Soybean sprout small molecule peptide and application thereof in weight losing and intestine conditioning

Abstract

The invention discloses a soybean sprout small molecule peptide and application thereof in weight losing and intestine conditioning, and belongs to the technical field of small molecule peptides, and the amino acid sequence of the soybean sprout small molecule peptide is shown as SEQ ID No.6, SEQ ID No.17 or SEQ ID No.20. The invention proves that the soybean sprout peptide SSP has the effects of reducing fat and losing weight in obese mice induced by high fat diet; the sequence of the SSP is identified by using a UPLC-MS / MS technology, and the pathway and mechanism of the SSP playing an anti-obesity role through an immunomodulatory function are explored by using network pharmacology and a molecular docking method; according to the invention, active components of soybeans are deeply studied, the soybean sprout small molecule peptide with more accurate and effective functions is developed, the defects of existing obesity treatment drugs can be overcome, the soybean sprout small molecule peptide is beneficial to the health of patients, the resource utilization value of soybeans can be improved, and related benefits can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of small molecule peptides, and in particular relates to a soybean sprout small molecule peptide and application thereof in weight loss and intestinal regulation. Background Art

[0002] Obesity is a non-communicable chronic disease caused by the interaction of multiple factors such as genetic factors, environmental factors, and different behavioral lifestyles. It is manifested as excessive accumulation of body fat. Obesity can have a negative impact on patients' health and quality of life, and increase the risk of death. Therefore, it is urgent to clarify the causes of obesity, explore the key pathogenesis, and find effective methods to prevent and treat obesity.

[0003] Soybeans have a history of more than 5,000 years of cultivation in Asia. Since they were introduced to Western countries in the mid-twentieth century, they have also been extremely popular and have become the main cash crop in many countries. Soybeans have high nutritional value and are rich in a variety of active ingredients. They can produce anti-inflammatory, antioxidant, anti-cancer, and immune regulation functions. They are emerging functional food ingredients with great development value. At present, most of the research on soybeans is still focused on soybeans themselves or crude extracts, and the specific functional components and mechanisms of action are still unclear. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes a soybean sprout small molecule peptide and its application in weight loss and bowel regulation. The soybean sprout small molecule peptide can play a role in weight loss and bowel regulation through immune regulation function.

[0005] To achieve the above object, the present invention provides a soybean sprout small molecule peptide with weight loss and intestinal regulation effects, the amino acid sequence of which is shown in SEQ ID No.6, SEQ ID No.17 or SEQ ID No.20.

[0006] Furthermore, the medicine is used for weight loss and bowel regulation.

[0007] The present invention also provides a soybean sprout small molecule peptide composition with weight loss and bowel regulation effects, the amino acid sequences of which are shown as SEQ ID No.6, SEQ ID No.17 and SEQ ID No.20.

[0008] Furthermore, the medicine is used for weight loss and bowel regulation.

[0009] The present invention also provides the use of the above-mentioned soybean sprout small molecule peptide or the above-mentioned soybean sprout small molecule peptide composition in the preparation of a drug, wherein the drug is used to regulate the expression level of AKT1, SRC, STAT3, FOS, ESR1 and / or NFKB1 target points.

[0010] The present invention also provides the use of the above-mentioned soybean sprout small molecule peptide or the above-mentioned soybean sprout small molecule peptide composition in the preparation of medicines, and the medicines are used to relieve high fat, high inflammation, metabolic disorders, intestinal damage and intestinal imbalance.

[0011] Furthermore, the drug is used to lower blood lipids and inhibit inflammation, the blood lipid indicators include TC and LDL-C, and the inflammation indicators include TNF-α;

[0012] And / or, the drug is used to alleviate jejunal damage, increase the content of intestinal digestive enzymes and inhibit intestinal inflammatory state, the intestinal digestive enzymes include trypsin and lipase, and the indicator of intestinal inflammatory state is IL-6;

[0013] And / or, the drug is used to increase the levels of OXR1 and OXR2 in the jejunum and hypothalamus;

[0014] And / or, the drug is used to enhance the Leptin content in the jejunum and hypothalamus and improve the Leptin resistance phenomenon;

[0015] And / or, the drug is used to increase the abundance of beneficial bacteria related to the decomposition of carbohydrates and proteins, and reduce the abundance of pathogenic bacteria, the beneficial bacteria include Fimicutes and Bacteroidota, and the pathogenic bacteria include Proteobacteria and Fusobacteriota.

[0016] The present invention also provides a medicine, comprising the above-mentioned soybean sprout small molecule peptide or the above-mentioned soybean sprout small molecule peptide composition, and pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects:

[0018] The present invention confirms the potential function of SSP in weight loss and bowel regulation through in vivo efficacy research, clarifies its mechanism of action, and provides data support and theoretical support for in-depth research on the physiological regulation function of seed plant-derived peptides in the germination period in the human body.

[0019] This invention is the first to comprehensively discuss the physiological functions of SSP in vivo by regulating three types of neuropeptides related to energy and metabolism, namely OXR1, OXR2 and Leptin.

[0020] The present invention reveals the signal pathways by which soybean sprout peptides FPFPRPP, SPHWNLN, EIPRPRP, NEGALFLPH and QDSHQKIRHF exert anti-obesity effects through network pharmacology and molecular docking methods, providing a reference for exploring nutritional factors with anti-obesity effects in soybeans during the sprout period. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a comparison of the body sizes of mice in each group; Figure 2 Figure 2 is the weight change of mice in each group; Figure 3 HE staining images of the jejunum of mice in each group; Figure 4 The figure is a comparison chart of serum lipid metabolism indexes of mice in each group; A is the TC content of mouse serum, B is the TG content of mouse serum, C is the HDL-C content of mouse serum, and D is the LDL-C content of mouse serum; Figure 5 The figure is a comparison chart of the levels of pro-inflammatory factors in the serum of mice in each group; A is the level of IL-1β in mouse serum, B is the level of IL-6 in mouse serum, C is the level of IL-8 in mouse serum, and D is the level of TNF-α in mouse serum; Figure 6 The figure is a comparison chart of the anti-inflammatory factor content in the serum of each group of mice; A is the IL-1 content in the serum of mice, B is the IL-4 content in the serum of mice, and C is the IL-10 content in the serum of mice; Figure 7 The figure is a comparison of the digestive enzyme content in the jejunum of each group of mice; A is the trypsin content in the jejunum of mice, B is the lipase content in the jejunum of mice, and C is the α-amylase content in the jejunum of mice; Figure 8 The figure is a comparison chart of the content of pro-inflammatory factors in the jejunum of mice in each group; A is the content of IL-1β in the jejunum of mice, B is the content of IL-6 in the jejunum of mice, C is the content of IL-8 in the jejunum of mice, and D is the content of TNF-α in the jejunum of mice; Fig. 9 The figure is a comparison chart of the anti-inflammatory factor content in the jejunum of each group of mice; A is the IL-1 content in the jejunum of mice, B is the IL-4 content in the jejunum of mice, and C is the IL-10 content in the jejunum of mice; Fig.10 The figure is a comparison of the contents of OXR1, OXR2 and Leptin in the jejunum of each group of mice; A is the content of OXR1 in the jejunum of mice, B is the content of OXR2 in the jejunum of mice, and C is the content of Leptin in the jejunum of mice; Fig.11 The figure is a comparison of the OXR1, OXR2 and Leptin contents in the hypothalamus of mice in each group; A is the OXR1 content in the hypothalamus of mice, B is the OXR2 content in the hypothalamus of mice, and C is the Leptin content in the hypothalamus of mice; Fig.12 It is the OTU RANK curve of each group of mice; Fig.13 The diversity diagrams of the intestinal flora of mice in each group; A is a Venn diagram and B is a petal diagram; Fig.14 This is the β diversity analysis of the intestinal flora of each group of mice; A is the principal coordinate analysis method, and B is the non-metric multidimensional calibration method; Fig.15 The UPGMA analysis diagram of the intestinal flora structure of mice in each group; Fig.16 This is a stacked diagram of species at the phylum level of the intestinal flora of each group of mice; Fig.17 This is a stacked diagram of species at the genus level of the intestinal flora of each group of mice; Fig.18 Venn diagram for peptides and common disease targets; Fig.19 It is a GO biological function annotation map; Fig. 20 This is a KEGG pathway enrichment analysis diagram. DETAILED DESCRIPTION

[0022] Example 1 Effect of soybean sprout peptide on alleviating obesity caused by high-fat diet

[0023] 1. Experimental Materials

[0024] 1.1 Soybean sprout peptide

[0025] Soybean sprouting peptide (SSP) comes from preliminary laboratory preparation and has been publicly reported. Technical personnel in this field can prepare it by themselves according to the literature. The literature is as follows: QIQ, ZHANG G, WANG W, et al. Preparation and Antioxidant Properties of Germinated Soybean Protein Hydrolysates [J]. Frontiers in Nutrition, 2022, 9.

[0026] 1.2 Experimental animals

[0027] Forty-eight SPF-grade 8-week-old male C57BL / 6J mice weighing 20-25 g were housed in an SPF-grade environment after passing quarantine at the Experimental Animal Center of Zhejiang Academy of Agricultural Sciences.

[0028] 1.3 Experimental animal feed

[0029] The purified high-fat feed with 60% fat energy supply (XTHF60, calorie ratio: 20% protein, 20% carbohydrate, 60% fat, total calorie 5.22 kcal / g) and the purified feed with 60% fat energy supply control group (XTCON50J, calorie ratio: 20% protein, 70% carbohydrate, 10% fat, total calorie 3.83 kcal / g) were purchased from Jiangsu Collaborative Medicine Bioengineering Co., Ltd. The specific composition of the feed is shown in Table 1.

[0030] Table 1 Composition of high-fat purified feed and low-fat purified feed (g / kg)

[0031]

[0032] 2. Test methods

[0033] 2.1 Animal Experiment Design

[0034] After one week of adaptive feeding, the mice were randomly divided into four groups, each with 6 mice, for feeding, namely, normal diet control group (ND), high-fat control group (HFD), normal diet + 1% SSP intervention group (ND+SSP), and high-fat diet + 1% SSP intervention group (HFD+SSP). During the feeding period, the weight of the mice and the weight of the feed were weighed once a week, and the body weight and food intake were recorded; the feces of the mice were collected once a week and frozen at -80℃ for testing. After 13 weeks of feeding, the mice were fasted for 12h, the eyeballs were removed and blood was collected, and the mice were killed. The hypothalamus and jejunum were removed and frozen at -80℃ for testing.

[0035] 2.2 Histopathological analysis

[0036] After the mouse was dissected, part of the jejunum was taken, the contents in the jejunum were removed, and the tissue was fixed in 4% paraformaldehyde for more than 24 hours and embedded in paraffin. The embedded tissue wax blocks were sliced ​​using a microtome, and then stained with hematoxylin and eosin (HE). The tissue degeneration of the jejunum was analyzed by observation and photography using an inverted microscope.

[0037] 2.3 Serum biochemical index determination

[0038] The blood samples obtained in 2.1 were kept at room temperature for 2 hours, and then centrifuged at 3000 rpm and 4°C for 15 minutes. The upper serum was taken to detect basic biochemical indicators: TC, TG, LDL-C and HDL-C; anti-inflammatory activity: IL-1, IL-4, IL-10; pro-inflammatory activity: IL-6, IL-1β, IL-8 and TNF-α.

[0039] 2.4 Jejunal function indexes and determination of OX1R, OX2R and Leptin

[0040] A 10% homogenate of jejunum was prepared and basic biochemical indices were measured: trypsin, lipase, and α-amylase;

[0041] Anti-inflammatory activity: IL-1, IL-4, IL-10; pro-inflammatory activity: IL-6, IL-1β, IL-8 and TNF-α; appetite and metabolism related: OXR1, OXR2 and Leptin.

[0042] 2.5 Determination of hypothalamic OX1R, OX2R and Leptin

[0043] A 10% homogenate of hypothalamus was prepared, and the appetite and metabolism-related indices: OXR1, OXR2 and Leptin were detected.

[0044] 2.6 Analysis of mouse intestinal microbiome

[0045] 2.6.1 Sample collection

[0046] The jejunum of mice in each group was collected into a clean centrifuge tube.

[0047] 2.6.2 Test process

[0048] 30 ng of DNA was extracted from the jejunal sample for PCR amplification. The PCR amplification product was purified using AgencourtAMPure XP magnetic beads and dissolved in Elution Buffer. Labels were attached to complete library construction. The fragment range and concentration of the library were tested using Agilent2100Bioanalyzer. The qualified libraries were sequenced using a sequencer according to the size of the inserted fragment.

[0049] 2.6.3 Data Analysis

[0050] The data is filtered off the machine, and the remaining high-quality clean data is used for later analysis; the reads are spliced ​​into tags through the overlap relationship between reads; the tags are clustered into OTUs and compared with the database and species annotated; based on the OTU and annotation results, sample species complexity analysis, inter-group species difference analysis, association analysis and model prediction are performed.

[0051] 2.7 Data processing and analysis

[0052] To determine the effect of SSP on obese mice, this study used two-way repeated measures analysis of variance (Two-wayRepeated MeasuresAnova) to study the difference in weight between groups of mice. Two-way ANOVA was used to compare the significance of the differences between the indicators in serum, jejunum and hypothalamus, that is, the histopathological morphology, serum basal indicators, serum inflammatory factors, jejunal digestive enzyme activity, jejunal inflammatory indicators, obesity-related hormones in the jejunum and obesity-related hormones in the hypothalamus of the four groups of mice, ND, HFD, ND+SSP and HFD+SSP, were analyzed with dietary pattern (d) and SSP intervention (p) as two influencing factors. The effects of dietary pattern and SSP intervention and their interaction (d×p) on the relevant indicators of mice were evaluated. One-way ANOVA was performed for those with significant interactions to evaluate the differences between groups of mice. All data were expressed as mean ± standard deviation (X±S), and the data processing software SPSS26 was used to complete. When p < 0.05, it was considered to have a statistically significant effect.

[0053] 3. Results and Analysis

[0054] 3.1 Feeding conditions of mice in each group

[0055] During the 13-week dietary intervention, the hair of mice in the ND and ND+SSP groups was shiny and glossy, they were more active and agile, and their feces were smaller and harder; while the hair of mice in the HFD and HFD+SSP groups was greasy and dull, their exercise volume was significantly reduced, their movements were sluggish, and their feces were larger, softer, and stickier. Figure 1 As shown, compared with the ND group, the mice in the HFD group were significantly larger in size; compared with the ND and NFD groups, the mice in the ND+SSP and HFD+SSP groups were slightly thinner in size.

[0056] 3.2 Body weight changes of mice in each group

[0057] Body weight changes of mice during 13 weeks Figure 2 As shown in the figure, before the experiment, there was no significant difference in the body weight of mice in each group (P>0.05). However, after 1 week of intervention, the body weight of mice in the HFD and HFD+SSP groups increased significantly compared with the ND and ND+SSP groups (P<0.05), and this significant difference lasted until the end of feeding. There was no significant difference in body weight between the ND and ND+SSP groups within 13 weeks (P>0.05), and the body weight of mice in the HFD group was slightly higher than that in the HFD+SSP group within 13 weeks, but there was no statistically significant difference (P>0.05).

[0058] 3.3 Effects of SSP on mouse histopathological morphology

[0059] The jejunal tissue morphology of mice in each group was as follows Figure 3 As shown. Histopathological analysis showed that the jejunal tissue structure of mice in the ND group and NFD+SSP group was intact, the number of villi was abundant and regularly arranged, and there was no obvious pathological damage. The jejunum of mice in the HFD group was severely damaged, with a large number of broken villi and inflammatory cell infiltration. After SSP intervention, the jejunal damage of obese mice was alleviated, the villi structure returned to normal, and the inflammatory infiltration was also improved. The results of histopathology showed that SSP can effectively improve the inflammation and intestinal damage caused by HFD, and has a significant protective effect on the jejunal tissue structure.

[0060] 3.4 Effect of SSP on serum lipid levels in mice

[0061] The results of serum lipid metabolism indexes of mice in each group are as follows Figure 4 As shown. Figure 4 As shown in A, the main effects of dietary type and SSP intervention on serum TC content in mice were significant (Pd<0.001, Pp<0.001), but there was no significant interaction (Pd×p=0). Figure 4As shown in B and C, the main effects of dietary type and SSP intervention on the serum TG (Pd = 0.764, Pp = 0.287) and HDL-C (Pd = 0.832, Pp = 0.652) levels of mice were not significant, and there was no significant interaction (Pd × p > 0.001). Figure 4 As shown in Figure D, the main effects of dietary type and SSP intervention on serum LDL-C in mice were significant (Pd<0.001, Pp<0.001), and the interaction between the two factors was significant (Pd×p<0.001); One-Way analysis showed that HFD significantly increased the LDL-C content in the serum of mice, and after SSP intervention, the LDL-C content in the serum of mice in the HFD+SSP group was significantly decreased; compared with the ND group, there was no significant difference in the LDL-C content in the serum of mice in the SSP-intervention ND+SSP group.

[0062] Effects of 3.5SSP on serum inflammatory factors in mice

[0063] The results of the determination of serum pro-inflammatory factors in each group of mice are as follows Figure 5 As shown. Figure 5 As shown in A and B, the main effects of dietary type and SSP intervention on the levels of pro-inflammatory indicators IL-1β (Pd=0.532, Pp=0.762) and IL-6 (Pd=0.652, Pp=0.678) in mouse serum were not significant, and there was no significant interaction (Pd×p). Figure 5 As shown in Figure C, the main effect of diet type on the pro-inflammatory index IL-8 content in mouse serum was not significant (Pd=0.164), the main effect of SSP intervention was significant (Pp<0.001), and the interaction between the two factors was not significant (Pd×p=0.238). Figure 5 As shown in Figure D, the main effects of dietary type and SSP intervention on the pro-inflammatory index TNF-α content in mouse serum were significant (Pd less than 0.001, Pp<0.001), and the interaction between the two factors was significant (Pd×p<0.001); One-Way analysis showed that HFD significantly increased the TNF-α content in mouse serum, and after SSP intervention, the TNF-α content in the serum of mice in the HFD+SSP group was significantly decreased; compared with the ND group, there was no significant difference in the TNF-α content in the serum of mice in the SSP-intervention ND+SSP group.

[0064] The results of the determination of serum anti-inflammatory factors in each group of mice are as follows Figure 6 As shown. Figure 6As shown in A, B, and C, the main effects of dietary type and SSP intervention on the levels of anti-inflammatory factors IL-1 (Pd=0.078, Pp=0.376), IL-4 (Pd=0.436, Pp=0.477), and IL-10 (Pd=0.549, Pp=0.761) in mouse serum were not significant (Pd>0.001, Pp>0.001), and the interactions between the two factors were not significant (Pd×p).

[0065] Effects of 3.6SSP on jejunal digestive enzymes in mice

[0066] The results of the determination of digestive enzyme indexes in the jejunum of each group of mice are as follows Figure 7 As shown. Figure 7 A dietary pattern and soybean SSP intervention had significant main effects on the trypsin content in the jejunum of mice (Pd < 0.001, Pp < 0.001), but the interaction between the two factors was not significant (Pd × p = 0.143). Figure 7 As shown in Figure B, the main effects of dietary pattern and SSP intervention on the lipase content in the jejunum of mice were significant (Pd < 0.001, Pp < 0.001), but the interaction between the two factors was not significant (Pd × p = 0.083). Figure 7 As shown in C, the main effects of dietary pattern and SSP intervention on the α-amylase content in the jejunum of mice were not significant (Pd=0.437, Pp=0.747), and the interaction between the two factors was not significant (Pd×p=0.896).

[0067] Effects of 3.7SSP on jejunal inflammatory parameters in mice

[0068] The results of the determination of pro-inflammatory factors in the jejunum of each group of mice are as follows Figure 8 As shown. Figure 8 As shown in A, the main effects of dietary type and SSP intervention on the IL-1β content in the jejunum of mice were not significant (Pd=0.648, Pp=0.437), and the interaction between the two factors was not significant (Pd×p=0.374). Figure 8 As shown in Figure B, the main effects of dietary type and SSP intervention on the IL-6 content in the jejunum of mice were significant (Pd<0.001, Pp<0.001), but the interaction between the two factors was not significant (Pd×p=0.096). Figure 8 As shown in Figure C, the main effect of dietary type on the IL-8 content in the jejunum of mice was significant (Pd < 0.001), but the main effect of SSP intervention was not significant (Pp = 0.972), and the interaction between the two factors was not significant (Pd × p = 0.259). Figure 8As shown in D, the main effects of dietary type and SSP intervention on the TNF-α content in the jejunum of mice were not significant (Pd=0.372, Pp=0.873), and the interaction between the two factors was not significant (Pd×p=0.173).

[0069] The results of the determination of anti-inflammatory factors in the jejunum of each group of mice are as follows Fig. 9 As shown. Fig. 9 As shown in A, B, and C, the main effects of dietary patterns on the contents of anti-inflammatory factors IL-1 (Pd=0.762, Pp=0.514), IL-4 (Pd=0.351, Pp=0.563), and IL-10 (Pd=0.154, Pp=0.215) in the jejunum of mice were not significant, and the interactions between the two factors were not significant (Pd×p).

[0070] Effects of 3.8SSP on OX1R, OX2R and Leptin Contents in Mouse Jejunum

[0071] The results of the determination of OX1R, OX2R and Leptin in the jejunum of each group of mice are shown in Fig.10 As shown. Fig.10 As shown in A, the main effects of dietary pattern and SSP intervention on the jejunal OXR1 content of mice were significant (Pd<0.001, Pp<0.001), but the interaction between the two factors was not significant (Pd×p=0.164). Fig.10 As shown in B, the main effects of dietary pattern and SSP intervention on the jejunal OXR2 content of mice were significant (Pd<0.001, Pp<0.001), and the interaction between the two factors was significant (Pd×p<0.001); One-Way analysis showed that HFD significantly reduced the jejunal OXR2 content of HFD group mice, but there was no significant difference between the HFD+SSP group and the HFD group after SSP intervention, and there was no significant difference between the ND and ND+SSP groups. Fig.10 As shown in C, the main effects of dietary pattern and SSP intervention on Leptin content in the jejunum of mice were significant (Pd<0.001, Pp<0.001), but the interaction between the two factors was not significant (Pd×p=0.081).

[0072] Effects of 3.9SSP on OX1R, OX2R and Leptin Content in Mouse Hypothalamus

[0073] The results of the determination of OXR1, OXR2 and Leptin in the hypothalamus of each group of mice are shown in Fig.11 As shown in A. Fig.11As shown in A, the main effects of dietary pattern and SSP intervention on the OXR1 content in the hypothalamus of mice were significant (Pd<0.001, Pp<0.001), and the interactions between the two factors were significant (Pd×p<0.001). One-Way analysis showed that HFD significantly reduced the OXR1 content in the hypothalamus of mice in the HFD group, but there was no significant difference between the HFD+SSP group and the HFD group after SSP intervention, and there was no significant difference between the ND and ND+SSP groups. Fig.11 As shown in Figure B, the main effects of dietary pattern and SSP intervention on the content of OXR2 in the hypothalamus of mice were significant (Pd<0.001, Pp<0.001), but the interaction between the two factors was not significant (Pd×p=0.076). Fig.11 As shown in C, the main effects of dietary pattern and SSP intervention on the leptin content in the hypothalamus of mice were significant (Pd<0.001, Pp<0.001), but the interaction between the two factors was not significant (Pd×p=0.094).

[0074] 3.1016SrRNA intestinal flora test results

[0075] 3.10.1 Sequencing statistics of intestinal flora of each group of mice

[0076] The OTURANK curve can show the abundance of species in different samples, and can explain both the abundance and uniformity of sample diversity. Calculate the relative abundance of each OTU in each sample, and then arrange them from large to small according to abundance, with the OTU level as the horizontal axis and the relative abundance of OTU as the vertical axis for plotting. Fig.12 As shown, with the increase of data amount, the curves of the four groups of mice gradually flattened, indicating that the number of sample sequencing was appropriate and the species composition in each sample was highly uniform, which can be used for further analysis.

[0077] from Fig.13 The Venn diagram and petal diagram show that there are significant differences in the intestinal flora of each group of mice. Fig.13 As shown in the Venn diagram of A, the numbers of OTUs in the five groups of ND, ND+SSP, HFD and HFD+SSP were 151, 91, 271 and 211, respectively, and the five groups had 134 OTUs in common. The number of OTUs in common between the ND group and the HFD group was 15, and the number of OTUs in common between ND and HFD+SSP was 43, which was greater than the number of OTUs in common between ND and HFD. The number of OTUs in common between ND+SSP and ND and HFD was 55 and 7, respectively. The above situation shows that there are significant differences in the composition and distribution of intestinal flora between ND and HFD, but SSP intervention can improve this situation.

[0078] 3.10.2 Analysis of β-diversity of intestinal flora in each group of mice

[0079] β diversity analysis can be used to reflect the similarities and differences in the intestinal flora structure between groups of mice. Principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) can reflect the composition differences of the intestinal flora of each group of mice, such as Fig.14 As shown in A, the degree of dispersion between the ND group and the ND+SSP group was low, indicating that the intestinal flora composition between the two groups was similar; compared with the HFD group, the NFD+SSP group after SSP intervention was closer to the ND group as a whole, indicating that HFD can cause intestinal flora disorders in mice, and SSP has an improving effect on this situation.

[0080] 3.10.3 UPGMA analysis of intestinal flora of mice in each group

[0081] The unweighted group average method (UPGMA) can draw a dendrogram showing the similarity of each sample. Fig.15 As shown in the figure, the distance between the ND group and the ND+SSP group was small, indicating that the intestinal flora of the two groups was highly similar; the distance between the HFD group and the ND group was large, indicating that the intestinal flora of the two groups was quite different. After SSP intervention, the overall distance between the ND group and the ND group was shortened, indicating that SSP can improve the distribution of intestinal flora in obese mice caused by HFD.

[0082] 3.10.4 Analysis of intestinal flora at the phylum level in each group of mice

[0083] To further clarify the effect of SSP on the species composition of the intestinal flora of mice, the differences in the intestinal flora of each group were compared at the phylum level, such as Fig.16 As shown. At the phylum level, the intestinal flora of each group was mainly composed of Fimicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Desulfobacterota. Compared with the ND group, the abundance of Fimicutes and Bacteroidota in the intestine of the HFD group mice was reduced, and the abundance of Proteobacteria and Fusobacteriota was increased. After SSP intervention, all the above changes were reversed except for Actinobacteriota.

[0084] 3.10.5 Analysis of intestinal flora at the genus level in each group of mice

[0085] like Fig.17As shown in the figure, at the genus level, the intestinal flora of mice in each group was mainly composed of Muribaculaceae (unclassified genus), Ileibacterium (Ileibacterium), Lactobacillus (Lactobacillus), Clostridium (Clostridium), Faecalibaculum (Faecalibaculum), Escherichia-Shigella (Escherichia-Shigella), Romboutsia (Romboutsia), Bifidobacterium (Bifidobacterium), Lachnospiraceae-NK4A136 (Lachnospiraceae-NK4A136 group) and Dubosiella (Durosiella).

[0086] Compared with the ND group, the abundance of Muribaculaceae, lleibacterium, Roseburia, Dubosiella and Lachnospiraceae-NK4A136 in the intestine of HFD mice was decreased, and the abundance of Romboutsia, Streptococcus, Lactobacillus, Faecalibaculum and Escherichia-Shigella was increased. After SSP intervention, the abundance of Roseburia and Lachnospiraceae-NK4A136 in the intestine of mice was increased, and the abundance of Escherichia-Shigella was decreased.

[0087] In summary, SSP can effectively alleviate inflammation, excessive lipid production and accumulation, metabolic disorders, etc. in mice caused by HFD. SSP can effectively regulate the intestinal flora disorder in HFD-induced obese mice, increase the beneficial bacteria involved in metabolism and protection of the intestinal barrier, protect the intestinal damage caused by HFD, and regulate the metabolic capacity of mice. At the same time, SSP can regulate the energy balance in mice by significantly increasing the secretion of Orexin receptors and reducing the secretion of Leptin. Considering the important role of the microbial-gut-brain axis in physiological regulation, SSP may regulate the secretion of related neuropeptides in the hypothalamus and intestines through this axis in vivo, affect the energy regulation of mice, improve the intestinal structure and flora composition, regulate adverse conditions in the body, and exert anti-obesity effects through multiple pathways.

[0088] Example 2 Identification, screening and anti-obesity activity study of SSP

[0089] 1. Test methods

[0090] 1.1 SSP amino acid sequence identification

[0091] 1.1.1 Sample processing

[0092] Take the SSP and use a C18 column to desalt. After desalting, freeze-dry the solution and wait for detection.

[0093] 1.1.2 HPLC-MS / MS detection

[0094] The amino acid sequence of SSP was detected by HPLC-MS / MS. The sample was dissolved, centrifuged at 13500 rpm and 4°C for 20 min, and 8 μL of supernatant was taken for mass spectrometry identification. The mobile phase A used was 0.1% formic acid, and the mobile phase B was 60.1% formic acid and 80% acetonitrile. The specific mobile phase parameters are shown in Table 2.

[0095] Table 2 Mobile phase parameters

[0096]

[0097] The mass spectrometer setting parameters are shown in Table 3.

[0098] Table 3 Mass spectrometry setting parameters

[0099]

[0100]

[0101] 1.1.3 Data processing

[0102] PEAKS software was used to search the database, and the specific search parameters are shown in Table 4.

[0103] Table 4 Identification parameters

[0104]

[0105] 1.2 Screening of potential active peptide sequences

[0106] According to the analysis of active peptide sequences, low molecular weight peptides with less than 10 amino acid residues are more active than high molecular weight peptides, so sequences containing 2-10 amino acid residues are screened out.

[0107] The physicochemical properties of each sequence were evaluated by an online analysis platform, and the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) was used to verify the novelty of the peptide. PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to score and predict the biological activity of the peptide. The activity threshold of PeptideRanker was set to 0.5. Peptide sequences with a prediction score of >0.5 were analyzed for physicochemical properties (molecular weight, hydrophobicity, isoelectric point, etc.) using Ppetide2.0 (https: / / www.peptide2.com / ). In the field of functional foods, the safety of peptides is also crucial. Toixinpred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) is used to predict whether the peptide sequence has potential toxicity, and Allergen FP (http: / / ddg-pharmfac.net / AllergenFP / ) is used to predict whether the peptide sequence has allergenicity.

[0108] 1.3 Discovering common targets between peptides and obesity

[0109] The peptide sequences screened by the above method were input into the SwissADME database (http: / / www.swissadme.ch) to obtain the SMILES number of each peptide segment, and then each SMILES number was imported into the Swiss TargetPrediction database (http: / / www.swisstargetprediction.ch) to predict the target.

[0110] The keyword "obesity" was entered into the OMIM (https: / / omim.org), TTD (http: / / db.idrblab.net / ttd / ) and Genecards (https: / / www.genecards.org) databases to collect information on targets related to obesity, and the retrieved target results were exported and integrated with the predicted targets to obtain common targets.

[0111] 1.4 GO biological function annotation and KEGG pathway enrichment analysis of common targets

[0112] The Metascape database (https: / / metascape.org) was used to perform GO biological function annotation and KEGG pathway enrichment analysis on the common targets screened above, and the analysis results were visualized.

[0113] 1.5 Analysis of PPI network construction between targets

[0114] The STRING database (https: / / string-db.org / ) was used to construct the PPI network interaction diagram of the above common targets, and the Cytoscape software was used to analyze and visualize the PPI network interaction diagram to screen out the core targets.

[0115] 2. Results and Analysis

[0116] 2.1 Peptide sequence identification and identification results

[0117] A total of 438 sequences were obtained by HPLC-MS / MS identification, and 20 sequences with scores > 0.5 were identified after PeptideRander scoring. The physicochemical properties of these 20 potential bioactive peptide sequences are shown in Table 5.

[0118] Table 5 Screened peptide sequences

[0119]

[0120] The novelty, toxicity and sensitization of these 20 sequences were then predicted. In order to further screen out peptide sequences with strong immune activity, combined with the analysis of existing research results, it was found that the characteristics related to strong immunity are:

[0121] 1. Hydrophobic amino acids (Trp, Phe, Ala, etc.), branched-chain amino acids (Val, Ile, Leu, etc.) and basic amino acids (Lys, Arg) often appear in immune peptide sequences.

[0122] 2. It has high hydrophobicity and isoelectric point.

[0123] Based on the above characteristics, five sequences with high potential immune activity, non-toxicity, non-sensitization and unreported were screened out, namely FPFPRPP (SEQ ID No. 1), SPHWNLN (SEQ ID No. 3), EIPRPRP (SEQ ID No. 6), NEGALFLPH (SEQ ID No. 17), and QDSHQKIRHF (SEQ ID No. 20).

[0124] 2.2 Target screening

[0125] The 5 peptide sequences screened were input into the SwissADME database to obtain SMILES numbers. The SMILES numbers of the 5 amino acid sequences were input into the Swiss TargetPrediction database for target prediction, and the prediction results were integrated and deduplicated to obtain 332 relevant component targets.

[0126] After entering the keyword "obesity" in the OMIM, TTD, and Genecards databases, we searched for disease-related targets, integrated and removed all the queried targets, and obtained 1,863 related disease targets. We entered the component targets and disease targets into the VENNY online website to obtain common targets with intersections. The analysis showed that there were 135 intersection targets between components and diseases, such as Fig.18 shown.

[0127] 2.3 GO enrichment analysis of common targets

[0128] The results of GO enrichment analysis are as follows Fig.19 (Showing the top ten in each category).

[0129] 2.4 KEGG enrichment analysis of common targets

[0130] To further explore how the targets of SSP regulate immune function and play a role in reducing fat and losing weight in signal pathways, KEGG enrichment analysis was performed on common targets. The analysis results showed that a total of 171 signal pathways were involved. The top 10 pathways were screened based on the target enrichment number and P value, and the relevant bubble chart was drawn, as shown in the figure below. Fig. 20 As shown, the horizontal axis is Generatio, which represents the ratio of the number of target genes enriched in the target pathway genes to the total number of target genes. The larger the value, the greater the degree of enrichment; the larger the bubble, the more enriched targets; the redder the bubble, the smaller the q-value and the more obvious the enrichment.

[0131] The top ten signaling pathways were cancer pathway (has05200), interaction between neuroactive ligands and receptors (has04080), chemical carcinogenesis-receptor activation (has05207), toxoplasmosis (has05145), apoptin signaling pathway (has04371), renin-angiotensin system (has04614), glucagon signaling pathway (has04922), multiple cell apoptosis (has04215), renin secretion (has04924) and bladder cancer (has05219).

[0132] 2.5PPI network analysis

[0133] To study the interaction between targets, the STRING database was used to create a PPI network diagram, with a total of 134 interacting targets and 1,601 interaction lines. The results were imported into the Cytoscape software for visualization, and further topological analysis was performed using the CytoNCA plug-in. The medians of the betweenness centrality (BC), closeness centrality (CC), and degree centrality (DC) greater than or equal to all targets were used as screening criteria, and core targets were obtained after three screenings. After the PPI network was integrated by the Cytoscape software, the median of BC was 48.683522, the median of closeness to CC was 0.50666851, and the median of DC was 18.5. After three screenings, relevant information of 52 targets was obtained. Generally speaking, targets that are connected to more proteins are more important in the network and are more likely to play a key role in the disease. Therefore, the 52 targets were sorted from high to low according to DC, and the top 6 genes with DC values ​​were selected as core targets, namely AKT1, SRC, STAT3, FOS, ESR1, and NFKB1, as shown in Table 6, and these 6 targets were used as receptors for molecular docking.

[0134] Table 6 Information of the 6 targets screened

[0135]

[0136] In summary, the present invention screened out peptides with potential immune activity and anti-obesity in SSP through the method of network pharmacology, including FPFPRPP, SPHWNLN, EIPRPRP, NEGALFLPH and QDSHQKIRHF, and predicted and screened out 6 action targets, including AKT1, SRC, STAT3, FOS, ESR1 and NFKB1. The action mechanism was predicted by GO and KEGG analysis, and it was found that these targets were involved in biological processes such as hormone response, behavior regulation, regulatory system process, tube morphogenesis and response to extracellular stimulation; it was found that these targets were mainly enriched in multiple signal pathways related to hormone regulation, such as apoptin signaling pathway, renin-angiotensin system, glucagon signaling pathway, and renin secretion.

[0137] Example 3 Study on the molecular mechanism of SSP against obesity

[0138] In this experiment, a peptide library for molecular docking was constructed through the two-dimensional structures of five peptides: FPFPRPP, SPHWNLN, EIPRPRP, NEGALFLPH and QDSHQKIRHF.

[0139] The five screened immune peptides were molecularly docked with the six core targets, and the binding energy was used to evaluate the binding ability between the two.

[0140] The molecular docking results showed that the binding energy values ​​between the five sequences and the key targets were all less than -6Kcal / mol, with good binding activity, and some binding energy values ​​were less than -10Kcal / mol, proving that the peptides had strong binding activity with the targets. And after visual analysis of the docking results, it was found that the peptides and target proteins had at least two pairs of interaction relationships in the final binding mode, indicating that the five peptides can regulate immune function through targets such as AKT1, SRC, STAT3, FOS, ESR1 and NFKB1, and then play an anti-obesity role, providing a certain theoretical basis for the subsequent research on anti-obesity functional foods.

Claims

1. A soybean sprout small molecule peptide with weight loss and bowel regulation effects, characterized in that: Its amino acid sequence is shown as SEQ ID No.6, SEQ ID No.17 or SEQ ID No.

20.

2. The use of the soybean sprout small molecule peptide according to claim 1 in the preparation of medicines, characterized in that: The medicine is used for reducing weight and regulating the intestine.

3. A soybean sprout small molecule peptide composition with weight loss and intestinal regulation effects, characterized in that: The amino acid sequences thereof are shown in SEQ ID No.6, SEQ ID No.17 and SEQ ID No.

20.

4. The use of the soybean sprout small molecule peptide composition as claimed in claim 3 in the preparation of medicines, characterized in that: The medicine is used for reducing weight and regulating the intestine.

5. Use of the soybean sprout small molecule peptide according to claim 1 or the soybean sprout small molecule peptide composition according to claim 3 in the preparation of medicines, characterized in that: The drug is used to regulate the expression levels of AKT1, SRC, STAT3, FOS, ESR1 and / or NFKB1 targets.

6. Use of the soybean sprout small molecule peptide according to claim 1 or the soybean sprout small molecule peptide composition according to claim 3 in the preparation of medicines, characterized in that: The drug is used to alleviate high fat, high inflammation, metabolic disorders, intestinal damage and intestinal imbalance.

7. The use according to claim 6, characterized in that: The drug is used to lower blood lipids and inhibit inflammation. The blood lipid indicators include TC and LDL-C, and the inflammation indicators include TNF-α. And / or, the drug is used to alleviate jejunal damage, increase the content of intestinal digestive enzymes and inhibit intestinal inflammatory state, the intestinal digestive enzymes include trypsin and lipase, and the indicator of intestinal inflammatory state is IL-6; And / or, the drug is used to increase the levels of OXR1 and OXR2 in the jejunum and hypothalamus; And / or, the drug is used to enhance the Leptin content in the jejunum and hypothalamus and improve the Leptin resistance phenomenon; And / or, the drug is used to increase the abundance of beneficial bacteria related to the decomposition of carbohydrates and proteins, and reduce the abundance of pathogenic bacteria, the beneficial bacteria include Fimicutes and Bacteroidota, and the pathogenic bacteria include Proteobacteria and Fusobacteriota.

8. A drug, characterized in that It comprises the soybean sprout small molecule peptide according to claim 1 or the soybean sprout small molecule peptide composition according to claim 3, and pharmaceutically acceptable excipients.

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