Spina date seed active peptide extract as well as preparation method and application thereof

Abstract

The invention provides a spina date seed active peptide extract as well as a preparation method and application thereof, and belongs to the field of bioactive peptides. The preparation method of the spina date seed active peptide extract comprises the following steps: crushing spina date seeds, degreasing with petroleum ether, extracting spina date seed protein by adopting an acetone-ammonium sulfate precipitation method, fermenting the spina date seed protein through lactobacillus reuteri LRSY523, carrying out ultrafiltration to intercept components less than 3kD, and desalting to obtain the spina date seed active peptide extract named as FZSSP-L. The FZSSP-L prepared by the invention can obviously improve the ulcerative colitis symptoms of mice, for example, the weight and colon length of the mice are obviously recovered, the DAI score is obviously reduced, the expression of intestinal tight junction protein is obviously increased, and the like; meanwhile, the FZSSP-L treatment obviously inhibits the inflammatory phenotype of mouse colonic macrophages M1 and promotes the anti-inflammatory phenotype of M2. Therefore, the FZSSP-L is expected to be researched and developed into related medical products for preventing and / or treating the inflammatory bowel disease.

Description

Technical Field

[0001] The invention belongs to the field of bioactive peptides, and specifically relates to a method for preparing a jujube seed active peptide extract (FZSSP-L), and application of FZSSP-L in preparing related pharmaceutical products for preventing and / or treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) refers to a series of chronic inflammatory diseases that occur in the gastrointestinal tract. In recent years, due to rapid urbanization, industrialization and changes in lifestyle, the incidence of IBD has gradually increased in low-incidence areas such as Asia. It has now become a common digestive system disease in my country. Due to a series of problems such as long course of disease, difficulty in cure, high cost, difficulty in control, easy deterioration and even disability, it has brought a huge burden to human survival and life. At present, the clinical treatment of IBD mainly relies on surgery, lifestyle intervention or drug therapy. There are many types of IBD therapeutic drugs used in clinical practice, such as aminosalicylic acid, glucocorticoids, immunosuppressants and biological agents, but these drugs have limited effects or serious adverse reactions and cannot meet the treatment needs of patients. Therefore, screening new therapeutic drugs with significant efficacy, affordable price and few side effects remains a research hotspot in the medical community.

[0003] Plant peptides are biologically active peptides with physiological functions isolated from plants. They are widely available, low in cost, high in nutritional value, and have many biological functions, such as promoting immunity, antibacterial, antiviral, lowering blood lipids, lowering blood pressure, and hormone regulation. They are also easy to digest and absorb, and have become a type of active factor that is beneficial to human health and can be used as an important treasure house for drug screening. Ziziphus jujuba seeds (scientific name: Ziziphus jujuba Mill.var.spinosa) are the dried mature seeds of the rhamnaceae plant Ziziphus jujuba, which are mainly produced in the northwest region of my country and the Yellow River Basin. Ziziphus jujuba seeds are authentic Chinese medicinal resources with both medicinal and edible properties, and have extremely high medicinal and economic values. Modern pharmacological studies have shown that Ziziphus jujuba seeds contain a variety of effective ingredients and have pharmacological activities such as antioxidant, anti-inflammatory and anti-tumor. Related studies have shown that Ziziphus jujuba seeds contain rich protein resources, with a protein content of about 27%. However, there are few studies on Ziziphus jujuba seeds protein, and there has been no in-depth study on its functional characteristics and biological activities. At the same time, research on Ziziphus jujuba seeds protein hydrolysates is relatively lacking.

[0004] The present invention prepares a jujube seed active peptide extract with significant efficacy in improving IBD through probiotic fermentation, which is expected to be further developed into a new type of pharmaceutical product for preventing and controlling IBD, and has important guiding significance for improving and fully utilizing the medicinal value of jujube seed protein resources. Summary of the invention

[0005] Based on the above problems, the purpose of the present invention is to provide a jujube seed active peptide extract (FZSSP-L) and a preparation method thereof, as well as to use FZSSP-L in the preparation of related pharmaceutical products for preventing and / or treating inflammatory bowel disease.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a wild jujube seed active peptide extract comprises the following steps:

[0008] Step 1, preparing a protein extract from Chinese date kernels by using an acetone-ammonium sulfate precipitation method;

[0009] Step 2, the protein extract of Chinese jujube kernel is fermented by Lactobacillus reuteri LRSY523 and the components less than 3 kDa are retained by ultrafiltration tube, and FZSSP-L is obtained after desalting.

[0010] The Lactobacillus reuteri LRSY523 can be found in patent ZL202111221173.2 (the strain collection number is CGMCC No. 22953).

[0011] Furthermore, the specific steps of step 1 are as follows:

[0012] (1) Take the Chinese jujube seed medicinal material, grind it in a grinder, and then pass it through a 60-mesh sieve, add petroleum ether in a ratio of W / V=1:4-6, stir for 24-36 hours to defat, centrifuge, and after the residual petroleum ether is precipitated and volatilized, grind and sieve again to obtain the Chinese jujube seed defatted powder, and store it in a refrigerator at 4°C for later use;

[0013] (2) Weigh an appropriate amount of defatted jujube kernel powder, add a protein extract solution, i.e., a 20 mM Tris-HCl solution containing 0.85% NaCl and a pH of 8.0, at a ratio of W / V = 1:4-6, extract at 4°C with stirring for 24-36 hours, and centrifuge to obtain the supernatant;

[0014] (3) Add 2-3 times the volume of -20°C precooled 80% acetone, precipitate for 1-2 hours, centrifuge, discard the supernatant, keep the precipitate, place the precipitate at -20°C to allow the acetone to completely evaporate; dissolve the precipitate with 20 mM Tris-HCl solution at pH = 8.0, centrifuge and take the supernatant;

[0015] (4) According to the volume of the supernatant, gradually add ammonium sulfate solid to make the saturation reach 90%, stand at 4°C for 30-45 minutes, then centrifuge, discard the supernatant and keep the precipitate;

[0016] (5) The precipitate was dissolved with a 20 mM Tris-HCl solution at pH 8.0, and the supernatant was retained after centrifugation. After desalting by dialyzing with a 100 Da dialysis bag, the resulting solution was the protein extract of the jujube kernel, which was freeze-dried and stored for later use.

[0017] Furthermore, the specific steps of step 2 are:

[0018] (1) Seed solution preparation

[0019] Lactobacillus reuteri LRSY523 was inoculated into MRS seed medium at a volume ratio of 2%, cultured at 37°C and 120 r / min for about 22-26 h, and activated 2-3 times to a bacterial concentration of 1×10 8 cfu / mL or more;

[0020] The seed culture medium formula is: MRS broth 48.3 g, distilled water 1000 mL, pH 6.2±0.2;

[0021] (2) Liquid fermentation

[0022] Inoculate 10% of the fermentation strain seed liquid into the fermentation medium, and ferment at 37°C and 120 r / min for 46-50 hours; after the fermentation is completed, heat with 100°C boiling water for 5-10 minutes, centrifuge the fermentation liquid, and keep the supernatant;

[0023] The fermentation medium formula is: 10-20g of jujube kernel protein, 10-25g of glucose, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 5g of sodium acetate, 1000mL of distilled water, pH value 6.2±0.2;

[0024] (3) Using a 3 kDa ultrafiltration device to retain polypeptide components with a molecular weight less than 3 kDa, and after desalting with 200 Da dialysis, FZSSP-L was obtained and freeze-dried for future use.

[0025] The active peptide extract FZSSP-L prepared by the present invention was used to intervene in the DSS-induced ulcerative colitis mouse model. The results showed that FZSSP-L could significantly improve the symptoms of ulcerative colitis in mice, such as: significant recovery of mouse weight and colon length, significant reduction of DAI score, significant increase in intestinal tight junction protein expression, etc.; at the same time, FZSSP-L treatment significantly inhibited the M1 inflammatory phenotype of mouse colon macrophages and promoted the M2 anti-inflammatory phenotype. The experiment showed that FZSSP-L has the potential to be developed as a related pharmaceutical product for the prevention and / or treatment of inflammatory bowel disease.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The active peptide extract FZSSP-L of Chinese jujube seeds obtained by fermentation of Lactobacillus reuteri LRSY523 of the present invention has high yield, good anti-inflammatory activity, small molecular weight, easy to be absorbed and utilized by human body, high bioavailability, and easy to develop related pharmaceutical products. Microorganisms have an efficient protein hydrolysis system, which is conducive to the release of bioactive peptides. The active peptides obtained by microbial fermentation have the advantages of low cost, simple preparation process, safety, non-toxicity and good taste, and are suitable for large-scale, low-cost and diversified protein peptide production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 FZSSP-L powder morphology image;

[0029] Figure 2 Effects of FZSSP-L on body weight changes and DAI index of colitis mice (A: changes in mouse body weight; B: changes in mouse DAI index);

[0030] Figure 3 Effect of FZSSP-L on colon length of colitis mice (A: picture of mouse colon; B: length of mouse colon);

[0031] Figure 4 Effects of FZSSP-L on the thymus index and spleen index of colitis mice (A: thymus index of mice; B: spleen index of mice);

[0032] Figure 5 Effects of FZSSP-L on cytokines in the serum of colitis mice (AD: relative levels of TNF-α, IL-1β, IL-6, and IL-10 in mouse serum);

[0033] Figure 6 Effects of FZSSP-L on HE staining and PAS staining of colon tissues in colitis mice (A: HE staining results; B: PAS staining results);

[0034] Figure 7 Effects of FZSSP-L on the intestinal barrier of colitis mice (WB detection of the expression of ZO-1, Occludin, and Claudin-1 proteins in colon tissues);

[0035] Figure 8 Effect of FZSSP-L on macrophage polarization in colon tissue of colitis mice (WB detected the expression of M1 and M2 macrophage polarization markers in colon tissue). DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail in conjunction with specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the method, step or condition of the present invention shall fall within the scope of protection of the present invention.

[0037] Example 1: Preparation of active peptide extract from Ziziphus jujuba seeds (FZSSP-L)

[0038] 1. Preparation of Ziziphus jujuba seed protein extract

[0039] Weigh 100g of spiny jujube kernel, crush it, pass it through a 60-mesh sieve, add 500mL of petroleum ether, stir for 24h for defatting, centrifuge at 3000rpm for 10min, precipitate under ventilation conditions, volatilize the residual petroleum ether, crush it again and sieve it to obtain 83g of spiny jujube kernel defatted powder, add 415mL of protein extract, i.e., 20mM Tris-HCl solution with pH=8.0 containing 0.85% NaCl, stir at 4℃ for 36h, centrifuge at 4℃ 11000rpm for 30min, retain the supernatant, and filter with gauze; collect the filtrate and add 2 times the volume of 80% acetone precooled at -20℃, precipitate at -20℃ for 2 hours, centrifuge at 4℃ 11000rpm for 30min, discard the supernatant, retain the precipitate, place the precipitate at -20℃ to completely volatilize the acetone; dissolve the precipitate with 20mM Tris-HCl solution with pH=8.0, centrifuge at 4℃ 11000rpm for 30min, rpm centrifuge for 30 min, retain the supernatant; accurately measure the volume of the supernatant, calculate the amount of ammonium sulfate according to the ammonium sulfate saturation usage table, gradually add ammonium sulfate solid to it to make its saturation reach 90%, let it stand at 4°C for 30 min, centrifuge at 4°C 11000 rpm for 30 min, discard the supernatant, and retain the precipitate; dissolve the precipitate with 20mM Tris-HCl solution with pH=8.0, centrifuge at 4°C 11000 rpm for 30 min, retain the supernatant, put it into a 100Da dialysis bag, dialyze and desalt at 4°C, and obtain 2.194g of Ziziphus jujuba seed protein extract, which is freeze-dried and stored for later use.

[0040] 2. Preparation of active peptide extract from Ziziphus jujuba seeds

[0041] Lactobacillus reuteri LRSY523 was inoculated into 5 mL MRS liquid culture medium at a volume ratio of 2%, cultured at 37 °C and 120 r / min for 22 h, and activated twice to make the bacterial concentration reach 1×10 8cfu / mL or more; prepare a fermentation medium without a nitrogen source according to the formula in the invention content of the specification, weigh 200 mg of the above-mentioned freeze-dried Chinese jujube kernel protein extract, dissolve it in 10 mL of the fermentation medium without a nitrogen source, filter and sterilize it with a 0.22 μm filter membrane, inoculate 10% of the fermentation strain seed liquid into the fermentation medium, and ferment it at 37°C and 120 r / min for 48 hours; after the fermentation is completed, heat it with 100°C boiling water for 5 minutes, and centrifuge the fermentation liquid at 4°C and 11000rpm for 30 minutes, and keep the supernatant; use a 3kDa ultrafiltration device to retain polypeptide components with a molecular weight less than 3kDa, use a 200Da dialysis bag, dialyze and desalt to obtain FZSSP-L20.17 mg, which is freeze-dried and stored for later use.

[0042] Figure 1 This is the powder morphology of FZSSP-L, which is white.

[0043] Example 2: Effects of FZSSP-L on related indicators of ulcerative colitis mice

[0044] 1. Construction of ulcerative colitis mouse model

[0045] (1) 50 male C57BL / 6J mice, weighing 20-22 g, were raised and tested in an SPF rodent laboratory (China Institute of Radiation Protection), with an ambient temperature of 25±1°C, humidity of 60±5%, and a 12-h cycle.

[0046] (2) After one week of adaptive feeding with normal drinking water, the experiment was started. 50 mice were weighed and randomly divided into control group (group C), model group (group M), 5-ASA positive control group (group PC), FZSSP-L low-dose group (group L) and FZSSP-L high-dose group (group H), with 10 mice in each group. After the adaptive feeding, except for the normal control group, the drinking water of the other groups was changed to 2% DSS. The mice drank 2% DSS drinking water continuously (7 days) to induce colitis and then switched back to normal drinking water until the 13th day. During this period, 5-ASA was gavaged at a dose of 200 mg / kg per mouse per day in the positive control group, and FZSSP-L low-dose group and high-dose group were gavaged at doses of 40 mg / kg and 80 mg / kg, respectively.

[0047] (3) During the experiment, the weight, stool hardness and rectal bleeding of the mice were recorded daily, the weight change rate of the mice was calculated, and the scores were calculated according to Table 1 to calculate the disease activity index (DAI). The results are shown in Table 1. Figure 2A-2B. On the 14th day, each experimental mouse was euthanized and dissected. The colon segment was cut and the length of the colon was measured with a ruler and photographed for preservation (eg Figure 3 A-3B), and calculated the thymus index and spleen index of each group of mice. The results are shown in Figure 4 A-4B.

[0048] Table 1 DAI scoring criteria

[0049]

[0050] Studies have shown that colitis is often accompanied by weight loss. The weight changes of mice showed that with the extension of time, the weight of the mice in the control group showed a relatively stable upward trend. The weight of the mice in other groups showed a trend of first decreasing and then increasing to varying degrees with the increase of DSS treatment time. Among them, the weight loss in the model group was the most obvious. Compared with the model group, both PC and FZSSP-L could restore the weight loss caused by colitis, and the effect of FZSSP-L was better.

[0051] In colitis research, DAI is often used to reflect the severity of colitis. The DAI score results showed that compared with the model group, the DAI scores of the PC group and the FZSSP-L group were lower, indicating that the severity of colitis in the PC and different concentrations of FZSSP-L groups was lower, that is, PC and FZSSP-L have the effect of inhibiting colitis, and FZSSP-L has a better effect.

[0052] Colon shortening is also one of the typical characteristics of colitis. The changes in colon length of mice in each group showed that the colon length of mice in the model group was significantly shorter than that in the control group, while the colon length of the PC group and different concentrations of FZSSP-L groups was significantly restored compared with the model group, indicating that both PC and FZSSP-L treatments can restore the colon shortening caused by DSS, and the effect of FZSSP-L is better and concentration-dependent. This indicates that FZSSP-L has a significant inhibitory effect on colitis.

[0053] Immune factors play an important role in the pathogenesis of colitis. The thymus and spleen are important immune organs. The thymus index and spleen index can roughly reflect the immune function and inflammation of mice. The results of the thymus index and spleen index of each group of mice showed that compared with the control group, the thymus index of the model group mice was significantly reduced and the spleen index was significantly increased; after treatment with PC and FZSSP-L, these phenomena were restored, and the effect of FZSSP-L was better and concentration-dependent. It shows that FZSSP-L can enhance the immune function of colitis mice and reduce the inflammatory response caused by splenomegaly in colitis mice.

[0054] 2. Detection of cytokines in mouse serum

[0055] (1) The mice were fasted for 14 hours before being sacrificed. The animals were anesthetized with isoflurane, their left whiskers were trimmed, and blood was collected from the medial canthal vein. During the operation, hair was avoided to avoid hemolysis. The blood sample was collected into a 1.5 mL EP tube, placed on ice for 30 minutes, and centrifuged at 3000 rpm, 4°C, and 15 minutes. The upper light yellow clear liquid was carefully collected as serum, and stored at -80°C after aliquoting.

[0056] (2) Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression level of cytokines in mouse serum. The mouse ELISA kit of Jiangsu Enzyme-Free Industrial Co., Ltd. was used to measure the content of TNF-α, IL-1β, IL-6, and IL-10 in the serum samples of each group of mice. The experimental operation was carried out according to the steps in the product manual. The results are shown in the figure. Figure 5 As shown in A-5D.

[0057] Cytokine detection results showed that compared with the control group, the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in the serum of the model group mice were significantly increased, and the level of anti-inflammatory cytokine IL-10 was significantly reduced; after treatment with PC and FZSSP-L, the above indicators of the model mice were significantly restored, and the effect of FZSSP-L was better and concentration-dependent. These results suggest that FZSSP-L can significantly alleviate DSS-induced inflammatory response in mice.

[0058] 3. Pathological staining of colon tissue sections

[0059] (1) Hematoxylin-eosin (H&E) staining: Mouse colon tissue samples were fixed with 4% paraformaldehyde for 24 h, embedded in paraffin, and sliced. Eosin stain was used to stain the extracellular space and extracellular matrix, and hematoxylin stain was used to stain the cell nucleus. After dehydration, the sections were observed under a microscope and images were collected. Figure 6 As shown in A.

[0060] Colitis can directly lead to colon damage and inflammation. The results of H&E staining of colon tissue showed that the pathological characteristics of mice in the control group were normal, while the pathological characteristics of mice in the model group changed significantly, including the disappearance of crypts, a large reduction in goblet cells, a large number of inflammatory cell infiltrations, mucosal erosions and submucosal edema, indicating that the DSS-induced mouse colitis model was successfully established. Compared with the model group, the above-mentioned colitis symptoms in the PC group and FZSSP-L group were alleviated to varying degrees, indicating that both PC and FZSSP-L treatments can inhibit DSS-induced colon damage and inflammatory response, and FZSSP-L has a better effect and is concentration-dependent. Explanation: FZSSP-L can significantly alleviate colon damage and inflammatory response in colitis mice.

[0061] (2) PAS staining: Mouse colon tissue samples were fixed with 4% paraformaldehyde for 24 hours, then embedded in paraffin and sliced, dewaxed and hydrated. They were first stained with dyes B, A, and C in the PAS dye kit (dye A should be kept away from light). Then, they were differentiated and blued with hydrochloric acid and ammonia water. Finally, the samples were dehydrated and sealed, and the images were observed and collected under a microscope. Figure 6 As shown in B.

[0062] Goblet cells form a physical barrier of the intestinal mucosa by secreting mucus covering the outermost layer of the intestinal epithelium. Mucus (a glycoprotein) is the main component of the intestinal mucosal barrier, which is mainly secreted by goblet cells and continuously distributed on the surface of intestinal epithelial cells. In the body of colitis, due to the large reduction in the number of goblet cells, mucus secretion is greatly reduced, causing the mucosal layer to become thinner or even partially missing. The PAS staining results of colon tissue showed that compared with the control group, the number of goblet cells in the model group was significantly reduced, the secreted mucus was also significantly reduced, and the intestinal mucosal barrier was severely damaged, indicating that DSS caused intestinal mucosal barrier damage in mice. Compared with the model group, the number of goblet cells in the PC group and the FZSSP-L group increased, and the secreted mucus also increased, indicating that both PC and FZSSP-L treatments can inhibit the intestinal mucosal barrier damage caused by DSS, and the effect of FZSSP-L is better and concentration-dependent. Explanation: FZSSP-L can inhibit the intestinal mucosal damage of mice caused by DSS.

[0063] 4. Western blot analysis

[0064] (1) Protein extraction: Take out the colon tissue from the -80℃ ultra-low temperature freezer, add appropriate amount of RIPA lysis buffer and PMSF, grind the colon tissue sample with an automatic sample freezing grinder at 4℃, 65Hz for 2min, let it stand on ice for 30min, and centrifuge it at 13000rpm for 15min at 4℃ to collect the supernatant, and use the BCA protein concentration assay kit to detect the sample concentration. Finally, take an appropriate amount of the diluted supernatant and mix it with 5× reduced protein loading buffer and denature it (100℃, 10min). After completion, place it on ice for rapid cooling and store it in a -20℃ refrigerator for later use.

[0065] (2) Immunoblotting experiment: Equal amounts of protein samples were separated using 10% SDS-PAGE, and the protein bands were transferred to a PVDF membrane. The PVDF membrane was incubated at room temperature for 30 minutes using a protein-free rapid blocking solution (1×) to block the impurities. After the incubation, the PVDF membrane was washed with 1× TBST buffer. The primary antibody was incubated overnight at 4°C. After the incubation, the PVDF membrane was washed with 1× TBST buffer and incubated with a horseradish peroxidase-labeled secondary antibody at room temperature for 2 hours. The expression of the protein bands was detected using a basic ECL chemiluminescence detection kit.

[0066] Using β-actin antibody as a control, the effect of FZSSP-L on the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 in the colon tissue of colitis mice was detected. Figure 7 As shown; and the effect of FZSSP-L on the expression of M1 and M2 polarization markers in macrophages of colitis mice, as shown Figure 8 shown.

[0067] Tight junction proteins of intestinal epithelial cells include intracellular proteins (such as ZO-1) and cell surface proteins (such as Occludin and Claudin-1). WB results showed that the expression levels of ZO-1, Occludin, and Claudin-1 proteins in the colon of the normal group were high, while the expression levels of ZO-1, Occludin, and Claudin-1 proteins in the colon of the model group were significantly reduced, indicating that DSS can destroy the integrity of the intestinal barrier function by reducing the expression of ZO-1, Occludin, and Claudin-1 proteins. Compared with the model group, the expression levels of ZO-1, Occludin, and Claudin-1 proteins in the PC and FZSSP-L groups were increased, and the effect of FZSSP-L was better, which was concentration-dependent. Explanation: FZSSP-L can better inhibit the intestinal barrier damage caused by DSS.

[0068] Macrophage polarization disorder is of great significance to the occurrence and development of IBD. WB results showed that compared with the control group, the expression levels of M1 macrophage markers (CD86 and iNOS) in the model group were significantly increased, indicating that there were more M1 macrophages in the intestinal tissue and the intestinal tissue inflammation was more severe. Compared with the model group, the expression levels of CD86 and iNOS in the FZSSP-L groups with different concentrations were reduced, indicating that FZSSP-L could significantly inhibit the expression of CD86 and iNOS in colitis mice, and it was concentration-dependent. On the contrary, compared with the model group, the expression levels of M2 macrophage markers (CD206 and Arg1) in the FZSSP-L groups with different concentrations were increased, indicating that FZSSP-L could promote the expression of CD206 and Arg1 in colitis mice, and it was concentration-dependent. It shows that FZSSP-L can inhibit the polarization of macrophages to M1 and promote the polarization of macrophages to M2.

[0069] The present invention uses the active peptide extract FZSSP-L of Chinese jujube seeds prepared by the above embodiment to intervene in the DSS-induced ulcerative colitis mouse model. The results show that compared with the positive control PC, FZSSP-L can more significantly improve the symptoms of ulcerative colitis in mice, such as: the weight and colon length of mice are significantly restored, the DAI score is significantly reduced, and the expression of intestinal tight junction proteins is significantly increased; at the same time, FZSSP-L treatment significantly inhibits the inflammatory phenotype of M1 colon macrophages in mice and promotes the anti-inflammatory phenotype of M2. Therefore, the FZSSP-L extracted by the present invention can be used as an ideal choice for preparing related pharmaceutical products for the prevention and / or treatment of inflammatory bowel disease.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of protection of the invention. It should be pointed out that, for those skilled in the art, the above-mentioned implementation methods can also be subjected to several deformations, combinations and improvements without departing from the concept of the present invention, and these all belong to the scope of protection of this patent.

Claims

1. A method for preparing a jujube seed active peptide extract having the efficacy of preventing and / or treating inflammatory bowel disease, characterized in that: The steps include: Step 1, preparing a protein extract from Chinese date kernels by an acetone-ammonium sulfate precipitation method; Step 2, the protein extract of Chinese jujube kernel is fermented by Lactobacillus reuteri LRSY523 and the components less than 3 kDa are retained by ultrafiltration tube, and the Chinese jujube kernel active peptide extract FZSSP-L is obtained after desalting.

2. The method for preparing the active peptide extract of Semen Ziziphi Spinosae according to claim 1, characterized in that: The specific steps of step 1 are: (1) Take the Chinese jujube seed medicinal material, grind it in a grinder, and then pass it through a 60-mesh sieve, add petroleum ether in a ratio of W / V=1:4-6, stir for 24-36 hours to defat, centrifuge, and after the residual petroleum ether is precipitated and volatilized, grind and sieve again to obtain the Chinese jujube seed defatted powder, and store it in a refrigerator at 4°C for later use; (2) Weigh an appropriate amount of defatted jujube kernel powder, add a protein extract solution, i.e., a 20 mM Tris-HCl solution containing 0.85% NaCl and a pH of 8.0, at a ratio of W / V = 1:4-6, extract at 4°C with stirring for 24-36 hours, and centrifuge to obtain the supernatant; (3) Add 2-3 times the volume of -20°C precooled 80% acetone, precipitate for 1-2 hours, centrifuge, discard the supernatant, keep the precipitate, place the precipitate at -20°C to allow the acetone to completely evaporate; dissolve the precipitate with 20 mM Tris-HCl solution at pH = 8.0, centrifuge, and take the supernatant; (4) According to the volume of the supernatant, gradually add ammonium sulfate solid to make the saturation reach 90%, stand at 4°C for 30-45 minutes, then centrifuge, discard the supernatant and keep the precipitate; (5) The precipitate was dissolved with a 20 mM Tris-HCl solution at pH 8.0, and the supernatant was retained after centrifugation. After desalting by dialyzing with a 100 Da dialysis bag, the resulting solution was the protein extract of the jujube kernel, which was freeze-dried and stored for later use.

3. The method for preparing the active peptide extract of Semen Ziziphi Spinosae according to claim 1, characterized in that: The specific steps of step 2 are: (1) Seed solution preparation Lactobacillus reuteri LRSY523 was inoculated into MRS seed medium at a volume ratio of 2%, cultured at 37°C and 120 r / min for about 22-26 h, and activated 2-3 times to a bacterial concentration of 1×10 8 cfu / mL or more; The seed culture medium formula is: MRS broth 48.3 g, distilled water 1000 mL, pH 6.2±0.2; (2) Liquid fermentation Inoculate 10% of the fermentation strain seed liquid into the fermentation medium, and ferment at 37°C and 120 r / min for 46-50 hours; after the fermentation is completed, heat with 100°C boiling water for 5-10 minutes, centrifuge the fermentation liquid, and keep the supernatant; The fermentation medium formula is: 10-20g of jujube seed protein extract, 10-25g of glucose, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 5g of sodium acetate, 1000mL of distilled water, pH value 6.2±0.2; (3) Using a 3 kDa ultrafiltration device to intercept polypeptide components with a molecular weight less than 3 kDa, and after desalting with 200 Da dialysis, the active peptide extract of the jujube kernel was obtained, which was freeze-dried and stored for later use.

4. The active peptide extract of Semen Ziziphi Spinosae prepared according to any one of claims 1 to 3.

5. Use of the active peptide extract of Semen Ziziphi Spinosae according to claim 4 in the preparation of functional foods or health products for preventing inflammatory bowel disease.

6. Use of the active peptide extract of Semen Ziziphi Spinosae according to claim 4 in the preparation of a medicament for preventing and / or treating inflammatory bowel disease.

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