sections (liver, stomach, kidney, colon) were observed. By detecting the ethanol content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities in the mouse serum, the contents of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), malondialdehyde (MDA) in the liver, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH), the anti-drunkenness effect and liver-protecting efficacy of corn peptides were studied. The simple experimental route is as Figure 1 shown.

[0054] Experimental materials

[0055] The corn oligopeptides prepared in the above Examples 1-3 (hereinafter also simply referred to as "corn peptides");

[0056] AST kit, ALT kit, total superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH) assay kit, malondialdehyde (MDA) assay kit, alcohol dehydrogenase (ALD) assay kit, aldehyde dehydrogenase (ALDH) assay kit: Nanjing Jiancheng Bioengineering Institute;

[0057] Ethanol content assay kit: Hangzhou Leyi Biotechnology Co., Ltd.;

[0058] BCA protein concentration assay kit: Shanghai Beyotime Biotechnology Co., Ltd.;

[0059] Phosphate buffer solution (PBS), sodium chloride (NaCl): Beijing Solarbio Science & Technology Co., Ltd.;

[0060] Paraformaldehyde: Shanghai Macklin Biochemical Co., Ltd.;

[0061] Carnoy's fixative: Wuhan Sevier Biotechnology Co., Ltd.;

[0062] 56% vol white liquor: Beijing Red Star Co., Ltd.;

[0063] RU-21 Antip Alcohol Natural Plant Extract Composite Tablets (RU-21): American Psychiatric Sciences Co., Ltd.

[0064] Male ICR mice (SPF grade), body weight 18 ± 2 g: Hangzhou Qizhen Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe) 2022-0005.

[0065] Experimental methods

[0066] In this experiment, male ICR mice weighing 20±2 g were selected. The breeding environment conditions for the mice were maintained at a room temperature of 25±2 °C, a relative humidity of 50±5%, and a 12 h / 12 h light-dark cycle. They were conventionally fed with standard rodent feed and had free access to food and water. After a one-week adaptation period, the mice were randomly divided into groups (see Table 1), with 10 mice in each group. They were gavaged with drugs corresponding to their body weights once a day for 15 days. Before the last gavage, they were fasted (but not water-deprived) for 12 h and then weighed. 30 min after gavaging with distilled water, positive drugs, and corn peptides corresponding to their body weights for the last time, except for the mice in the blank group and the normal group, the remaining groups were gavaged with 0.14 mL / 10 g·BW of Red Star Erguotou. Corn oligopeptides and the positive RU-21 drug were both dissolved in PBS, and the volume of each gavage was approximately 0.2 mL.

[0067] Table 1 Grouping of experimental animals

[0068]

[0069]

[0070] 4.1 Drunkenness and hangover time

[0071] The drunken state of the mice was observed and judged using the righting test, and the time required for the mice to lose the righting reflex (drunkenness) from gavage and the time required for the righting reflex to disappear (drunkenness) to recovery (hangover) were recorded.

[0072] The results are shown in Table 2. The drunkenness time of the model group (MC) treated only with alcohol gavage was 6.39±1.25 min, and the positive drug group was extended to 23.47±2.02 min. The drunkenness time of the mice in each group gavaged with corn peptide samples for a certain period was significantly extended compared with the model group. Among them, the high-dose groups YP-H1, YP-H2, and YP-H3 extended the drunkenness time to 16.79±1.91 min, 13.09±1.52 min, and 12.33±1.45 min respectively, indicating that each group of corn oligopeptides could increase the drunkenness time of the mice, but obvious inter-group differences were also shown (comparison of the corn oligopeptides in Example 1 with those in Examples 2 and 3, p<0.01). The corn oligopeptides prepared by the method of Example 1 had a stronger anti-drunkenness effect.

[0073] The alcohol awakening time of the model group (MC) treated with intragastric administration of alcohol was 259.79 ± 14.41 min. Compared with the model group, the awakening times of the positive drug group (PC) and the mice with different doses of corn oligopeptides were shortened. Among them, the high-dose groups YP-H1, YP-H2, and YP-H3 shortened the alcohol awakening time of the mice to 132.44 ± 17.00 min, 188.61 ± 20.03 min, and 175.67 ± 17.58 min, respectively. This indicates that corn oligopeptides can shorten the alcohol awakening time of mice and alleviate the drunken state of mice. The three corn oligopeptide samples prepared in the present invention also showed significant inter-group differences in terms of the alcohol awakening time. The corn oligopeptide prepared by the method of Example 1 had a better alcohol awakening effect (compared with the corn oligopeptides of Example 2 and 3, high-dose group, p < 0.001), and was even superior to the positive drug RU-21 at medium and high doses.

[0074] In addition, for the corn oligopeptides prepared in the present invention, among the three groups with the same sample but different doses, the high dose showed a better effect than the low and medium doses in prolonging the drunken time and shortening the awakening time of mice, and there were significant differences among the groups (p < 0.01), indicating a dose-dependent effect within a certain dose range.

[0075] Table 2 Effects of corn peptides on the alcohol awakening and drunken times of mice (mean ± standard deviation)

[0076]

[0077] 4.2 Body weight and organ indices of mice

[0078] After the start of the experiment, the body weights of the mice were weighed and recorded every day. At 6 h after alcohol administration, blood was collected from the eye socket (for the determination of biochemical indices below), and the mice were immediately sacrificed. The liver, kidney, stomach, and colon were dissected, rinsed with physiological saline (the colon was not placed in physiological saline), and the water was blotted with filter paper, and then their weights were accurately measured. The formula for calculating the organ index is as follows: Organ index (%) = Organ weight (g) / Body weight of mouse (g).

[0079] The results of body weight and liver index are shown in Table 3. Compared with the model group (MC), the liver indices of the low-, medium-, and high-dose groups of corn oligopeptides decreased, and the positive drug group (PC) and the medium- and high-dose groups showed significant decreases (p < 0.05), indicating that corn oligopeptides can, to a certain extent, present a dose-dependent prevention of alcohol-induced liver edema and reduce the damage to the liver caused by alcohol.

[0080] By comparing the differences among the groups of corn oligopeptides prepared in the present invention, it can be found that the corn oligopeptide prepared by the method of Example 1 had a better effect, and there were significant differences compared with the same-dose groups of Example 2 and 3 (p < 0.05). This is consistent with the above results of drunkenness and awakening time.

[0081] Table 3 Effects of corn peptides on body weight and liver index of mice (mean ± standard deviation)

[0082]

[0083] Since the corn oligopeptides prepared in Example 1 are superior to those prepared in Examples 2 and 3 in terms of hangover time, drunkenness time, and liver index, the following experiments were only carried out on the sample groups of Example 1 (different doses, denoted as YP-L, YP-M, and YP-H respectively).

[0084] 4.3 Determination of biochemical indexes

[0085] After blood was collected from the eyes of mice, serum was prepared, and an ethanol content assay kit was used to measure the ethanol concentration in the mouse serum. Kits for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used to detect the AST and ALT concentrations in the mouse serum. An appropriate amount of normal saline was added to the mouse liver obtained by dissection, and it was ground with a tissue homogenizer to prepare 10% mouse tissue homogenate. After centrifugation at 4000 rpm for 20 min, the supernatant was taken, and a kit was used to measure the activity in the mouse liver tissue, and the contents of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), malondialdehyde (MDA) in the mouse liver and the enzyme activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH) were detected.

[0086] 4.3.1 Ethanol content in serum

[0087] The ethanol concentration in the blood can reflect the severity of alcohol poisoning. As Figure 2 shown, compared with 3.20 ± 0.12 μmol / mL in the MC group, the alcohol concentration in the serum of the PC group decreased to 1.14 ± 0.16 μmol / mL; the serum ethanol content in the low-dose group decreased to 2.66 ± 0.39 μmol / mL, and there were extremely significant differences in the decrease of serum ethanol content in the medium- and high-dose groups of corn peptides (YP-M, YP-H); the ethanol content in the medium-dose group (YP-M) was 1.96 ± 0.21 μmol / mL; the ethanol content in the high-dose group (YP-H) decreased to 0.66 ± 0.17 μmol / mL. It can be seen that the serum ethanol content of drunken mice decreased in a dose-dependent manner with intragastric administration of corn peptides.

[0088] 4.3.2 Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)

[0089] The results of ALT activity and AST activity in different dose groups are as Figure 3As shown in the figure. In terms of ALT activity, compared with 26.74 ± 0.74 U / L in the MC group, the ALT activity in the PC group decreased extremely significantly (p < 0.001), being 19.89 ± 1.21 U / L. Compared with MC, the decrease in ALT activity in the low-dose group (YP-L) was significantly different (p < 0.05), dropping to 23.34 ± 1.28 U / L; the AST activities in the medium- and high-dose groups (YP-M, YP-H) decreased extremely significantly (p < 0.001), dropping to 19.50 ± 0.55 and 16.37 ± 0.99 U / L respectively; it can be seen that the serum ALT content in drunken mice decreased in a dose-dependent manner with intragastric administration of corn peptide.

[0090] In terms of AST activity, the value in the MC group was 44.08 ± 1.14 U / L, and the AST activity in the PC group decreased extremely significantly, being 25.99 ± 1.33 U / L. Figure 3 It shows that compared with the MC group, the AST activity in the low-dose group (YP-L) decreased to a certain extent, dropping to 39.92 ± 2.04 U / L; the AST activities in the medium- and high-dose groups (YP-M, YP-H) decreased extremely significantly (p < 0.001), dropping to 25.30 ± 0.84 and 16.38 ± 1.25 U / L respectively. This result indicates that corn peptide can improve the permeability of the hepatic cell membrane in a dose-dependent manner to a certain extent and has a certain protective effect on the liver.

[0091] 4.3.3 Activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH)

[0092] The results of ADH activity and ALDH activity in different dose groups are as Figure 4 shown. In terms of ADH activity, compared with the NC group, the alcohol dehydrogenase (ADH) activity in the normal group (NC-YP) was not significantly different (p > 0.05), and the ADH activity in the MC group was significantly different from that in the NC group (p < 0.001), indicating that the model was successfully established. Figure 4 It shows that compared with 2.81 ± 0.55 U / mgprot in MC, the ADH activities in the YP-L, YP-M, and YP-H groups all increased, increasing to 5.52 ± 1.92, 8.41 ± 1.70, and 9.90 ± 0.98 U / mgprot respectively, and the ADH activity in the YP-H group increased extremely significantly (p < 0.001).

[0093] In terms of ALDH activity, compared with the 11.56 ± 1.21 U / mgprot of the MC group, the corn peptide sample group could increase the ALDH activity. The YP-L, YP-M, and YP-H groups were increased to 12.45 ± 1.66, 14.24 ± 1.71, and 17.84 ± 2.16 U / mgprot respectively. The YP-H group significantly increased the ALDH activity (p < 0.01). The results showed that corn peptides could increase the activities of ADH and ALDH in the liver of acutely alcohol-consuming mice, and there was a certain dose-effect relationship with the activities of ADH and ALDH in the mouse liver.

[0094] 4.3.4 Determination of malondialdehyde content

[0095] Alcohol poisoning can directly produce toxic effects on cells, causing cells to generate a large amount of free radicals, resulting in oxidative stress, damaging tissues and cells, and making the liver function indicators, body weight, and liver index in the serum abnormal. The disorder of lipid metabolism in the body can cause fatty liver degeneration in the liver, leading to enhanced lipid peroxidation and more serious oxidative stress. Malondialdehyde (MDA) is one of the metabolites in the lipid peroxidation process of cell membrane degradation. Therefore, MDA is an important indicator reflecting the level of oxidative stress in organisms.

[0096] As Figure 5 shown, compared with the MDA content (1.52 ± 0.09 nmol / mgprot) of the NC group, the MDA content (1.59 ± 0.08 nmol / mgprot) of the normal group NC-YP had no significant difference (p > 0.05), while the MDA activity of the MC group increased extremely significantly (p < 0.001) to 3.66 ± 0.13 nmol / mgprot, indicating that the modeling was successful. Compared with the MC group, the MDA content of the PC group decreased extremely significantly (p < 0.001) to 2.63 ± 0.06 nmol / mgprot. Corn oligopeptides could also significantly reduce the MDA content in the liver tissue of mice. All three doses extremely significantly reduced the MDA content in the liver (p < 0.001), and the low, medium, and high dose groups were 2.99 ± 0.21, 2.71 ± 0.08, and 2.19 ± 0.07 nmol / mgprot respectively. It was shown that corn peptides had a certain protective effect on mice with alcoholic liver injury.

[0097] 4.3.5 Determination of superoxide dismutase (SOD) activity

[0098] Superoxide dismutase (SOD) is an antioxidant metalloenzyme existing in organisms. It can catalyze the dismutation of superoxide anion free radicals to generate oxygen and hydrogen peroxide, playing a crucial role in the oxidation and antioxidant balance of the body.

[0099] As Figure 6As shown, compared with the SOD enzyme activity of 87.88 ± 2.58 U / mgprot in the NC group, the SOD activity of 84.60 ± 1.97 U / mgprot in the normal group (NC-YP) had no significant difference (p > 0.05). However, the SOD activity in the MC group decreased extremely significantly (p < 0.001), dropping to 66.76 ± 2.73 U / mgprot, proving that the model was successfully established. Compared with the MC group, the SOD activity in the PC group increased significantly (p < 0.01), rising to 75.14 ± 2.07 U / mgprot; the MDA content in the YP-L group changed significantly, rising to 74.41 ± 2.69 U / mgprot; medium and high doses (YP-M, YP-H) could significantly increase the SOD activity in the liver tissue of mice, and the differences were extremely significant (p < 0.001), increasing to 80.95 ± 1.79 and 84.16 ± 1.95 U / mgprot respectively; they were higher than the positive drug group and close to the normal group, indicating that they had a certain protective effect on the livers of mice. The results showed that corn oligopeptides at different doses could all increase the content of SOD in the liver tissue of mice with alcoholic liver injury, had a certain liver protection effect, and showed a dose-dependence.

[0100] 4.3.6 Determination of Glutathione Peroxidase (GSH) Activity

[0101] Glutathione peroxidase (GSH) is an important antioxidant and free radical scavenger in the body. As Figure 7 shown, compared with the NC group (45.78 ± 2.11 μmol / gprot), the GSH activity in the NC-YP group (44.85 ± 1.72 μmol / gprot) had no significant difference (p > 0.05). However, the GSH activity in the MC group decreased extremely significantly (p < 0.001), with a value of 26.18 ± 1.46 μmol / gprot, indicating that the model was successfully established. Compared with the MC group, the GSH activity in the PC group increased significantly (p < 0.01), with a value of 34.54 ± 1.94 μmol / gprot; the GSH activity in the low-dose corn peptide group (YP-L) was 27.48 ± 1.43 μmol / gprot, showing a slight increase, but there was no significant difference compared with the MC group (p > 0.05); the GSH activity in the medium-dose corn peptide group (YP-M) increased significantly (p < 0.01), being 34.98 ± 2.70 μmol / gprot; the GSH activity in the high-dose corn peptide group (YP-H) increased extremely significantly (p < 0.001), being 41.96 ± 1.77 μmol / gprot.

[0102] The results showed that corn oligopeptides at different doses could all increase the content of GSH in the liver tissue of mice with alcoholic liver injury to varying degrees, relieve the liver injury caused by alcohol in mice to a certain extent, and showed a dose-dependence.

[0103] 4.4. Histopathological Observation of Tissue Morphology

[0104] After dissecting the obtained mouse liver, kidney and stomach samples, a piece was quickly taken and fixed in 4% formalin for 24 h, and the colon was fixed in Carnoy's fixative for 24 h. Then it was dehydrated in a series of gradient ethanol from 70% to 100%, and embedded in paraffin using tissue embedding technology. The tissue was cut into 4-μm sections with a microtome. The liver, kidney and stomach tissues were stained with hematoxylin and eosin, and the colon was stained with AB-PAS. Photographs were taken using an optical microscope equipped with a camera.

[0105] 4.4.1 Liver Tissue Morphology

[0106] Figure 8 The representative results of H&E staining of mouse liver tissue sections are shown. In the blank group (NC) and the normal group (NC-YP) without alcohol gavage but gavaged with corn oligopeptides, the liver tissue cells had normal morphology, uniform size, and neat arrangement. The hepatic cord structure (red arrow) surrounded the central vein and was arranged radially. The hepatic sinusoids (yellow arrow) were clear, and the separation between the liver cell nucleus and cytoplasm was obvious, without inflammatory infiltration. In the model group (MC), the arrangement of mouse liver cells was chaotic, the hepatic cord structure was disordered, and there was obvious inflammatory infiltration (blue arrow). In the positive group (PC) and the low, medium, and high dose groups of corn oligopeptides alone, there was no obvious inflammatory infiltration, and the arrangement of liver cells was relatively neat, showing a radial shape. Compared with the low and medium dose groups (YP-L, YP-M), the hepatic cord structure in the high dose group (YP-H) was more neat, and the hepatic sinusoids were relatively clearer. The results showed that corn oligopeptides could improve the pathological condition of alcoholic liver injury, reduce the inflammation of the damaged liver tissue, make the arrangement of liver cells tend to be neat, and with the increase of the dose, the recovery effect was more ideal.

[0107] 4.4.2 Stomach Tissue Morphology

[0108] Figure 9 The representative results of H&E staining of mouse stomach tissue sections are shown. In the blank group (NC) and the normal group (NC-YP) without alcohol gavage but gavaged with corn oligopeptides, the morphology of the gastric antrum mucosal epithelial cells was normal, and the glandular structure (black arrow) was arranged neatly, without mucosal exfoliation, congestion, edema, or inflammatory cell infiltration. In the model group (MC), surface epithelial damage and exfoliation (black arrow) were seen in the gastric antrum mucosa. In the positive group (PC) and the low and medium dose groups of corn oligopeptides, the exfoliation of gastric antrum mucosal epithelial cells decreased, and no disorder of glandular structure was seen. In the high dose group, the glandular structure was arranged neatly and the gastric antrum mucosa of the mouse was basically normal, indicating that within a certain range, the morphological recovery of mouse stomach tissue was better with the increase of the corn oligopeptide dose.

[0109] 4.4.3 Kidney Tissue Morphology

[0110] Figure 10 The representative results of H&E staining of mouse kidney tissue sections are shown. In the blank group (NC) and the normal group (NC-YP) of mice that were not given alcohol and were gavaged with corn oligopeptides, the glomeruli (black arrows) had normal structures, the renal tubules (yellow arrows) had clear structures, the renal tubular epithelial cells were intact and arranged regularly. In the model group (MC), the volume of the glomeruli in the mice increased, the renal corpuscles shrank, and the cytoplasmic boundaries were unclear. In the positive group (PC) and the low, medium, and high-dose groups of corn oligopeptides, compared with the model group, there were obvious improvements. Moreover, as the dose of corn peptide increased, the arrangement of renal tubular epithelial cells became more regular and tended to be complete, and the cytoplasmic boundaries of the glomeruli became clearer.

[0111] 4.4.4 Colon tissue morphology

[0112] Goblet cells in the colon maintain the integrity of the intestinal mucosa by secreting mucin, preventing dysbacteriosis from leading to aggravated liver injury. AB-PAS staining can detect various mucins, and the staining results are as Figure 11 shown. In the blank group (NC) and the normal group (NC-YP) of mice that were not given alcohol and were gavaged with corn peptides, the number of goblet cells was large and relatively plump. In the model group (MC), the intestinal mucosa of the mice was damaged, and the number of goblet cells decreased significantly. In the positive group (PC) and the low, medium, and high-dose groups of corn peptides, compared with the model group, the number of goblet cells increased significantly, and it increased in a dose-dependent manner with the increase of corn peptide dose. The results show that corn oligopeptides can, to a certain extent, reduce the damage of alcohol to the colon mucosa and thus relieve the damage to the liver.

[0113] In this paper, the in vivo anti-alcoholism activities of various samples were evaluated by establishing animal models, and their possible mechanisms of action were explored. Some conclusions are as follows:

[0114] 1. Treat corn protein powder with protease alcalase for a short time (within 1 h), then centrifuge, and take the precipitate for further enzymatic hydrolysis treatment, which can enhance the anti-alcoholism and / or hangover effects of the obtained corn oligopeptides.

[0115] 2. After 15 days of corn peptide gavage intervention, 56% vol white wine at 0.014 mL / g was given according to the body weight of the mice, and an in vivo anti-alcoholism animal model was successfully established.

[0116] 3. Regarding the effects of corn peptides on the drunkenness and hangover of mice, compared with the model group, gavage of mice with corn peptide products could all prolong the drunkenness latency period and shorten the hangover time. In the medium and high-dose groups, the drunkenness latency period was extremely significantly prolonged, and the hangover time was extremely significantly shortened, with statistical significance (p < 0.001).

[0117] 4. Intragastric administration of corn peptides can improve the pathological condition of alcoholic liver injury, reduce liver tissue inflammation, and with the increase of dose, the recovery effect is more ideal; intragastric administration of corn peptides can reduce the exfoliation of gastric antrum mucosal epithelial cells and alleviate the disorder of glandular structure. The gastric antrum mucosa of mice in the high-dose group is basically normal, indicating that within a certain range, the morphological recovery of mouse gastric tissue is better with the increase of dose; compared with the model group, the low, medium, and high-dose groups of corn peptides have obvious improvements, and with the increase of dose, the arrangement of renal tubular epithelial cells gradually becomes complete, and the cytoplasmic boundary of glomeruli is more distinct; corn peptides can, to a certain extent, reduce the damage of alcohol to the colonic mucosa and relieve the damage to the liver accordingly. In summary, intragastric administration of corn peptides can dose-dependently reduce the damage of alcohol to various tissues to achieve the effect of anti-intoxication.

[0118] 5. Regarding the effect of corn peptides on ethanol metabolism in the livers of mice, intragastric administration of different doses of corn peptides can significantly reduce the ethanol content, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the bodies of alcohol-gavaged mice. At the same time, it can increase the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the livers of acutely alcohol-consuming mice, and shows a dose-dependent relationship.

[0119] 6. The results of the effect of corn peptides on the antioxidant capacity of mouse liver tissues show that it can dose-dependently scavenge reactive oxygen free radicals, inhibit lipid peroxidation reactions, and then reduce the malondialdehyde (MDA) content in the bodies of drunken mice, increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH) in the body, reduce its oxidative stress, and relieve liver damage caused by alcohol, thus achieving the function of anti-intoxication.

Claims

1. A method for preparing corn peptides, comprising: 1) enzymatically hydrolyzing corn gluten powder with protease alcalase, and collecting the precipitate after centrifugation; 2) suspending the precipitate with water, then enzymolyzing it with papain and trypsin, and collecting the supernatant after centrifugation; as well as 3) The supernatant is concentrated, decolorized and dried to obtain a light yellow powder.

2. The method according to claim 1, wherein the enzymolysis in step 1) is carried out at 60-70°C, preferably 65°C; the enzymolysis time does not exceed 1 hour, preferably 40 minutes.

3. The method according to claim 1 or 2, wherein the enzymolysis in step 2) is carried out at 35-45°C, preferably 40°C; and the enzymolysis time is 4-5h, preferably 4.5h.

4. The method according to any one of claims 1 to 3, wherein the amount of the protease alcalase is 2.5% by mass of the corn gluten meal.

5. The method according to any one of claims 1 to 4, wherein the amount of papain used is 1% by mass of the corn gluten meal.

6. The method according to any one of claims 1 to 5, wherein the amount of trypsin used is 0.5% of the mass of the corn gluten meal.

7. The method according to any one of claims 1 to 6, wherein the method further comprises subjecting the supernatant to an enzyme inactivation treatment before step 3).

8. The method according to any one of claims 1 to 7, wherein the method further comprises filtering the supernatant with a filter membrane with a pore size of 20 nm before step 3) to collect the filtrate.

9. Corn peptide prepared by the method according to any one of claims 1 to 8.

10. Use of the corn peptide according to claim 9 in the preparation of food, beverage or medicine for preventing drunkenness and / or sobering up and / or protecting the liver.

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