Glutamine peptide with anti-inflammatory effect and application thereof

Abstract

The invention provides glutamine peptide with an anti-inflammatory effect and application of the glutamine peptide, and belongs to the technical field of anti-inflammatory active peptide. The invention provides four glutamine peptides with an anti-inflammatory effect, which can be used for preparing medicines or feed additives with an anti-inflammatory function or a function of preventing and treating inflammatory bowel diseases. The glutamine peptide with the anti-inflammatory function is developed by taking the corn protein powder as a raw material, so that the additional value of corn processing byproducts can be increased, and the glutamine peptide has important significance on maintaining sustainable and stable development of the livestock industry and the feed industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-inflammatory active peptides, and in particular relates to a glutamine peptide with anti-inflammatory effects and applications thereof. Background Art

[0002] As the body's largest immune organ, the intestines protect against pathogen invasion, promote nutrient digestion and absorption, and maintain overall health. Inflammatory bowel disease (IBD) can disrupt nutrient digestion and absorption, slow growth, reduce disease resistance, and increase susceptibility to pathogens, leading to a variety of other diseases. Therefore, finding alternatives to antibiotics to protect animal intestinal health is crucial for maintaining the sustainable development of the animal husbandry and feed industries.

[0003] Glutamine (Gln) is a conditionally essential amino acid for the body. It can play an anti-inflammatory role by participating in the energy metabolism of immune cells and regulating inflammatory signaling pathways, thereby ensuring the integrity of intestinal function. However, the short half-life and low bioavailability of free glutamine in the body limit its clinical application. Studies have found that glutamine-binding peptides not only retain the multiple physiological functions and nutrition of Gln, but also have the advantages of good absorbability, low sensitization, and strong stability. They can be used as a substitute for Gln stabilization. However, only two artificially synthesized alanyl-glutamine dipeptide and glycyl-glutamine dipeptide have been applied to production, and the production cost is high, and the product price is expensive. Therefore, it is very necessary to actively develop glutamine peptides with anti-inflammatory functions. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a glutamine peptide with anti-inflammatory effect and its application.

[0005] A second object of the present invention is to provide a fermentation product containing the above-mentioned glutamine peptide and its application.

[0006] The third object of the present invention is to provide an extract containing the above-mentioned glutamine peptide and its application.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a glutamine peptide with anti-inflammatory effect, wherein the glutamine peptide comprises at least one of amino acid sequences such as SEQ ID NO.1 to SEQ ID NO.4.

[0009] The present invention also provides a fermentation product containing the above-mentioned glutamine peptide, which is prepared by the following method: mixing a fermentation substrate, corn steep liquor, a composite bacterial agent, an acidic protease and water to make the water content of the mixture 45%, and fermenting and culturing the mixture at 30-35°C for 3-5 days to obtain a fermentation product; the fermentation substrate is obtained by mixing corn gluten powder, bran, soybean meal and corn germ meal in a dry basis ratio of 4:1:1:4; the composite bacterial agent is composed of Bacillus subtilis YY-12, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-7.

[0010] Preferably, each 3 mL of the composite bacterial agent contains (1.0-2.0)×10 8 CFU Bacillus subtilis YY-12, (1.0~2.0)×10 7 CFU of Saccharomyces cerevisiae YY-14 and (1.0~3.0)×10 7 CFU Lactobacillus plantarum YY-7.

[0011] Preferably, the mass volume ratio of the fermentation substrate to the corn steep liquor is 100 g:10 mL; the mass volume ratio of the fermentation substrate to the composite bacterial agent is 100 g:1 mL.

[0012] Preferably, 25 U of acid protease is added per 1 g of fermentation substrate.

[0013] The present invention also provides an extract containing the glutamine peptide, which is prepared by the following steps: drying the fermented product, mixing it with water for extraction, and collecting the supernatant to obtain the extract.

[0014] Preferably, the leaching includes stirring leaching, the stirring speed is 200 r / min, and the stirring leaching time is 30 min.

[0015] The present invention also provides the use of any one of the following in the preparation of an anti-inflammatory product or a product for preventing and treating inflammatory bowel disease: (1) the above-mentioned glutamine peptide; (2) the above-mentioned fermentation product; (3) the above-mentioned extract.

[0016] Preferably, the product comprises a medicine or a feed additive.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides four glutamine peptides with anti-inflammatory effects, which can be used to prepare medicines or feed additives with anti-inflammatory functions or functions of preventing and treating inflammatory bowel disease.

[0019] The present invention uses corn protein powder as raw material to develop glutamine peptides with anti-inflammatory function, which can not only increase the added value of corn processing by-products, but also has important significance for maintaining the sustainable and stable development of the animal husbandry and feed industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of glutamine peptide on the proliferation of IEC-6 cells;

[0021] Figure 2 The effect of glutamine peptide on the survival rate of IEC-6 cells, among which ####p<0.0001, compared with the CON group; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, compared with the LPS model group; ××××p<0.0001, ×p<0.05, compared with the Ala-Gln positive control group;

[0022] Figure 3 The effect of glutamine peptide on NO concentration in LPS-induced IEC-6 cells, among which ####p<0.0001, compared with CON group; ****p<0.0001, compared with LPS injury model group; ××××p<0.0001, compared with Ala-Gln positive control group;

[0023] Figure 4 Figure 2 shows the effects of glutamine peptide on LPS-induced inflammatory factors in IEC-6 cells, where (a) is the result of IL-10, (b) is the result of IL-1β, (c) is the result of IL-6, and (d) is the result of TNF-α. ####p<0.0001, compared with the CON group; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, compared with the LPS injury model group; ××××p<0.0001, ×××p<0.001, ×p<0.05, compared with the Ala-Gln positive control group.

[0024] Figure 5 The effect of different groups of extracts on the survival rate of IEC-6 cells;

[0025] Figure 6 The results of the in vitro anti-inflammatory effects of the extracts from different groups are shown. The left figure shows the cell survival rate, and the right figure shows the NO concentration detection results. Among them, ####p<0.0001, compared with CON; ****p<0.0001, ***p<0.001, **p<0.01, compared with the LPS model group. DETAILED DESCRIPTION

[0026] The present invention provides a glutamine peptide with anti-inflammatory effect, wherein the glutamine peptide comprises at least one of QQQCCHQIR (SEQ ID NO.1), QQCCHQIR (SEQ ID NO.2), QPQPQFP (SEQ ID NO.3) and QPQPP (SEQ ID NO.4).

[0027] The present invention uses corn gluten meal as the main fermentation substrate and adopts a bacterial enzyme collaborative fermentation method to prepare a fermentation product with a high peptide content. The fermentation product is extracted and vacuum freeze-dried to obtain a mixture of the glutamine peptides, and the amino acid sequence of the glutamine peptides is obtained by mass spectrometry sequencing. In the present invention, the glutamine peptides can be synthesized by a solid-phase synthesis method. Corn gluten meal is the main by-product of the wet production of corn starch and has the advantages of a wide source, high yield, and low price. However, corn gluten meal is poorly soluble in water, and the physiological functions of glutamine peptides cannot be exerted. The present invention adopts a bacterial enzyme collaborative fermentation method to hydrolyze corn gluten meal, which can effectively release corn glutamine peptides with anti-inflammatory activity. The present invention uses corn gluten meal as a raw material to develop glutamine peptides with anti-inflammatory function, which not only increases the added value of corn processing by-products, but also has important significance for maintaining the sustainable and stable development of the animal husbandry and feed industries.

[0028] The present invention also provides a fermentation product containing the above-mentioned glutamine peptide, which is prepared by the following method: mixing a fermentation substrate, corn steep liquor, a composite bacterial agent, an acidic protease and water to make the water content of the mixture 45%, and fermenting and culturing the mixture at 30-35°C for 3-5 days to obtain a fermentation product; the fermentation substrate is obtained by mixing corn gluten powder, bran, soybean meal and corn germ meal in a dry basis ratio of 4:1:1:4; the composite bacterial agent is composed of Bacillus subtilis YY-12, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-7.

[0029] The present invention does not specifically limit the specific sources of the above-mentioned raw materials. In the present invention, the corn gluten meal is preferably puffed corn gluten meal. The method for preparing the puffed corn gluten meal preferably includes extruding the corn gluten meal at 150-160°C for 5-10 seconds, more preferably at 155°C, and for 7-8 seconds. After the extrusion, the process preferably includes pulverizing the corn gluten meal; the pulverized particle size is preferably 30-50 mesh, more preferably 40 mesh.

[0030] In the present invention, each 3 mL of the composite bacterial agent preferably contains (1.0-2.0)×10 8 CFU Bacillus subtilis YY-12, (1.0~2.0)×10 7 CFU of Saccharomyces cerevisiae YY-14 and (1.0~3.0)×10 7 CFU Lactobacillus plantarum YY-7, more preferably containing 1.5×10 8 CFU Bacillus subtilis YY-12, 1.5×10 7 CFU of Saccharomyces cerevisiae YY-14 and (1.5~2.5)×10 7CFU Lactobacillus plantarum YY-7. In the present invention, the deposit number of the Bacillus subtilis YY-12 is CGMCC No. 24696, the deposit number of the Saccharomyces cerevisiae YY-14 is CGMCC No. 24698, and the deposit number of the Lactobacillus plantarum YY-7 is CGMCC No. 24691.

[0031] In the present invention, the mass volume ratio of the fermentation substrate to the corn steep liquor is preferably 100g:10mL; the mass volume ratio of the fermentation substrate to the composite bacterial agent is preferably 100g:1mL ； Preferably, 25 U of acid protease is added per 1 g of fermentation substrate. In the present invention, the fermentation temperature is preferably 31-34° C., more preferably 32-33° C., and the fermentation time is preferably 3.5-4.5 days.

[0032] The present invention also provides an extract containing the glutamine peptide, which is prepared by the following steps: drying the fermented product, mixing it with water for extraction, and collecting the supernatant to obtain the extract.

[0033] In the present invention, the drying method is preferably oven drying, the oven drying temperature is preferably 65°C, and the oven drying time is preferably 72 hours; the mass volume ratio of the dried fermented product to water is preferably 1g:10mL. In the present invention, the extraction preferably includes stirring extraction, the stirring speed is preferably 200r / min, and the stirring extraction time is preferably 30min. In the present invention, after the extraction, a centrifugation step is preferably included, the centrifugation speed is preferably 12000r / min, and the centrifugation time is preferably 30min.

[0034] The present invention also provides the use of any one of the following in the preparation of an anti-inflammatory product or a product for preventing and treating inflammatory bowel disease: (1) the above-mentioned glutamine peptide; (2) the above-mentioned fermentation product; (3) the above-mentioned extract. In the present invention, the product preferably comprises a medicine or a feed additive.

[0035] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] In the following examples, unless otherwise specified, all methods are conventional.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The corn steep liquor in the following examples was purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd., and the acidic protease was purchased from Novozymes Biotechnology Co., Ltd. (acidic protease: 50,000 U / g; pH 2.0-4.0).

[0039] Example 1

[0040] A fermentation product containing the glutamine peptides QQQCCHQIR (SEQ ID NO. 1), QQCCHQIR (SEQ ID NO. 2), QPQPQFP (SEQ ID NO. 3) and QPQPP (SEQ ID NO. 4) is prepared as follows:

[0041] (1) Bacterial culture

[0042] Bacillus subtilis YY-12 was inoculated into LB medium at a volume percentage of 1%, the fermentation temperature was 37°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 3.5, the number of bacteria was 1.0×10 8 CFU / mL, the seed culture was completed, and the Bacillus subtilis YY-12 seed liquid was obtained.

[0043] Saccharomyces cerevisiae YY-14 was inoculated into YPD medium at a volume percentage of 1%, the fermentation temperature was 35°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 7.8, the number of bacteria was 1.0×10 7 CFU / mL, the seed culture was completed, and the cerevisiae YY-14 seed liquid was obtained.

[0044] Lactobacillus plantarum YY-7 was inoculated into MRS medium at a volume percentage of 1%, and the fermentation temperature was 37°C. The culture was allowed to stand for 2 h. 600 The absorbance was measured at 400 nm. When the OD value was 5.2, the number of bacteria was 1.0×10 7 CFU / mL, the seed culture was completed, and the Lactobacillus plantarum YY-7 seed liquid was obtained.

[0045] The seed liquid of Bacillus subtilis YY-12, the seed liquid of Saccharomyces cerevisiae YY-14 and the seed liquid of Lactobacillus plantarum YY-7 were uniformly mixed in a volume ratio of 1:1:1 to obtain a composite bacterial agent.

[0046] (2) The corn gluten powder was extruded and puffed at 155°C for 8s and crushed to a particle size of 40 mesh to obtain puffed corn gluten powder; the puffed corn gluten powder, bran, soybean meal, and corn germ meal were mixed uniformly at a dry weight ratio of 4:1:1:4 to obtain a fermentation matrix; corn slurry and composite bacterial agent were added to the fermentation matrix, the mass volume ratio of the fermentation matrix to the corn slurry was 100g:10mL, and the mass volume ratio of the fermentation matrix to the composite bacterial agent was 100g:1mL, and then 25U of acid protease was added per gram of fermentation matrix, and the above substances were mixed uniformly. After hydration, the mixture was adjusted to a water content of 45% and a natural pH value; and then placed in a one-way valve breathable bag and cultured at 30°C for 5 days to obtain a fermentation product.

[0047] Example 2

[0048] A fermentation product containing the glutamine peptides QQQCCHQIR (SEQ ID NO. 1), QQCCHQIR (SEQ ID NO. 2), QPQPQFP (SEQ ID NO. 3) and QPQPP (SEQ ID NO. 4) is prepared as follows:

[0049] (1) Bacterial culture

[0050] Bacillus subtilis YY-12 was inoculated into LB medium at a volume percentage of 1%, the fermentation temperature was 37°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 3.7, the number of bacteria was 2.0×10 8 CFU / mL, the seed culture was completed, and the Bacillus subtilis YY-12 seed liquid was obtained.

[0051] Saccharomyces cerevisiae YY-14 was inoculated into YPD medium at a volume percentage of 1%, the fermentation temperature was 35°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 8.5, the number of bacteria was 2.0×10 7 CFU / mL, the seed culture was completed, and the cerevisiae YY-14 seed liquid was obtained.

[0052] Lactobacillus plantarum YY-7 was inoculated into MRS medium at a volume percentage of 1%, and the fermentation temperature was 37°C. The culture was allowed to stand for 2 h. 600 The absorbance was measured at 400 nm. When the OD value was 6.9, the number of bacteria was 3.0×10 7 CFU / mL, the seed culture was completed, and the Lactobacillus plantarum YY-7 seed liquid was obtained.

[0053] The seed liquid of Bacillus subtilis YY-12, the seed liquid of Saccharomyces cerevisiae YY-14 and the seed liquid of Lactobacillus plantarum YY-7 were uniformly mixed in a volume ratio of 1:1:1 to obtain a composite bacterial agent.

[0054] (2) Extruding corn gluten powder at 160°C for 6s and crushing it to a particle size of 40 mesh to obtain puffed corn gluten powder; mixing puffed corn gluten powder, bran, soybean meal, and corn germ meal in a dry weight ratio of 4:1:1:4 to obtain a fermentation matrix; adding corn slurry and composite bacterial agent to the fermentation matrix, the mass volume ratio of the fermentation matrix to the corn slurry being 100g:10mL, and the mass volume ratio of the fermentation matrix to the composite bacterial agent being 100g:1mL, and then adding acid protease in an amount of 25U of acid protease per gram of fermentation matrix, mixing the above substances evenly, and adding water to make the mixture have a water content of 45% and a natural pH value; then placing the mixture in a one-way valve breathable bag and culturing it at 35°C for 3 days to obtain a fermentation product.

[0055] Example 3

[0056] A fermentation product containing the glutamine peptides QQQCCHQIR (SEQ ID NO. 1), QQCCHQIR (SEQ ID NO. 2), QPQPQFP (SEQ ID NO. 3) and QPQPP (SEQ ID NO. 4) is prepared as follows:

[0057] (1) Bacterial culture

[0058] Bacillus subtilis YY-12 was inoculated into LB medium at a volume percentage of 1%, the fermentation temperature was 37°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 3.6, the number of bacteria was 1.5×10 8 CFU / mL, the seed culture was completed, and the Bacillus subtilis YY-12 seed liquid was obtained.

[0059] Saccharomyces cerevisiae YY-14 was inoculated into YPD medium at a volume percentage of 1%, the fermentation temperature was 35°C, the shaking speed was 180 r / min, and the OD 600 The absorbance was measured at 400 nm. When the OD value was 8.2, the number of bacteria was 1.5×10 7 CFU / mL, the seed culture was completed, and the cerevisiae YY-14 seed liquid was obtained.

[0060] Lactobacillus plantarum YY-7 was inoculated into MRS medium at a volume percentage of 1%, and the fermentation temperature was 37°C. The culture was allowed to stand for 2 h. 600 The absorbance was measured at 400 nm. When the OD value was 6.1, the number of bacteria was 2.0×10 7CFU / mL, the seed culture was completed, and the Lactobacillus plantarum YY-7 seed liquid was obtained.

[0061] The seed liquid of Bacillus subtilis YY-12, the seed liquid of Saccharomyces cerevisiae YY-14 and the seed liquid of Lactobacillus plantarum YY-7 were uniformly mixed in a volume ratio of 1:1:1 to obtain a composite bacterial agent.

[0062] (2) The corn gluten powder was extruded and puffed at 160°C for 6s and crushed to a particle size of 40 mesh to obtain puffed corn gluten powder; the puffed corn gluten powder, bran, soybean meal, and corn germ meal were mixed uniformly at a dry weight ratio of 4:1:1:4 to obtain a fermentation matrix; corn slurry and composite bacterial agent were added to the fermentation matrix, the mass volume ratio of the fermentation matrix to the corn slurry was 100g:10mL, and the mass volume ratio of the fermentation matrix to the composite bacterial agent was 100g:1mL, and then acid protease was added in an amount of 25U of acid protease per gram of fermentation matrix, and the above substances were mixed uniformly. After hydration, the mixture was adjusted to a water content of 45% and a natural pH value; and then placed in a one-way valve breathable bag and cultured at 32°C for 4 days to obtain a fermentation product.

[0063] Example 4

[0064] An extract containing glutamine peptides QQQCCHQIR (SEQ ID NO. 1), QQCCHQIR (SEQ ID NO. 2), QPQPQFP (SEQ ID NO. 3) and QPQPP (SEQ ID NO. 4) is prepared as follows:

[0065] The fermented product obtained in Example 1 was dried at 65°C for 72 h and crushed. 2.5 g of the dried and crushed fermented product was accurately weighed and the volume was made up to 25 mL with distilled water. The product was stirred and extracted at 200 r / min at room temperature for 30 min, and then centrifuged at 12000 r / min for 30 min. The supernatant was collected to obtain the extract.

[0066] Example 5

[0067] The extract obtained in Example 4 was freeze-concentrated and dried at -80°C in a vacuum oven to obtain a lyophilized powder. 1.0 g of the lyophilized powder was dissolved in distilled water, and then the pH was adjusted to neutral with 0.1 mol / L NaOH, and the volume was made up to 10 mL. The solution was filtered through a 0.22 μm filter to obtain the test solution.

[0068] Take 2 mL of the test solution, add dithiothreitol (DTT) solution to a final concentration of 10 mmol / L, reduce in a 56°C water bath for 1 hour, add iodoacetamide (IAA) solution to a final concentration of 50 mmol / L, react in the dark for 40 minutes, desalt using a self-packed desalting column, and evaporate the solvent in a vacuum centrifuge at 45°C. The sample is then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0069] Liquid chromatography conditions are as follows:

[0070] 1) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 3 μm, 2) Mobile phase A: 0.1% formic acid; 3) Mobile phase B: 0.1% formic acid, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time per component: 66 min;

[0071] Table 1 Liquid chromatography time component program

[0072] time Phase B Phase A 0 4％ 96％ 2 8％ 92％ 45 28％ 72％ 55 40％ 60％ 56 95％ 5％ 66 95％ 5％

[0073] Mass spectrometry conditions are as follows:

[0074] 1) Primary mass spectrometry parameters:

[0075] Resolution: 70,000;

[0076] AGCtarget: 3e6;

[0077] MaximumIT: 100ms;

[0078] Scan range: 100 to 1500 m / z;

[0079] 2) Secondary mass spectrometry parameters

[0080] Resolution: 17,500;

[0081] AGCtarget: 1e5;

[0082] MaximumIT: 50ms;

[0083] TopN: 20;

[0084] NCE / steppedNCE: 28;

[0085] The mass spectrometry raw files were searched against the target protein database using the Byonic software. The search parameters were as follows:

[0086] 1) Fixed modifications: Carbamidomethyl (C);

[0087] 2) Variable modifications: Oxidation (M), Acetylation (N-term);

[0088] 3) Enzyme: Non specific;

[0089] 4) Maximum Missed Cleavages: 3;

[0090] 5) Peptide Mass Tolerance: 20 ppm;

[0091] 6) Fragment Mass Tolerance: 0.02 Da;

[0092] The raw files collected by mass spectrometry were searched using the Byonic database software. The peptides obtained were matched with the corn protein database, and four glutamine-rich peptides were obtained. The peptide information is shown in Table 2.

[0093] Table 2 Basic information of glutamine peptides

[0094]

[0095] Example 6

[0096] Solid phase synthesis of the glutamine peptide segments in Table 2 of Example 5, Q1: QQQCCHQIR, Q2: QQCCHQIR, Q3: QPQPQFP, Q4: QPQPP, was used for in vitro anti-inflammatory studies:

[0097] (1) Culture of rat small intestinal epithelial cells (IEC-6) and establishment of an LPS-induced cellular inflammation model

[0098] Cells were cultured in 25 cm 2 Cell flasks were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum and double-antibody antibodies at 37°C in a carbon dioxide incubator with 5% CO2, with the medium changed every other day. When the cells reached 80-90% confluency, they were subcultured at a 1:3 ratio. Cells from passages 20-30 were used for this experiment.

[0099] IEC-6 cells were cultured at a rate of 2 × 10 5 Cells / 100 μL were inoculated into a 96-well plate and cultured at 37°C for 24 h. Then, 100 μL of LPS solution with concentrations of 0, 10, 20, 50, 100, 150, 200, and 250 μg / mL was added to each well and cultured for another 24 h. Cell viability was determined by CCK-8 method, and the NO concentration of cells after LPS damage was determined by NO kit. The concentration of LPS damage was determined to be 100 μg / mL.

[0100] (2) Effect of glutamine peptide on the proliferation of IEC-6 cells

[0101] 100 μL IEC-6 cells (2×10 5 After culturing for 24 h, 100 μL of glutamine peptide solution with a final concentration of 0.001-1 mg / mL was added to each well. After further culturing for 24 h in a carbon dioxide incubator, 10 μL of CCK-8 reagent was added to each well. After further culturing for 4 h, the OD was measured on a microplate reader. 450 Alanyl-glutamine peptide (Ala-Gln) (A0550) was used as a control group. The Ala-Gln was purchased from Sigma-Aldrich (powder).

[0102] The results are as follows Figure 1 As shown, within the concentration range of 1 to 1000 μg / mL, the survival rates of IEC-6 cells treated with the four glutamine peptides and Ala-Gln were all above 90%, indicating that the four glutamine peptides and Ala-Gln had no significant toxicity to IEC-6 cells. When the concentration was greater than 10 μg / mL, the growth-promoting effect of glutamine peptide Q1 on IEC-6 cells was significantly greater than that of Ala-Gln, and all groups showed a pro-proliferation effect at concentrations greater than 50 μg / mL. The results showed that glutamine peptides Q1 to Q4 were non-toxic to IEC-6 cells and had a nutritional effect on IEC-6 cells, among which glutamine peptide Q1 had the best growth-promoting effect.

[0103] (3) Effect of glutamine peptide on LPS-induced IEC-6 cell survival

[0104] After multiple preliminary tests, we found that glutamine peptides Q1 to Q4 had a protective effect on IEC-6 cells at three doses of 25, 50, and 100 μg / mL. Glutamine peptides (Q1 to Q4 and Ala-Gln) and LPS (final concentration 100 μg / mL) were added to IEC-6 cells after 24 hours of culture and incubated for 24 hours. The IEC-6 cells cultured alone were used as the blank control group (CON). The effect of glutamine peptides on the survival rate of IEC-6 cells induced by LPS is shown in Figure 2. Figure 2 .Depend on Figure 2It can be seen that compared with the LPS damage model group, different concentrations of glutamine peptides Q1 and Q2 co-incubated with LPS can significantly increase the survival rate of IEC-6 cells, and with the increase of glutamine peptide concentration, the cell survival rate of IEC-6 showed an increasing trend; it is worth noting that when the concentration is 100 μg / mL, the IEC-6 cell survival rates of glutamine peptide Q1, Q2 and Q4 groups were 76.75%, 71.52% and 67.46%, respectively, which were significantly higher than the Ala-Gln positive control group (cell survival rate 65.99%); when the concentration is 25 μg / mL, peptide Q3 had no significant effect on cell survival rate (p>0.05); when the concentrations are 50 and 100 μg / mL, peptides Q3 and Q4 can significantly increase the cell survival rate of IEC-6 (p<0.0001), and the IEC-6 cell survival rate of peptide Q4 is higher than that of peptide Q3. The above results showed that the four glutamine peptides had a protective effect on LPS-induced IEC-6 cell damage, and peptide Q1 had the best protective effect on IEC-6 cells.

[0105] (4) Effect of glutamine peptide on NO content in LPS-induced IEC-6 cells

[0106] Five experimental groups were set up, namely blank control (CON), model group (LPS), Ala-Gln positive control group, Gln peptide low (25 μg / mL), medium (50 μg / mL) and high (100 μg / mL) dose groups. IEC-6 cells in the logarithmic growth phase were taken and 100 μL IEC-6 cells (2×10 5 After culturing for 24 hours, the culture medium was discarded. Subsequently, 100 μL of complete culture medium and LPS solution (100 μg / mL) were added to each well of the CON group and model group. 100 μL of peptide solution and LPS solution of different concentrations were added to each well of the low, medium, and high dose groups of Gln peptide to make the final LPS concentration of 100 μg / mL. After culturing for 24 hours, the OD was measured on a microplate reader using a NO detection kit. 540 The absorbance value was measured and the secretion amount of NO in the cell supernatant was calculated according to the NO standard curve.

[0107] The results are as follows Figure 3 As shown. Figure 3It can be seen that compared with the CON group, the NO content in IEC-6 cells was significantly increased after LPS treatment (p<0.0001); compared with the LPS model group, the Ala-Gln (50 μg / mL) positive control group could significantly alleviate the release of NO (p<0.0001); Glutamine peptide Q1 and Q2 could significantly reduce the release of NO at all concentrations, and showed a dose-dependent relationship with the peptide concentration. When the concentrations were 50 and 100 μg / mL, the NO concentration in the glutamine peptide Q1 group was lower than that in the glutamine peptide Q2 group. Peptide Q2 group; when the concentration of Q1 and Q2 was 100 μg / mL, the NO content in IEC-6 cells was significantly lower than that in the Ala-Gln positive control group; when the concentration of glutamine peptides Q3 and Q4 was 25 μg / mL, there was no significant effect on the NO content, indicating that at this concentration, glutamine peptides Q3 and Q4 could not alleviate the inflammatory damage of IEC-6 cells caused by LPS; when the concentration of glutamine peptides Q3 and Q4 was 50 μg / mL and 100 μg / mL, they could significantly reduce the release of NO. The above results show that glutamine peptides Q1 to Q4 can alleviate the inflammatory damage of IEC-6 cells caused by LPS.

[0108] (5) Effect of glutamine peptide on the content of inflammatory factors in IEC-6 cells induced by LPS

[0109] IEC-6 cells were seeded into 6-well plates (1×10 7 cells / 2mL / well) and perform the experiment according to the operation and grouping in (4). After the culture, the cells and cell supernatant were collected and centrifuged at 12000r / min for 5min. The supernatant was collected and the cytokine content was determined according to the instructions of the ELISA kit.

[0110] The results are as follows Figure 4As shown in the figure, compared with the CON group, LPS treatment significantly increased the concentrations of pro-inflammatory factors IL-6, TNF-α, IL-1β and anti-inflammatory factor IL-10 in IEC-6 cells (p<0.0001), indicating that LPS caused inflammatory damage to IEC-6 cells. Compared with the LPS model group, Ala-Gln significantly reduced the levels of IL-6, TNF-α, and IL-1β in IEC-6 cells and increased the level of IL-10, indicating that Ala-Gln alleviated the cell damage of IEC-6 caused by LPS to a certain extent. Compared with the LPS model group, glutamine peptides Q1 and Q2 at all concentrations could significantly reduce the increase of inflammatory factors IL-6, IL-1β and TNF-α in IEC-6 cells caused by LPS, and significantly increase the content of IL-10. When the peptide concentrations were 50 μg / mL and 100 μg / mL, the concentrations of IL-6, IL-1β and TNF-α in IEC-6 cells were significantly lower than those in the Ala-Gln positive control group, and the concentration of IL-10 was significantly higher than that in the positive control group, indicating that at this concentration, glutamine peptides Q1 and Q2 have good anti-inflammatory effects.

[0111] Comparative Example 1

[0112] The difference from Example 1 is that step (2) does not contain the step of "then placing in a one-way valve breathable bag and culturing at 30° C. for 5 days". The rest is the same as Example 1 to obtain a product; then the product is extracted according to the method of Example 4, and the supernatant is collected to obtain an unfermented extract;

[0113] Example 7

[0114] The extract obtained in Example 4 was recorded as glutamine peptide extract, and the following experiments on the effect on IEC-6 cell survival rate were carried out respectively with Comparative Example 1:

[0115] The cells were assessed by CCK-8 assay. 100 μL of IEC-6 cells (2×10 5 After culturing for 24 h, 100 μL of glutamine peptide extract (or extract before fermentation) prepared in DMEM with concentrations of 0, 0.01, 0.1, 1.0, 10, and 100 μg / mL was added to each well. After further culturing for 24 h, 10 μL of CCK-8 solution was added to each well. The cells were cultured for another 4 h, and the OD was measured on a microplate reader. 450 The absorbance value.

[0116] The results are as follows Figure 5As shown, when the concentration was less than 0.01 μg / mL, the survival rates of IEC-6 cells in the unfermented extract and glutamine peptide extract groups were both greater than 90%, indicating that the unfermented extract and glutamine peptide extract had no obvious toxicity to IEC-6 cells; when the concentration was greater than 0.01 μg / mL, glutamine peptide extract showed a pro-proliferation effect on IEC-6 cells, and the unfermented extract showed a pro-proliferation effect on IEC-6 when it was greater than 10 μg / mL.

[0117] Example 8

[0118] The extract obtained in Example 4 was recorded as glutamine peptide extract, and the following in vitro anti-inflammatory experiments were performed together with Comparative Example 1:

[0119] 100 μL of IEC-6 cells (2×10 5 cells / well), and after culturing for 24 hours, the culture medium was discarded. 100 μL of complete culture medium was added to each well of the blank control group, and 100 μL of LPS solution prepared with DMEM at a concentration of 100 μg / mL was added to each well of the model group. LPS solution and glutamine peptide samples (prepared with DMEM) of different concentrations were added to each well at the same time, so that the final concentration of LPS was 100 μg / mL, and the final concentrations of the extracts were 50, 100, and 200 μg / mL, respectively. After culturing for 24 hours, the OD was measured on a microplate reader using a NO detection kit. 540 The absorbance value was calculated and the secretion of NO in the cell supernatant was calculated according to the NO standard curve; at the same time, the OD value was measured on the microplate reader using the CCK-8 kit. 450 The cell survival rate was calculated by the absorbance value of the cells. The blank control group (CON) was used without the addition of LPS solution and extract, and the model group (LPS) was used with the addition of LPS solution but no extract.

[0120] The results are as follows Figure 6 As shown in the results, compared with the LPS injury model group, incubation with the unfermented extract had no significant difference in the IEC-6 cell survival rate and NO content (p>0.05), while incubation with different concentrations of glutamine peptide extract significantly increased the IEC-6 cell survival rate (p<0.05) and significantly reduced the NO release (p<0.05), indicating that the glutamine peptide extract of the present invention has a good in vitro anti-inflammatory effect.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A glutamine peptide with anti-inflammatory effect, characterized in that: The glutamine peptide comprises at least one of the amino acid sequences of SEQ ID NO.1 to SEQ ID NO.

4.

2. A fermentation product containing the glutamine peptide according to claim 1, characterized in that: The fermentation product is prepared by the following method: a fermentation matrix, corn steep liquor, a composite bacterial agent, an acidic protease and water are mixed to adjust the water content of the mixture to 45%, and the mixture is fermented and cultured at 30-35°C for 3-5 days to obtain a fermentation product; the fermentation matrix is obtained by mixing corn gluten powder, bran, soybean meal and corn germ meal in a dry basis ratio of 4:1:1:4; and the composite bacterial agent is composed of Bacillus subtilis YY-12, Saccharomyces cerevisiae YY-14 and Lactobacillus plantarum YY-7.

3. The fermentation product according to claim 2, characterized in that Each 3mL of compound bacterial agent contains (1.0~2.0)×10 8 CFU Bacillus subtilis YY-12, (1.0~2.0)×10 7 CFU of Saccharomyces cerevisiae YY-14 and (1.0~3.0)×10 7 CFU Lactobacillus plantarum YY-7.

4. The fermentation product according to claim 2, characterized in that The mass volume ratio of the fermentation matrix to the corn steep liquor is 100 g:10 mL; the mass volume ratio of the fermentation matrix to the composite bacterial agent is 100 g:1 mL.

5. The fermentation product according to claim 2, characterized in that Add 25 U of acid protease per 1 g of fermentation substrate.

6. An extract containing the glutamine peptide according to claim 1, characterized in that: The fermentation product is prepared by the following steps: drying the fermentation product according to any one of claims 2 to 5, mixing the mixture with water for extraction, and collecting the supernatant to obtain the extract.

7. The extract according to claim 6, characterized in that The leaching includes stirring leaching, the stirring speed is 200r / min, and the stirring leaching time is 30min.

8. Use of any one of the following in the preparation of an anti-inflammatory product, characterized in that: (1) The glutamine peptide according to claim 1; (2) The fermentation product according to claims 2 to 5; (3) The extract according to claim 6 or 7.

9. Use of any one of the following in the preparation of a product for preventing and treating inflammatory bowel disease, characterized in that: (1) The glutamine peptide according to claim 1; (2) The fermentation product according to claims 2 to 5; (3) The extract according to claim 6 or 7.

10. The use according to claim 8 or 9, characterized in that: The products include medicines or feed additives.

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