Quinoa bran polypeptide as well as preparation method and application thereof

Abstract

The invention discloses quinoa bran polypeptide as well as a preparation method and application thereof, and belongs to the technical field of food processing. The quinoa bran polypeptide provided by the invention is collected after quinoa bran is subjected to degreasing, alkali extraction, acid precipitation, enzymolysis and ultrafiltration interception, and the molecular weight of the quinoa bran polypeptide is 1t; a 3KD active ingredient; and further performing LC-MS / MS analysis and bioinformatics screening on the quinoa bran polypeptide to obtain the quinoa bran anti-inflammatory active single peptide with a specific amino acid sequence. Experimental results show that the quinoa bran polypeptide and the quinoa bran anti-inflammatory active single peptide provided by the invention do not influence the activity of macrophages, can significantly reduce the NO release amount of the macrophages and regulate the expression amounts of inflammatory factors TNF-alpha, IL-1beta and IL-6, and have a significant inflammation inhibition effect. According to the invention, chenopodium quinoa willd processing byproducts are effectively utilized, a novel material with low toxic and side effects is provided for the preparation of anti-inflammatory drugs, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, in particular to a quinoa bran polypeptide and a preparation method and application thereof. Background Art

[0002] Quinoa (Chenopodium quinoa), an annual herbaceous flowering plant, belongs to the genus Chenopodium in the family Chenopodiaceae. It has outstanding cold-, drought-, and salt-tolerance, and can adapt to a variety of natural environments, which is why it is widely cultivated. Quinoa is known as "nutritional gold" and is extremely nutritious. It is rich in various bioactive substances such as protein, dietary fiber, saponins, polysaccharides, polyphenols, and flavonoids, making it a high-quality nutritional supplement. Compared to most grains, quinoa has a significant advantage in protein content, far exceeding its peers. Quinoa bran, the outer seed coat of quinoa, also contains extremely high nutritional value and a considerable protein content.

[0003] However, quinoa bran, a byproduct of current quinoa processing, although rich in protein, is mostly discarded as industrial waste, resulting in a significant waste of resources. Fortunately, studies have found that enzymatic hydrolysis of quinoa bran protein can yield antioxidant peptides with small molecular weight, strong activity, and easy absorption by the human body. These antioxidant peptides have important physiological functions such as scavenging free radicals and inhibiting peroxidation, opening the door to the high-value utilization of quinoa bran. Ultrafiltration, an emerging technology that can effectively separate and purify substances, is expected to further improve the utilization efficiency of quinoa bran and unlock more potential value if applied to the subsequent processing of quinoa bran protein hydrolysates.

[0004] Inflammation is an autoimmune response triggered by the body to protect itself from harmful stimuli (such as pathogens, irritants, or infections). It is also the core pathological process of many diseases, including chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, cardiovascular disease, diabetes, etc. The incidence of these diseases is on the rise worldwide, posing a huge threat to human health. Currently, although traditional anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs and glucocorticoids can relieve inflammation to a certain extent, they have problems such as large side effects. For example, non-steroidal anti-inflammatory drugs may cause gastrointestinal bleeding, and glucocorticoids may cause immunosuppression and other adverse reactions.

[0005] Quinoa itself is a safe food, and the active peptides extracted from quinoa bran may have better safety and lower toxic side effects than chemically synthesized anti-inflammatory drugs, which makes them more advantageous for long-term use in the future. Summary of the Invention

[0006] The purpose of the present invention is to provide a quinoa bran polypeptide and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The quinoa bran polypeptide provided by the present invention and the quinoa bran anti-inflammatory active single peptide obtained by screening the quinoa bran polypeptide can significantly reduce the NO release of macrophage RAW264.7 cells, regulate the expression of inflammatory factors TNF-α, IL-1β and IL-6, and have significant anti-inflammatory activity.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a quinoa bran polypeptide, and the preparation method of the quinoa bran polypeptide comprises the following steps:

[0009] The quinoa bran is crushed, sieved, mixed with ethanol, soaked, and air-dried to obtain defatted quinoa bran powder;

[0010] Mixing the defatted quinoa bran powder with water, extracting the mixture, centrifuging the mixture after extraction, separating the supernatant and the precipitate, repeatedly extracting the precipitate once, centrifuging the mixture, combining the two supernatants, and obtaining an extract;

[0011] adjusting the pH of the extract, allowing the extract to stand, collecting the precipitate by centrifugation, washing, and drying to obtain quinoa bran protein;

[0012] The quinoa bran protein is mixed with water, enzymatically hydrolyzed, and intercepted using an ultrafiltration tube to retain components with a molecular weight less than 3KD, thereby obtaining the quinoa bran polypeptide.

[0013] Preferably, the mass volume ratio of the defatted quinoa bran powder to water is 1 g:5 mL; the pH of the extraction is 11.0, the temperature is 45° C., and the time is 3 h.

[0014] Preferably, the pH of the extract is adjusted to 4.5 using hydrochloric acid; and the standing time is 30 minutes.

[0015] Preferably, the mass volume ratio of the quinoa bran protein to water is 1g:12mL; the mass volume ratio of the quinoa bran protein to water is 1g:12mL; the enzymatic hydrolysis uses a composite enzyme preparation of neutral protease and bromelain in a mass ratio of 1:1; the total enzyme addition amount of the composite enzyme preparation is 5000U / g; and the enzymatic hydrolysis time is 3h.

[0016] The present invention also provides a quinoa bran anti-inflammatory active single peptide, which is a quinoa bran anti-inflammatory active single peptide obtained from the above-mentioned quinoa bran polypeptide through LC-MS / MS analysis and bioinformatics screening.

[0017] Preferably, the quinoa bran anti-inflammatory active single peptide is QBAI-Ⅰ, QBAI-Ⅲ or QBAI-Ⅳ;

[0018] Wherein, the amino acid sequence of QBAI-Ⅰ is shown in SEQ ID NO.1;

[0019] The amino acid sequence of QBAI-III is shown in SEQ ID NO.3;

[0020] The amino acid sequence of QBAI-IV is shown in SEQ ID NO.4.

[0021] Preferably, the quinoa bran anti-inflammatory active single peptide is QBAI-III with an amino acid sequence as shown in SEQ ID NO.3.

[0022] The present invention also provides a use of the quinoa bran polypeptide or the quinoa bran anti-inflammatory active single peptide in the preparation of anti-inflammatory drugs.

[0023] The present invention also provides an anti-inflammatory drug, which uses the quinoa bran polypeptide or the quinoa bran anti-inflammatory active single peptide as a main active ingredient.

[0024] Preferably, a pharmaceutically acceptable excipient is also included.

[0025] The present invention discloses the following technical effects:

[0026] The quinoa bran polypeptide provided by the present invention is an active ingredient with a molecular weight of less than 3KD collected after quinoa bran is subjected to defatting, alkali extraction, acid precipitation, enzymatic hydrolysis, and ultrafiltration interception; the quinoa bran polypeptide is further subjected to LC-MS / MS analysis and bioinformatics screening to obtain a quinoa bran anti-inflammatory active single peptide with a specific amino acid sequence. Experimental results show that the quinoa bran polypeptide and quinoa bran anti-inflammatory active single peptide provided by the present invention do not affect the activity of macrophages, can significantly reduce the amount of NO released by macrophages, regulate the expression of inflammatory factors TNF-α, IL-1β, and IL-6, and have significant inflammation-inhibiting effects. The present invention effectively utilizes quinoa processing byproducts, provides a new type of material with low toxicity and side effects for the manufacture of anti-inflammatory drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 The figure shows the experimental results of the effects of different concentrations of quinoa bran polypeptides QBPP-Ⅰ, QBPP-Ⅱ and QBPP-Ⅲ and quinoa bran crude protein on the activity of macrophage RAW264.7;

[0029] Figure 2 This is the experimental result of the effect of different concentrations of lipopolysaccharide on the activity of macrophage RAW264.7;

[0030] Figure 3 The results of the lipopolysaccharide (LPS) modeling concentration screening experiment are shown in Figure 1. * indicates P < 0.05, and ** indicates P < 0.01.

[0031] Figure 4 This is the experimental result of the effect of quinoa bran polypeptides QBPP-Ⅰ, QBPP-Ⅱ and QBPP-Ⅲ on NO release; among them, ** indicates P < 0.01;

[0032] Figure 5 This is the experimental result of the effects of quinoa bran single peptides QBAI-Ⅰ, QBAI-Ⅲ and QBAI-Ⅳ on the activity of macrophage RAW264.7 cells;

[0033] Figure 6 The figure shows the experimental results of the effects of quinoa bran peptides QBAI-Ⅰ, QBAI-Ⅲ and QBAI-Ⅳ on NO release; ** indicates P < 0.01;

[0034] Figure 7 The figure shows the experimental results of the effect of QBAI-Ⅲ on the inflammatory factors TNF-α and IL-1β in LPS-induced macrophage RAW264.7 cells; different lowercase letters indicate significant differences;

[0035] Figure 8 The figure shows the experimental results of the effect of QBAI-Ⅲ on the inflammatory factor IL-6 in LPS-induced macrophage RAW264.7 cells; different lowercase letters indicate significant differences. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] In the present specification, quinoa bran polypeptide (QBPP) refers to a mixture of peptides with different amino acid sequences within a specific molecular weight range obtained from quinoa bran protein after enzymatic hydrolysis and separation; quinoa bran anti-inflammatory peptide (QBAI) or quinoa bran single peptide refers to a single peptide with a clear amino acid sequence obtained from quinoa bran polypeptide after bioinformatics analysis.

[0042] Example 1: Extraction and preparation of quinoa bran polypeptide (QBPP)

[0043] The quinoa bran is crushed and sieved, mixed with 95% ethanol, soaked and air-dried to obtain defatted quinoa bran powder.

[0044] Defatted quinoa bran powder was mixed with water at a ratio of 1 g:5 mL. The pH was adjusted to 11.0 with sodium hydroxide solution and the mixture was extracted at 45°C for 3 hours. The extract was transferred to a centrifuge tube and centrifuged at 6000 rpm for 20 minutes. The supernatant was collected and a second extraction was performed under the same conditions. The supernatant was then centrifuged and collected.

[0045] The pH of the supernatant was adjusted to 4.5 with hydrochloric acid solution, allowed to stand for 30 minutes, centrifuged at 6000 rpm for 20 minutes, the precipitate was collected, washed with distilled water to remove soluble salts, and dried to constant weight to obtain quinoa bran protein.

[0046] Quinoa bran protein powder was mixed with distilled water at a material-liquid ratio of 1g:12mL to obtain a protein solution. This solution was then enzymatically hydrolyzed with 5000 U / g of bromelain at a 1:1 mass ratio of neutral protease and 3 hours of enzymes to obtain quinoa bran peptides. The calculated protein concentration was 39.31 mg / mL, and the protein content was 19.675 mg / g.

[0047] Ultrafiltration tubes were used for interception to obtain active ingredients with different molecular weight components: QBPP-Ⅰ (>10KD), QBPP-Ⅱ (3-10KD), and QBPP-Ⅲ (<3KD).

[0048] Example 2: Experiment on the effect of quinoa bran polypeptide on the activity of macrophage RAW264.7

[0049] Select RAW264.7 cells in the logarithmic growth phase, wait until the RAW264.7 cells grow to about 90%, blow off the adherent cells, centrifuge at 1000 rpm for 5 min, resuspend the RAW264.7 cells in 1 mL of culture medium, and 5 The cells were seeded at a density of 100 μL / well in a 96-well plate, with 100 μL per well, and cultured in a constant temperature incubator.

[0050] The next day, quinoa bran peptides were diluted in complete culture medium to the following concentrations: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL. The supernatant was carefully aspirated, and different concentrations of the three components of QBPP-I (>10 kD), QBPP-II (3-10 kD), and QBPP-III (<3 kD) were added, along with quinoa bran crude protein. Complete culture medium was used as a control, with 200 μL added to each well. Five replicates were set up for each group, and the cells were incubated for another 24 hours. Under dark conditions, 20 μL of MTT was added to each well, and the cells were incubated until purple crystals appeared. 150 μL of DMSO was added, and the cells were shaken for 10 minutes. After the crystals dissolved, the absorbance (OD) at 490 nm was measured using a microplate reader. The blank control value was subtracted from the OD value to calculate cell viability. The results were analyzed graphically using Origin software.

[0051] Experimental results: Figure 1 It can be seen that compared with the control group, different components had a certain effect on the viability of RAW264.7 cells, but when the active peptide concentration was 12.5-200 μg / mL, the four components of quinoa bran crude protein, quinoa bran polypeptide QBPP-Ⅰ, QBPP-Ⅱ, and QBPP-Ⅲ had no obvious inhibitory effect on the survival of RAW264.7 cells.

[0052] Example 3: Effect of lipopolysaccharide (LPS) on the activity of macrophage RAW264.7

[0053] Select RAW264.7 cells in the logarithmic growth phase, wait until the RAW264.7 cells grow to about 90%, blow off the adherent cells, centrifuge at 1000 rpm for 5 minutes, resuspend the RAW264.7 cells in 1 mL of culture medium, and 5 The cells were seeded at a density of 100 μL / well in a 96-well plate, with 100 μL per well, and cultured in a constant temperature incubator.

[0054] The next day, the 50 μg / mL LPS stock solution was diluted with PBS to the following concentrations: 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 2 μg / mL. The supernatant was carefully aspirated and LPS dilutions of varying concentrations were added sequentially. Complete culture medium was used as the control group, and 200 μL was added to each well. Five replicates were set up for each group and cultured for another 24 hours. Under dark conditions, 20 μL of MTT was added to each well and incubated until purple crystals appeared. 150 μL of DMSO was added and shaken for 10 minutes. After the crystals dissolved, the absorbance at 490 nm (OD value) was measured using a microplate reader. The blank control value was subtracted from all OD values to calculate cell viability. The experimental results were analyzed graphically using Origin software.

[0055] The experimental results are as follows Figure 2 As shown, compared with the control group, different concentrations of lipopolysaccharide had a certain effect on the viability of RAW264.7 cells, but when the lipopolysaccharide concentration was 0.1-2 μg / mL, there was no obvious inhibitory effect on the survival of RAW264.7 cells.

[0056] Example 4: Establishment of RAW.264.7 cell inflammation model

[0057] When the RAW264.7 cells grew to about 90%, the adherent cells were blown off, centrifuged at 1000 rpm for 5 min, and resuspended in 1 mL of culture medium. 6 The cells were seeded at a density of 1000 cells / well in a six-well plate, with 2 mL of DMEM medium per well, shaken and placed in a constant temperature incubator for culture.

[0058] The next day, a 50 μg / mL LPS stock solution was diluted with PBS to the following concentrations: 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 2 μg / mL. The supernatant was carefully aspirated and the LPS dilutions of varying concentrations were added sequentially. Complete culture medium served as the control. After 24 hours of treatment, NO release was measured using the Griess method. 50 μL of the culture supernatant and 50 μL of solution A were shaken and incubated in a 37°C incubator for 10 minutes. Then, 50 μL of solution B was added and shaken and incubated in a 37°C incubator for 10 minutes. The absorbance at 540 nm was measured to determine the concentration for modeling. The results were analyzed graphically using Origin software.

[0059] The experimental results are as follows Figure 3 As shown in the figure, compared with the control group, the release of NO in the cell culture medium of the 0.1-2μg / mL LPS treatment group continued to increase, and the increase in NO release at 1μg / mL was significantly more than that at 0.5μg / mL. When the LPS concentration was greater than 1μg / mL, even though the NO release increased, the increase was not significant. Therefore, 1μg / mL was selected as the experimental modeling concentration.

[0060] Example 5: Effect of Quinoa Bran Peptides on NO Content in LPS-Induced Macrophages RAW264.7

[0061] When the RAW264.7 cells grew to about 90%, the adherent cells were blown off, centrifuged at 1000 rpm for 5 min, and resuspended in 1 mL of culture medium. 5 The cells were seeded at a density of 100 cells / well in a 24-well plate, with 1 mL of DMEM medium per well, shaken and placed in a constant temperature incubator for culture.

[0062] The next day, complete culture medium was used to dilute the quinoa bran peptides QBPP-I, QBPP-II, and QBPP-III to 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively. The supernatant was carefully aspirated and various concentrations of the quinoa bran peptide dilutions were added sequentially. Complete culture medium was used as the control, with 1 mL added to each well. Two hours later, the cells were induced with LPS (1 μg / mL) for 24 hours.

[0063] After incubation, NO release was measured using the Griess method. 50 μL of the culture supernatant and 50 μL of Solution A were shaken and incubated at 37°C for 10 minutes. Then, 50 μL of Solution B was added and shaken, and the mixture was incubated at 37°C for 10 minutes. Absorbance was measured at 540 nm, and the results were analyzed graphically using Origin software.

[0064] NO release was used as an indicator to screen the anti-inflammatory activity of quinoa bran peptides on macrophage RAW264.7 cells.

[0065] Depend on Figure 4 It can be seen that the anti-inflammatory activity of different quinoa bran polypeptide components was analyzed using the LPS-induced macrophage inflammation model, and the effect of quinoa bran polypeptide on LPS-induced macrophage NO release was detected by the Griess method. Compared with the blank group, the LPS-induced NO release of RAW264.7 cells was significantly increased, indicating that the model was successful. The NO release of each component of quinoa bran polypeptide showed a downward trend compared with the model group as a whole, but the NO release of individual components was higher than that of the model group. Among the three components, the NO release of quinoa bran polypeptide QBPP-Ⅲ showed a concentration-dependent decrease, which indicated that this component exhibited good anti-inflammatory activity, so the quinoa bran polypeptide QBPP-Ⅲ was used in subsequent studies.

[0066] Example 6: Bioinformatics Analysis of Quinoa Bran Peptide QBPP-III

[0067] Mass spectrometry analysis of QBPP-Ⅲ was performed, and the peptide activity of QBPP-Ⅲ polypeptide was studied in combination with biological information, and the functions and physicochemical properties of the polypeptide peptides in QBPP-Ⅲ were predicted.

[0068] Through LC-MS / MS analysis, 14 short peptide sequences with anti-inflammatory activity were obtained from QBPP-Ⅲ based on the amino acid sites of YG, LPF, LLY, and anti-inflammatory activity from the BIOPEP database. The peptide length was limited to between 7 and 10 amino acids, and 9 peptides with a score > 0.5 were selected according to PeptideRanker. After further screening, 4 quinoa bran anti-inflammatory active single peptides (QBAI) with anti-inflammatory activity were finally synthesized. Their amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.4:

[0069] SEQ ID NO. 1: DAGRLPGYG;

[0070] SEQ ID NO. 2: PSFALLYS;

[0071] SEQ ID NO.3: VWYGGLAMA;

[0072] SEQ ID NO. 4: WSPSKVYG.

[0073] The four quinoa bran anti-inflammatory peptides were named QBAI-I (SEQ ID NO. 1), QBAI-II (SEQ ID NO. 2), QBAI-III (SEQ ID NO. 3), and QBAI-IV (SEQ ID NO. 4). A novelty check of these four peptides in EROPMoscow revealed that none of the four peptide sequences had been previously reported. Subsequently, physicochemical properties of these four sequences were analyzed, as shown in Table 1. All four peptides were non-toxic, with molecular weights ranging from 896 to 967 Da.

[0074] Table 1 Physicochemical properties of the anti-inflammatory active peptides of the present invention

[0075]

[0076] The chemical structures of the above four single peptides are shown below:

[0077] QBAI-Ⅰ(DAGRLPGYG):

[0078]

[0079] QBAI-Ⅱ(PSFALLYS):

[0080]

[0081] QBAI-Ⅲ(VWYGGLAMA):

[0082]

[0083] QBAI-Ⅳ(WSPSKVYG):

[0084]

[0085] Due to solubility issues with QBAI-Ⅱ (PSFALLYS), QBAI-Ⅱ was abandoned in subsequent experiments.

[0086] Example 7: Experiment on the effect of quinoa bran peptide on the activity of macrophage RAW264.7

[0087] The experimental method differs from that of Example 2 only in that quinoa bran single peptides QBAI-Ⅰ, QBAI-Ⅲ and QBAI-Ⅳ are used to replace quinoa bran polypeptides QBPP-Ⅰ, QBPP-Ⅱ and QBPP-Ⅲ.

[0088] The experimental results are given by Figure 5It can be seen that the effects of quinoa bran single peptides QBAI-Ⅰ, QBAI-Ⅲ, and QBAI-Ⅳ on the viability of RAW264.7 cells at different concentrations. Compared with the control, different components had a certain effect on the viability of RAW264.7 cells, but when the single peptide concentration was 12.5-200 μg / mL, these three components had no obvious inhibitory effect on the survival of RAW264.7 cells.

[0089] Example 8: Effect of Quinoa Bran Peptide on NO Content in LPS-Induced Macrophages RAW264.7

[0090] The experimental method differs from that of Example 5 only in that quinoa bran single peptides QBAI-Ⅰ, QBAI-Ⅲ and QBAI-Ⅳ are used to replace quinoa bran polypeptides QBPP-Ⅰ, QBPP-Ⅱ and QBPP-Ⅲ.

[0091] The experimental results are given by Figure 6 The anti-inflammatory activity of different single peptide components was analyzed using an LPS-induced macrophage inflammation model. The Griess assay was used to examine the effects of quinoa bran anti-inflammatory peptides on LPS-induced macrophage NO secretion. Compared with the blank control group, LPS-induced NO secretion in RAW264.7 cells was significantly increased, indicating the success of the model. While NO secretion from quinoa bran peptides decreased overall compared to the model group, some individual peptides exhibited higher NO secretion levels than the model group. Among the three peptides, QBAI-I quinoa bran peptide exhibited higher NO secretion than the model group at concentrations of 25 and 50 μg / mL; QBAI-IV quinoa bran peptide exhibited higher NO secretion than the model group at a concentration of 25 μg / mL. QBAI-III exhibited a concentration-dependent decrease in NO secretion, demonstrating its potent anti-inflammatory activity. Therefore, QBAI-III was used in subsequent studies.

[0092] Example 9: Effects of QBAI-III on LPS-induced inflammatory factors TNF-α, IL-1β, and IL-6 in macrophage RAW264.7 cells

[0093] The experiment was divided into control group, model group (M), LPS+QBAI-Ⅲ group (QBAI-Ⅲ concentration was 25, 50, 100, 200 μg / mL)

[0094] When the RAW264.7 cells grew to about 90%, the adherent cells were blown off, centrifuged at 1000 rpm for 5 min, and resuspended in 1 mL of culture medium. 6Cells were seeded at a density of 100 μg / well in a six-well plate, with 2 mL of DMEM medium per well, shaken, and cultured in a constant temperature incubator. The next day, QBAI-III was diluted to 200 μg / mL, 100 μg / mL, 50 μg / mL, and 25 μg / mL using complete culture medium. The supernatant was carefully aspirated, and different concentrations of QBAI-III were added sequentially. Complete culture medium was used as the control group, with 2 mL added to each well. After 2 hours, LPS (1 μg / mL) was induced for 24 hours. The OD values of TNF-α, IL-1β, and IL-6 were measured at 450 nm according to the kit instructions. The significance of the differences was tested using a one-way ANOVA test in SPSS, and the experimental results were analyzed graphically using Origin software.

[0095] The experimental results are as follows Figure 7 and Figure 8 As shown in the results, compared with the normal control group, the levels of TNF-α, IL-1β and IL-6 in the LPS RAW264.7 cells in the model group were significantly increased (P<0.05). Compared with the LPS model group, the levels of the above indicators in the RAW264.7 cells of quinoa bran single peptide QBAI-Ⅲ at all concentrations were significantly decreased (P<0.05).

[0096] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A quinoa bran polypeptide, characterized in that The preparation method of the quinoa bran polypeptide comprises the following steps: The quinoa bran is crushed, sieved, mixed with ethanol, soaked, and air-dried to obtain defatted quinoa bran powder; Mixing the defatted quinoa bran powder with water, extracting the mixture, centrifuging the mixture after extraction, separating the supernatant and the precipitate, repeatedly extracting the precipitate once, centrifuging the mixture, combining the two supernatants, and obtaining an extract; adjusting the pH of the extract, allowing the extract to stand, collecting the precipitate by centrifugation, washing, and drying to obtain quinoa bran protein; The quinoa bran protein is mixed with water, enzymatically hydrolyzed, and intercepted using an ultrafiltration tube to retain components with a molecular weight less than 3KD, thereby obtaining the quinoa bran polypeptide.

2. The quinoa bran polypeptide according to claim 1, wherein The mass volume ratio of the defatted quinoa bran powder to water is 1 g:5 mL; the pH of the extraction is 11.0, the temperature is 45° C., and the time is 3 h.

3. The quinoa bran polypeptide according to claim 1, wherein The pH of the extract is adjusted to 4.5 using hydrochloric acid; and the standing time is 30 minutes.

4. The quinoa bran polypeptide according to claim 1, wherein The mass volume ratio of the quinoa bran protein to water is 1 g:12 mL; the enzymatic hydrolysis uses a composite enzyme preparation of neutral protease and bromelain in a mass ratio of 1:1; the total enzyme addition amount of the composite enzyme preparation is 5000 U / g; and the enzymatic hydrolysis time is 3 h.

5. A single peptide with anti-inflammatory activity from quinoa bran, characterized in that: The quinoa bran anti-inflammatory active single peptide is a quinoa bran anti-inflammatory active single peptide obtained by LC-MS / MS analysis and bioinformatics screening of the quinoa bran polypeptide according to any one of claims 1 to 4.

6. The anti-inflammatory active single peptide of quinoa bran according to claim 5, characterized in that The quinoa bran anti-inflammatory active single peptide is QBAI-Ⅰ, QBAI-Ⅲ or QBAI-Ⅳ; Wherein, the amino acid sequence of QBAI-Ⅰ is shown in SEQ ID NO.1; The amino acid sequence of QBAI-III is shown in SEQ ID NO.3; The amino acid sequence of QBAI-IV is shown in SEQ ID NO.

4.

7. The anti-inflammatory active single peptide of quinoa bran according to claim 6, characterized in that The quinoa bran anti-inflammatory active single peptide is QBAI-III with an amino acid sequence as shown in SEQ ID NO.

3.

8. Use of the quinoa bran polypeptide according to any one of claims 1 to 4 or the quinoa bran anti-inflammatory active single peptide according to any one of claims 5 to 7 in the preparation of anti-inflammatory drugs.

9. An anti-inflammatory drug, characterized in that The main active ingredient is the quinoa bran polypeptide according to any one of claims 1 to 4 or the quinoa bran anti-inflammatory active single peptide according to any one of claims 5 to 7.

10. The anti-inflammatory drug according to claim 9, characterized in that Pharmaceutically acceptable excipients are also included.

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