Determination method for antioxidant activity of soybean bioactive peptide

Abstract

The invention belongs to the technical field of antioxidant activity products, and provides a method for determining the antioxidant activity of soybean bioactive peptide. Comprising the following steps: (1) a first step of feeding; (2) a second step of sample collection and treatment; (3) determining antioxidant enzymes and immune indexes: superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, nitric oxide and inducible nitric oxide synthase; gene expression determination: determining expression of hepatopancreas antioxidation and immune related genes of the aquatic animals by adopting real-time fluorescence quantitative analysis; observation of the hepatopancreas tissue structure: taking out and fixing the hepatopancreas tissue of the aquatic animals, carrying out gradient dehydration, degreasing and transparentizing, immersing in wax, embedding, spreading, dyeing, sealing, observing under an optical microscope, and shooting; and (4) processing the data. Compared with the prior art, the method has the advantages that the activity of the antioxidant enzyme and the gene expression quantity of the antioxidant enzyme are greatly improved, and the growth of aquatic animals is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant activity products, and in particular relates to a method for determining the antioxidant activity of soybean bioactive peptides. Background Art

[0002] At present, antioxidant enzymes can fight against oxidative stress caused by intracellular reactive oxygen species and maintain a good balance between intracellular oxidants and antioxidants. Once the production of reactive oxygen species exceeds the degradation, the accumulation of reactive oxygen species will occur, leading to lipid peroxidation, and its decomposition products include malondialdehyde (Sies, 1991; Khan and Khan, 2021). As the concentration of malondialdehyde increases, its damage to the body will increase.

[0003] Studies have shown that antioxidant enzymes such as superoxide dismutase may be regulated by key genes of the NF-κB signaling pathway, such as tlr, dorsal, imd, and relish (Yang et al., 2023). The tlr and imd signaling pathways play an indispensable role in regulating the response of invertebrates to resist bacterial and viral infections and invasion of the body (Li and Xiang, 2013a, 2013b), and are also upstream pathways for activating nitric oxide synthase activity (Lv et al., 2021). The above indicators are important indicators for measuring the level of antioxidant and immune levels in the body. In addition, as an important detoxification and metabolic organ, the changes in the structure of the hepatopancreas can be used as an important indicator to measure whether the physiological function of crustaceans is normal (Zheng Shanjian, 2007); however, low fish meal feeds in existing technologies will significantly inhibit the antioxidant capacity and immune status of aquatic animals, which is not conducive to the growth of aquatic animals.

[0004] To this end, the present invention provides a method for determining the antioxidant activity of soybean bioactive peptides. Summary of the invention

[0005] In view of the above problems existing in the prior art, the object of the present invention is to provide a method for determining the antioxidant activity of soybean bioactive peptides, which has the advantage of improving the activity of antioxidant enzymes and their gene expression levels.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The method for determining the antioxidant activity of soybean bioactive peptides comprises the following steps:

[0008] (1) The first step, feeding: taking an appropriate amount of aquatic animals and dividing them into six groups of breeding barrels to form a reference group, a comparative example, Example 1, Example 2, Example 3 and Example 4, respectively, adding different amounts of growth active peptides in the feed to the aquatic animals in Example 1, Example 2, Example 3 and Example 4;

[0009] (2) Step 2, sample collection and processing: After the breeding experiment, each group of experiments was fasted for 24 hours. After the hemolymph, hepatopancreas and other samples of aquatic animals were collected, the hepatopancreas tissue of aquatic animals was randomly collected from each breeding tank and fixed with 4% neutral formaldehyde. Four tubes of samples were collected in each group. The hepatopancreas samples were stored in a -20°C refrigerator for testing;

[0010] (3) Step 3, determination of antioxidant enzymes and immune indexes: First, the protein content in 10% hepatopancreas homogenate was determined; then various kits were used to detect antioxidant enzyme activities and immune indexes in the hepatopancreas, including superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, nitric oxide, and inducible nitric oxide synthase;

[0011] Gene expression determination: Real-time fluorescence quantitative analysis was used to determine the expression of antioxidant and immune-related genes in the hepatopancreas of aquatic animals;

[0012] Observation of hepatopancreas tissue structure: including the removal and fixation of hepatopancreas tissue of aquatic animals, gradient dehydration, defatting and transparency, wax immersion, embedding, slide spreading, staining, sealing, observation under optical microscope, and filming;

[0013] (4) Step 4: Data processing: Use Excel software to record data and calculate the average value of each breeding bucket for significant difference analysis; use SPSS20.0 software to analyze data; first use Levene's test to test the homogeneity of variance, and use one-way analysis of variance to compare the effects of different feeds; Duncan's multiple comparisons were used to compare the effects of different feeds, where p < 0.05 was considered significant; finally, the analyzed data were presented as mean ± standard error.

[0014] As a preferred technical solution of the present invention, the growth active peptide is a light brown paste obtained by fermenting soybeans as a substrate.

[0015] As a preferred technical solution of the present invention, the optical microscope used in step (3) is a 400× optical microscope.

[0016] As described above, the present invention relates to a method for determining the antioxidant activity of soybean bioactive peptides, which has the following beneficial effects:

[0017] The present invention applies to adding different levels of soybean bioactive peptides to fish meal feed and setting a normal fish meal reference group, which can show that soybean bioactive peptides can significantly reduce the malondialdehyde content in the hepatopancreas of aquatic animals, and it has the advantage of increasing the activity of antioxidant enzymes and their gene expression, which is helpful for the growth of aquatic animals.

[0018] The present invention will be further described below in conjunction with specific implementation modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the effects of soybean bioactive peptides on the expression of antioxidant enzyme related genes in the present invention;

[0020] Figure 2 This is an analysis diagram of the effects of soybean bioactive peptides on the expression of tlr, dorsal, imd and relish genes in the hepatopancreas in the present invention;

[0021] Figure 3 This is a diagram of the determination of the hepatopancreas tissue structure by soybean bioactive peptides applied for in the present invention; HE staining, 400× optical microscope. DETAILED DESCRIPTION

[0022] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions.

[0023] The examples are only used to explain the present invention and should not be construed as limiting the present invention.

[0024] The disclosure below provides many different embodiments or examples to realize different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplicity and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed. In addition, the present invention provides various specific examples of processes and materials, but those of ordinary skill in the art can be aware of the application of other processes and / or the use of other materials.

[0025] The present application provides a method for determining the antioxidant activity of soybean bioactive peptides. The determination method takes shrimp as an example of aquatic animals in the following reference examples, comparative examples and each embodiment. An appropriate amount of shrimp is divided into six groups of breeding barrels to constitute a reference example (R group), a comparative example (CT group), Example 1 (SBP1.25 group), Example 2 (SBP2.5 group), Example 3 (SBP5 group) and Example 4 (SBP10 group); it should be noted that: in the SBP1.25 group, 1.25 represents a soybean bioactive peptide content of 1.25 g / kg, in the SBP2.5 group, the soybean bioactive peptide content is 2.5 g / kg, in the SBP5 group, the soybean bioactive peptide content is 5 g / kg, and in the SBP10 group, the soybean bioactive peptide content is 10 g / kg.

[0026] The determination method of the present invention comprises the following steps:

[0027] (1) The first step, feeding: taking an appropriate amount of aquatic animals and dividing them into six groups of breeding barrels to form a reference group, a comparative example, Example 1, Example 2, Example 3 and Example 4, respectively, adding different amounts of growth active peptides in the feed to the aquatic animals in Example 1, Example 2, Example 3 and Example 4;

[0028] (2) Step 2, sample collection and processing: After the breeding experiment, each group of experiments was fasted for 24 hours. After the hemolymph, hepatopancreas and other samples of aquatic animals were collected, the hepatopancreas tissue of aquatic animals was randomly collected from each breeding tank and fixed with 4% neutral formaldehyde. Four tubes of samples were collected in each group. The hepatopancreas samples were stored in a -20°C refrigerator for testing;

[0029] (3) Step 3, determination of antioxidant enzymes and immune indexes: First, the protein content in 10% hepatopancreas homogenate was determined; then various kits were used to detect antioxidant enzyme activities and immune indexes in the hepatopancreas, including superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, nitric oxide, and inducible nitric oxide synthase;

[0030] The above-mentioned test kit can be produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China);

[0031] Gene expression determination: Real-time fluorescence quantitative analysis was used to determine the expression of antioxidant and immune-related genes in the hepatopancreas of aquatic animals;

[0032] Observation of the hepatopancreas tissue structure: including the removal and fixation of the hepatopancreas tissue of aquatic animals, gradient dehydration, defatting and transparentization, wax immersion, embedding, slide spreading, staining, sealing, observation under an optical microscope, and filming; and the optical microscope used in step (3) is a 400× optical microscope;

[0033] (4) Step 4: Data processing: Use Excel software to record data and calculate the average value of each breeding bucket for significant difference analysis; use SPSS20.0 software to analyze data; first use Levene's test to test the homogeneity of variance, and use one-way analysis of variance to compare the effects of different feeds; Duncan's multiple comparisons were used to compare the effects of different feeds, where p < 0.05 was considered significant; finally, the analyzed data were presented as mean ± standard error.

[0034] The components in step (3) are referred to as: superoxide dismutase (SOD, A001-3), malondialdehyde (MDA, A003-1), glutathione peroxidase (GPX, A005-1), catalase (CAT, A007-1-1), nitric oxide (NO, A012-1) and inducible nitric oxide synthase (i NOS, A014-1).

[0035] The specific primers used in the gene expression assay in step (3) are shown in Table 1 below:

[0036]

[0037] Table 2 is the determination table of antioxidant enzyme activity of soybean bioactive peptides in shrimp hepatopancreas

[0038]

[0039] Note: Data are mean ± standard error.

[0040] As shown in Table 2, (1) compared with the R group, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the CT group were significantly decreased (p<0.05), while the malondialdehyde (MDA) content was significantly increased (p<0.05), and there was no significant difference in catalase (CAT) activity (p>0.05); (2) compared with the CT group, the MDA content of each test group was significantly decreased (p<0.05), while the CAT activity was significantly increased (p<0.05), and there was no significant difference between them and the R group (p>0.05); (3) the SOD and GPX activities of the SBP1.25 group were significantly increased compared with the CT group (p<0.05), and there was no significant difference between them and the R group (p>0.05), and there was basically no significant difference between the other test groups and the CT group (p>0.05).

[0041] Table 3: Test results of soybean bioactive peptides on hepatopancreatic immune indexes of Macrobrachium nipponense

[0042]

[0043] Note: Data are mean ± standard error.

[0044] Combination Figure 1 As shown in the figure, compared with the R group, the gene expression of gpx in the CT group was significantly decreased (p<0.05), while there was no significant difference in the gene expression of sod and cat (p>0.05). Compared with the CT group, there was no significant difference in the gene expression of sod in each test group (p>0.05); the gene expression of cat and gpx in the SBP1.25 group was significantly upregulated (p<0.05), and there was no significant difference with the R group (p>0.05), and there was no significant difference in the gene expression of cat and gpx in other test groups (p>0.05).

[0045] Combination Figure 2 As shown, there was no significant difference in the relative expression levels of tlr, dorsal, imd and relish in the hepatopancreas of the CT group compared with the R group (p>0.05).

[0046] When the amount of SBP added exceeded 2.5 g / kg, the gene expression of tlr was significantly enhanced compared with the R and CT groups (p<0.05). The gene expression of dorsal in the SBP10 group was significantly reduced compared with the R and CT groups (p<0.05). The gene expression of imd and re l ish in the SBP1.25 group was significantly enhanced compared with the R and CT groups (p<0.05), and there was basically no significant difference in the gene expression of the other experimental groups compared with the CT group (p>0.05).

[0047] Combination Figure 3 As shown, the Figure 3 E represents embryonic cells, B represents secretory cells, F represents absorptive cells, and R represents storage cells; no obvious microscopic lesions were observed in the hepatopancreas of the reference example and the SBP1.25 group; under an optical microscope, the morphological structure of the hepatic tubules was normal and pentagonal, no obvious inflammatory cell infiltration, exudation and tissue hyperplasia were observed between the hepatic tubules, no inflammatory exudate was observed in the lumen of the hepatic tubules, no obvious necrosis of the epithelial cells, and no obvious shedding of the microvilli on their surface; the gaps between the hepatic tubules of the control example and other test groups (Examples 1-4) were reduced; among them, the B cells in the hepatopancreas of the SBP10 group were significantly enlarged, resulting in deformation and destruction of the hepatic tubules.

[0048] From Table 1-3 and Figures 1 to 3 Combined with comprehensive analysis, it can be seen that compared with the comparative example, after adding 1.25g / kg soybean bioactive peptide in Example 1, the malondialdehyde (MDA) content in the hepatopancreas of shrimp was significantly reduced, the antioxidant enzyme activity and its gene expression were significantly increased, and there was no significant difference with the reference example, which can indicate that soybean bioactive peptide can significantly reduce the malondialdehyde content in the hepatopancreas of aquatic animals;

[0049] (2) Compared with the reference feed group and the control group, the iNOS activity and NO content in the shrimp hepatopancreas of the SBP1.25 group were significantly increased, and the imd and re li sh gene expressions reached the highest level; it has the advantages of increasing the activity of antioxidant enzymes and their gene expressions, which is beneficial to the growth of aquatic animals;

[0050] (3) No obvious microscopic lesions were observed in the hepatopancreatic tissue structures of the reference example and Example 1 compared with the other groups. The morphological structure of the hepatic ducts under an optical microscope was normal and presented a pentagonal shape.

[0051] It should be noted that growth active peptide is a light brown paste obtained by fermenting soybeans as a base material.

[0052] In the description of this specification, the description with reference to the terms "one embodiment", "certain embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0053] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. The method for determining the antioxidant activity of soybean bioactive peptides is characterized in that: The following steps are included (1) The first step, feeding: taking an appropriate amount of aquatic animals and dividing them into six groups of breeding barrels to form a reference group, a comparative example, Example 1, Example 2, Example 3 and Example 4, respectively, adding different amounts of growth active peptides in the feed to the aquatic animals in Example 1, Example 2, Example 3 and Example 4; (2) Step 2, sample collection and processing: After the breeding experiment, each group of experiments was fasted for 24 hours. After the hemolymph, hepatopancreas and other samples of aquatic animals were collected, the hepatopancreas tissue of aquatic animals was randomly collected from each breeding tank and fixed with 4% neutral formaldehyde. Four tubes of samples were collected in each group. The hepatopancreas samples were stored in a -20°C refrigerator for testing; (3) Step 3, determination of antioxidant enzymes and immune indexes: First, the protein content in 10% hepatopancreas homogenate was determined; then various kits were used to detect antioxidant enzyme activities and immune indexes in the hepatopancreas, including superoxide dismutase, malondialdehyde, glutathione peroxidase, catalase, nitric oxide, and inducible nitric oxide synthase; Gene expression determination: Real-time fluorescence quantitative analysis was used to determine the expression of antioxidant and immune-related genes in the hepatopancreas of aquatic animals; Observation of hepatopancreas tissue structure: including the removal and fixation of hepatopancreas tissue of aquatic animals, gradient dehydration, defatting and transparency, wax immersion, embedding, slide spreading, staining, sealing, observation under optical microscope, and filming; (4) Step 4: Data processing: Excel software was used to record data and the mean value of each breeding barrel was calculated for significant difference analysis. SPSS 20.0 software was used for data analysis. The homogeneity of variance was first tested using Levene's test, and the effects of different feeds were compared using one-way ANOVA. Duncan's multiple comparisons were used to compare the effects of different feeds, with p < 0.05 being considered significant. Finally, the analysis data were presented as mean ± standard error.

2. The method for determining the antioxidant activity of soybean bioactive peptides according to claim 1, characterized in that: Growth active peptide is a light brown paste obtained by fermenting soybeans.

3. The method for determining the antioxidant activity of soybean bioactive peptides according to claim 1 or 2, characterized in that: The optical microscope used in step (3) is a 400× optical microscope.

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