Walnut polypeptide with the efficacy of improving cognitive impairment and regulating intestinal flora and its application

Abstract

The present invention discloses a walnut polypeptide having the efficacy of improving cognitive impairment and regulating intestinal flora and its application. The polypeptide obtained by enzymatically hydrolyzing walnut protein in walnut meal with alkaline protease is separated by a 1 kDa ultrafiltration membrane and the polypeptide sequence is identified by LC-MS / MS. The identification result simulates the interaction between acetylcholinesterase and the polypeptide through molecular docking, and 1 walnut polypeptide with the efficacy of improving cognitive impairment is screened out. Its amino acid sequence from the N-terminus to the C-terminus is Arg-Leu-Trp-Pro-Phe. Mouse experiments prove that the walnut polypeptide can effectively improve the brain damage caused by D-galactose, and the walnut polypeptide can restore the changes in the intestinal flora composition caused by D-galactose, and has broad market application prospects in the preparation of health products for assisting in regulating intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of health foods, and particularly relates to a walnut polypeptide having the functions of improving cognitive impairment and regulating intestinal flora. Background Art

[0002] With the continuous increase in the proportion of the elderly population globally, problems related to aging have increasingly become the main global health issues. Learning and memory abilities are one of the basic functions of the brain, and aging is inevitably associated with the decline of these cognitive abilities. At the same time, cognitive dysfunction is closely related to the occurrence and development of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and vascular dementia. So far, the main drugs for treating such diseases are acetylcholinesterase inhibitors such as rivastigmine, donepezil, and galantamine, etc. However, long-term use of these drugs will cause side effects such as gastrointestinal disorders, hepatotoxicity, and hypotension. Therefore, the development of natural bioactive substances that can enhance learning, memory, and cognitive abilities has become the focus of research.

[0003] Compared with synthetic drugs, small molecule peptides have the advantages of high biological activity, low toxicity, easy metabolism in the human body, etc., and are more beneficial to the nervous, immune, and gastrointestinal systems. At present, the efficacy of bioactive peptides from different sources in improving cognitive ability has been verified in various in vivo cognitive impairment animal models and in vitro related cell models. Previous studies have shown that oxidative stress and neuroinflammation are the main early pathological features of cognitive impairment and will show an up-regulated phenomenon during the disease. Recently, a large number of research results have shown that intestinal flora disorder plays an extremely important role in cognitive impairment, and it is pointed out that microbiota-targeted intervention is a promising treatment method for resisting aging-related decline in cognitive ability.

[0004] Walnut (Juglans regia L.) is a plant of the genus Juglans in the walnut family, rich in unsaturated fatty acids, proteins, polyphenols, vitamins, and minerals, and has important biological activities such as antioxidant, anti-tumor, anti-inflammatory, blood pressure lowering, and brain strengthening. It is a traditional Chinese food and medicine. However, except for a few walnuts that are directly eaten without processing, most walnuts are used for oil extraction, generating a large amount of walnut meal by-products. These walnut meals are used as feed or directly discarded, resulting in serious waste of resources and greatly restricting the development of China's walnut industry. Research has found that walnut meal is rich in proteins, with a complete variety of amino acids, especially the types and contents of amino acids with neuroprotective effects are relatively high, making it an excellent source of neuroprotective peptides. Although the polypeptides in walnut meal are rich and diverse, there are few related studies on the regulation of intestinal flora by neuroprotective peptides. Summary of the Invention

[0005] The purpose of the present invention is to provide a walnut polypeptide that can improve cognitive impairment by regulating the homeostasis of intestinal flora.

[0006] The amino acid sequence of the walnut polypeptide of the present invention from the N-terminus to the C-terminus is: arginine-leucine-tryptophan-proline-phenylalanine (Arg-Leu-Trp-Pro-Phe, abbreviated as RLWPF). The present invention uses defatted walnut meal as a raw material, extracts the protein in the defatted walnut meal by the alkali solution acid precipitation method, then enzymatically hydrolyzes it with alkaline protease to obtain a walnut polypeptide enzymatic hydrolysis product. After ultrafiltration with a 1 kDa ultrafiltration membrane, the polypeptide sequence of the peptide segments <1 kDa is identified by liquid chromatography-mass spectrometry (LC-MS / MS), and the walnut polypeptide with the function of improving cognitive impairment and regulating the intestinal flora is screened out by molecular docking technology and PeptideRanker activity prediction. The walnut polypeptide of the present invention can also be artificially synthesized by solid-phase synthesis.

[0007] The walnut polypeptide of the present invention can also be used to prepare health products for assisting in regulating the intestinal flora.

[0008] The beneficial effects of the present invention are as follows:

[0009] The present invention establishes a cognitive impairment model with D-galactose and measures the HE staining of brain tissue sections and the intestinal flora composition of mice. The results show that after ingesting the walnut polypeptide RLWPF of the present invention, it can significantly improve the apoptosis of hippocampal cells, and the neuronal distribution in these hippocampal regions is better than that in the model group. In terms of intestinal flora regulation, the walnut polypeptide RLWPF of the present invention can restore the diversity and richness of the microbiota after intervention. The RLWPF intervention improves cognitive impairment by regulating the Firmicutes / Bacteroidetes ratio and reducing the abundances of harmful bacteria including Helicobacter, Allobaculum, Alistipes, and Mucispirillum. Therefore, the walnut polypeptide RLWPF of the present invention has the functions of improving cognitive impairment and regulating intestinal microorganisms, and has extremely high application prospects in the preparation of health products (food or drugs) for assisting in regulating the intestinal flora. Description of the Drawings

[0010] Figure 1 It is a 2D map of the molecular docking of RLWPF and AChE.

[0011] Figure 2 It is a 3D map of the molecular docking of RLWPF and AChE.

[0012] Figure 3 It is a HE staining section map of the hippocampal region of the brain tissue.

[0013] Figure 4 It is a Venn diagram of the OTUs of the intestinal flora of mice.

[0014] Figure 5It is the percentage of the abundance of each community of the mouse intestinal flora at the phylum level.

[0015] Figure 6 The ratio (F / B) of Firmicutes and Bacteroidetes in the mouse intestine.

[0016] Figure 7 It is the percentage of the abundance of each community of the mouse intestinal flora at the genus level.

[0017] Figure 8 It is the phylogenetic tree (genus level) of the species classification of the mouse intestinal flora.

[0018] Figure 9 It is the bar chart of the LDA value distribution of significantly different species in the mouse intestinal flora (genus level). Detailed implementation mode

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0020] Example 1

[0021] (1) Pretreatment of walnut meal

[0022] After crushing the walnut meal, add n-hexane according to the solid-liquid ratio of 1 g:5 mL, stir and extract at room temperature for 40 min, filter by suction after precipitation, collect the residue, after continuous extraction twice, place the residue in a fume hood overnight, air dry the excess n-hexane, and obtain defatted walnut meal powder, which is stored at -4°C.

[0023] (2) Protein extraction by alkali solution and acid precipitation method

[0024] Mix the defatted walnut meal powder and distilled water according to the solid-liquid ratio of 1 g:10 mL, adjust the pH of the mixture to 9.0 with 1 mol / L NaOH aqueous solution, sonicate for 1 min and then have a 1-min interval, repeat 5 times, then stir at room temperature for 4 h, let it stand and cool, centrifuge at 8000 rpm for 10 min, collect the supernatant, adjust the pH to 4.5 with 1 mol / L HCl aqueous solution, let it stand at 4°C for 4 h, then centrifuge at 8000 rpm at 4°C for 10 min, collect the precipitate, adjust the pH to 7.0 with 1 mol / L NaOH aqueous solution, and freeze-dry to obtain walnut meal protein powder, which is stored at -80°C.

[0025] (3) Alkaline protease hydrolysis

[0026] The freeze-dried walnut meal protein powder and distilled water were mixed at a solid-liquid ratio of 1 g:25 mL, heated in a water bath at 85 °C for 20 min, cooled to room temperature, and then the pH was adjusted to 9.0 with 1 mol / L NaOH aqueous solution. Alkaline protease was added at a mass ratio of 1:25 of alkaline protease to walnut meal protein powder, and hydrolysis was carried out at 55 °C for 4 h. During the hydrolysis process, 1 mol / L NaOH aqueous solution was added to keep the pH of the reaction system around 9.0. After the enzymatic hydrolysis was completed, the system was placed in a water bath at 90 °C to terminate the enzymatic hydrolysis reaction. After cooling to room temperature, the pH of the solution was adjusted to 7.0 with 1 mol / L HCl aqueous solution, and centrifuged at 8000 rpm for 10 min to collect the supernatant.

[0027] (4) Separation and screening of polypeptides

[0028] The supernatant collected in step (3) was ultrafiltered using an ultrafiltration membrane with a molecular weight cut-off of 1 kDa, and the peptides <1 kDa were subjected to LC-MS / MS analysis. The raw data was screened, and 20 polypeptides were selected according to the confidence level (ALC% > 95%), relative content (Area > 1×10 7 ), activity score (PeptideRanker score > 0.5), and arginine-rich (Arg, R).

[0029] (5) Molecular docking

[0030] The interaction between the polypeptide and acetylcholinesterase (AChE) was simulated using molecular docking technology. AChE (PDBID: 1GQR) was downloaded from the PDB protein database. Using AChE as the receptor and the 20 polypeptides screened in step (4) as ligands respectively, molecular docking was carried out using Autodock tools 1.5.6 software. The docking results were reflected by the Vina score, which was generally negative. The smaller the value, the closer the binding between the ligand and the receptor protein, and the stronger the interaction. The docking results are shown in Table 1.

[0031] Table 1 Docking results of polypeptides with acetylcholinesterase

[0032] Polypeptide sequence Vina score (kcal / mol) Polypeptide sequence Vina score (kcal / mol) RLWPF -10.3 FVLRL -8.7 LRFPL -9.5 VLRLF -9.9 FLLR -9.6 LPLLR -9.3 LWDRPL -9.3 TDDRFL -8.1 SWPGSR -9.6 FVLR -9.8 FQLPR -9 LERF -8.8 FALR -9.1 LRLL -9.4 DADFLR -9.5 YYLR -9.7 TRWL -8.1 ALRL -8.7 RWLQ -9.3 FVLDLR -9.4

[0033] A polypeptide sequence with the best docking effect was screened from the docking results in Table 1. Its amino acid sequence from the N-terminus to the C-terminus is: Arg-Leu-Trp-Pro-Phe, abbreviated as RLWPF. The Vina score of the docking of RLWPF with AChE is -10.3 kcal / mol. By Figure 1 and Figure 2It can be seen that Trp84, Ser122, Phe331, Gln74, Tyr70, Tyr121, and Tyr334 of AChE can form hydrogen bonds with the H atoms of the upper side chains of RLWPF and the N and O atoms at the N-terminus and C-terminus; Phe330, Phe331, and Trp279 are connected to RLWPF by π-alkyl bonds; Trp84 is connected to RLWPF by π-π bonds.

[0034] (6) In vivo brain-boosting effect of walnut polypeptide RLWPF

[0035] RLWPF (purity ≥ 98%) was synthesized using Fmoc solid-phase peptide synthesis technology. The synthesized polypeptide was verified for its brain-boosting activity through mouse experiments. After one week of quarantine and adaptive feeding of the mice, they were randomly and equally divided into 3 groups: a control group, a model group, and an RLWPF group. The mice in the model group and the RLWPF group were first subjected to D-galactose (D-gal) modeling treatment. The two groups of mice received continuous subcutaneous injection at 400 mg / kg / d for 8 weeks. Since the 5th week, the model group was simultaneously gavaged with normal saline, and the RLWPF group was simultaneously gavaged with walnut polypeptide RLWPF. The control group was normally fed. After 8 weeks, the mice were anesthetized by inhalation with isoflurane at a concentration of 2% - 3%. After anesthesia, the mice were sacrificed by cervical dislocation. The brain tissues of the mice were carefully removed, rinsed with normal saline, and then placed in a 4% paraformaldehyde solution for fixation. After fixation, it was embedded with paraffin to obtain paraffin-embedded tissue blocks. Then, the paraffin blocks were sectioned with a section thickness of 5 μm. Finally, the tissue sections were stained using hematoxylin-eosin staining, and the distribution of neurons in the hippocampal region was observed under the bright field of the microscope. The results are as Figure 3 shown.

[0036] It can be Figure 3 seen that the cells in the DG region and CA3 region of the hippocampus of the mice in the model group were arranged loosely and disorderly, the cell membrane boundaries were unclear, and the staining was relatively deep, indicating cell apoptosis. Gavage with walnut polypeptide RLWPF could reverse this symptom and improve the neuronal morphology of the hippocampus.

[0037] (7) Regulation of walnut polypeptide RLWPF on the intestinal flora of mice

[0038] Based on experiment (6), mouse fecal samples were collected, total fecal DNA was extracted, and the 16S rRNA V3-V4 region of bacterial genes was amplified by PCR (the primer sequences were 338F: 5'-barcode + ACTCCTACGGGAGGCAGCA-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3'). After gel recovery, the library was constructed using the TruSeq Nano DNA LT Library Prep Kit and sequenced on a machine. Paired-end sequencing was performed based on the Illumina NovaSeq machine. After sequencing, QIIME2 was used to perform microbiome bioinformatics analysis on the 16S rRNA sequence data. As Figure 4 shown, the numbers of unique operational taxonomic units (OTUs) in the control group, model group, and RLWPF group were 13322, 4088, and 10737, respectively, and the number of OTUs shared by the three groups was 1952. The diversity of the gut microbiota decreased in D-gal-induced cognitive impairment mice, and the intervention of RLWPF could restore the diversity of the gut microbiota. As Figure 5 shown, Firmicutes, Bacteroidetes, and Proteobacteria were the dominant species at the phylum level. D-gal induction led to a significant decrease in the relative abundance of Firmicutes and a significant increase in the relative abundance of Bacteroidetes. The intervention of walnut polypeptide RLWPF helped restore the gut microbiota structure to the normal level. It has been reported that D-gal induction would lead to a decrease in the ratio of Firmicutes to Bacteroidetes (F / B) in the host gut microbiota. RLWPF treatment had a restorative effect on the decrease in the F / B value. The F / B values of the control group, model group, and RLWPF group were 2.13 ± 0.34, 1.01 ± 0.29, and 1.55 ± 0.26 (see Figure 6 ). In addition, to better understand the effect of RLWPF on the gut microbiota of mice, the species composition at a lower taxonomic level (genus level) was analyzed (see Figure 7 ). The results showed that compared with the control group, the relative abundance of Lactobacillus in the model group was significantly decreased, and the relative abundances of Helicobacter, Allobaculum, Alistipes, and Mucispirillum were significantly increased; compared with the model group, RLWPF had an obvious regulatory effect on multiple genera of the gut microbiota. RLWPF could significantly inhibit the increase in the abundances of Helicobacter, Allobaculum, Alistipes, and Mucispirillum induced by D-gal. Finally, through linear discriminant analysis effect size analysis (see Figure 8 and9 ) It was found that the signature flora of the model group were Rikenellaceae and Alistipes, and the signature flora of the RLWPF group were Erysipelotrichi, Erysipelotrichaceae, Erysipelotrichales, and Allobaculum, which were important reasons for the differences in the flora among groups.

[0039] The above results indicate that the walnut polypeptide RLWPF described in the present invention has the functions of improving cognitive impairment and regulating intestinal microbiota, and can be used in the preparation of health products for assisting in regulating intestinal flora.

Claims

1. A walnut polypeptide with the efficacy of improving cognitive impairment and regulating intestinal flora, characterized in that, The amino acid sequence of the walnut polypeptide from the N-terminus to the C-terminus is Arg-Leu-Trp-Pro-Phe.

2. Use of the walnut polypeptide according to claim 1 in the preparation of a health product for assisting in regulating the intestinal flora.

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