Preparation method and application of deep-grain walnut inner seed coat extract

Abstract

The invention discloses a preparation method of a deep-grain walnut inner seed coat extract, which specifically comprises the following steps: adding crushed deep-grain walnut inner seed coats and distilled water into a high-pressure reaction kettle, stirring and extracting for 10-20 minutes at 100-120 DEG C and 1-3 MPa, collecting an extracting solution, and carrying out rotary evaporation concentration and freeze drying to obtain a crude extract; dissolving the crude extract with water, adjusting the pH value to 2-3, purifying the crude extract solution with NKA-9 type macroporous resin, eluting with an ethanol solution with the volume concentration of 45-65%, collecting the eluent, and concentrating and drying to obtain the deep-grain walnut inner seed coat extract. After the extract is subjected to in-vitro simulated digestion fermentation, the abundance of beneficial bacteria can be increased, generation of flora metabolites, namely short-chain fatty acid and indolepropionic acid is promoted, and polyphenol can be converted into urolithin which is easier to absorb and has better activity under the action of flora; the method disclosed by the invention expands the activity of the deep-grain walnut endopleura polyphenol, and provides a new way for preventing intestinal microbe disorder by using the deep-grain walnut endopleura polyphenol as prebiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of extract preparation and application, and particularly relates to a preparation method of a deep-grained walnut inner seed coat extract and its application in regulating intestinal microbial diversity and flora metabolism. Background Art

[0002] Although the walnut inner seed coat only accounts for about 10% of the entire kernel mass, the polyphenols in the walnut kernel that are beneficial to the human body are mainly concentrated in the inner seed coat, accounting for more than 90% of the total polyphenols in the walnut kernel.

[0003] Yunnan is a deep-grained walnut ( Juglans sigillata D.) is the origin and distribution center of walnuts. Its planting area accounts for about 50% of the national walnut planting area, with an annual output of 148.58×10 4 However, as a species endemic to southwest my country, the research on the polyphenol content, components, and bioactivity of the endocarp of deep-grained walnut is still relatively lacking.

[0004] Traditional extraction methods for polyphenols suffer from limitations such as low extraction efficiency, loss of active substances, and the risk of organic solvent contamination. Subcritical water not only has a similar ability to organic solvents in dissolving bioactive substances, weakening the tight bonds between polysaccharides, polyphenols, and other substances, but is also non-toxic, pollution-free, and free of solvent residue. Furthermore, subcritical water modifies the structure of bioactive compounds, making it widely applicable for the extraction of bioactive substances.

[0005] Polyphenols are natural antioxidants found in plants and foods, especially condensed and hydrolyzable tannins, which have prebiotic effects, helping to increase the number of beneficial intestinal flora, reduce the abundance of harmful intestinal flora, and regulate the physiological metabolism of human tissues and organs, thereby improving or maintaining the health of the human body. Summary of the Invention

[0006] The invention provides a method for extracting a deep-grained walnut inner seed coat extract with subcritical water. The method comprises the following steps: uniformly mixing crushed deep-grained walnut inner seed coat with distilled water, adding the mixture into a high-pressure reactor, stirring and extracting at 100-120° C. and 1-3 MPa for 10-20 minutes, collecting the extract, concentrating it by rotary evaporation, and freeze-drying it to obtain a crude extract; dissolving the crude extract with water, adjusting the pH to 2-3, purifying the crude extract solution with an NKA-9 macroporous resin, eluting it with an ethanol solution with a volume concentration of 45-65%, collecting the eluate, concentrating it, and drying it to obtain the deep-grained walnut inner seed coat extract.

[0007] UPLC-Q-Orbitrap-MS 2The walnut endocarp extract was identified and found to have main components including HHDP-glucose, trigalloyl-HHDP-glucose, galloyl-HHDP-glucose, ellagic acid pentoside, ellagic acid hexoside, and monogalloyl-glucose isomer. The preparation method of the present invention is environmentally friendly, easy to operate, and low in cost. The obtained walnut endocarp polyphenol extract has high purity and good in vitro antioxidant activity.

[0008] Another object of the present invention is to use the deep-grained walnut inner seed coat polyphenol extract obtained by the above method in the preparation of a preparation for regulating intestinal microbial diversity and flora metabolism.

[0009] After in vitro simulated digestion and fermentation, the present invention found that the deep-grained walnut inner seed coat polyphenol extract can increase the beneficial bacteria Megamonas_rupellensis, Faecalibacterium_prausnitzii The abundance of the likes of phenols and polyphenols promotes the production of short-chain fatty acids such as propionic acid and indolepropionic acid, which are metabolites of the bacterial flora. In addition, polyphenol extracts can be converted by intestinal flora into urolithins, which are more beneficial to the human body.

[0010] The composition (or active ingredient) of the preparation for regulating intestinal microbial diversity and bacterial metabolism of the present invention is a polyphenol extract from the inner seed coat of deep-grained walnut. One or more excipients acceptable in pharmaceutical science or functional food may be added, or the preparation may be compounded with other active ingredients to exert therapeutic effects. The product can be made into pharmaceutical or functional food tablets, pills, powders, capsules, granules, etc., and can also be made into various forms such as oral liquid.

[0011] Advantages and technical effects of the present invention: The preparation process of the present invention is simple and easy to operate. The prepared walnut inner seed coat polyphenols have high purity and clear ingredients. Walnut inner seed coat is a major by-product of the walnut oil processing industry, is low in price, and the raw materials are easily available, so high-value utilization of the walnut inner seed coat can be achieved. The deep-grained walnut inner seed coat polyphenols prepared by the method have good effects in regulating intestinal flora diversity and promoting intestinal flora metabolism. The extract of the present invention is suitable for industrial production and market promotion and application, and has good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 1 shows the results of single-factor and response surface experiments on the subcritical water extraction conditions for polyphenols from the inner seed coat of deep-grained walnuts. Figure A shows the effect of extraction temperature on polyphenol content; Figure B shows the effect of extraction time on polyphenol content; Figure C shows the effect of solid-liquid ratio on polyphenol content; Figure D shows a 3D surface plot of extraction time and temperature; Figure E shows a 3D surface plot of solid-liquid ratio and extraction temperature; and Figure F shows a 3D surface plot of solid-liquid ratio and extraction time. Figure 2The results of total phenol content test of crude extract and purified polyphenol extract of deep-grained walnut inner seed coat; Figure 3 The results are the test results of DPPH free radical scavenging ability of crude extract and purified polyphenol extract of deep-grained walnut inner seed coat; Figure 4 The results are the test results of ABTS free radical scavenging ability of crude extract and purified polyphenol extract of deep-grained walnut inner seed coat; Figure 5 The results are the FRAP reducing ability test results of the crude extract and purified polyphenol extract of deep-grained walnut inner seed coat; Figure 6 This is the principal coordinate analysis (PCoA) of intestinal microorganisms after in vitro simulated fermentation of deep-grained walnut inner seed coat polyphenol extract; Figure 7 The composition of the intestinal microbiota at the phylum level after in vitro simulated fermentation of the polyphenol extract of the inner seed coat of deep-grained walnut; Figure 8 The composition of the intestinal microbiota at the genus level after in vitro simulated fermentation of the polyphenol extract of the inner seed coat of deep-grained walnut; Figure 9 The composition of intestinal microbiota at species level after in vitro simulated fermentation of polyphenol extracts from deep-grained walnut inner seed coat; Figure 10 This is the test result of the content of total short-chain fatty acids, a metabolite of bacterial flora, in the polyphenol extract of deep-grained walnut inner seed coat. Figure 11 The results of the test of acetic acid (Figure A), propionic acid (Figure B), butyric acid (Figure C), and isovaleric acid (Figure D) in the bacterial metabolites by the polyphenol extract of the deep-grained walnut inner seed coat; Figure 12 This is the result of the effect of the polyphenol extracts from the inner seed coat of deep-grained walnut on the indole derivatives of bacterial metabolites; Figure 13 These are the test results for urolithin M5 (Figure A); urolithin M6, urolithin A, and urolithin C (Figure B); and urolithin B and urolithin D (Figure C) produced through the interaction of deep-grained walnut inner seed coat polyphenol extract with bacterial flora. DETAILED DESCRIPTION

[0013] The present invention is further illustrated by the following examples, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified. Example 1: Extraction of polyphenols from deep-grained walnut seed coat 1. After drying and crushing the deep-grained walnut inner seed coat, the powder (10 g) was sieved through a 60-mesh sieve. 10 g of the walnut inner seed coat powder was mixed with 400 mL of distilled water in a high-pressure reactor. The temperature was raised to 100, 110, 120, 140, 160, and 180°C, respectively, with stirring at 65 r / min and a pressure of 2 MPa. After reacting for 20 min, the temperature was lowered and the pressure was released. The extract was collected and rotary evaporated at 55°C. The crude extract was freeze-dried to obtain the crude powder. The total phenol content of the crude extract obtained at different extraction temperatures was determined by the Folin & Ciocalteu method. The results are shown in Table 1. Figure 1 A; 2. Based on the results of step 1, with time as the single variable, heat at 110℃ for 5, 10, 15, 20, 25, and 30 min respectively to prepare crude powder. The rest of the operation is the same as step 1. The total phenol content results are shown in Figure 1 B; 3. Based on the results of step 1, the crushed walnut inner seed coat was mixed with distilled water at a material-liquid ratio of 1:30, 1:40, 1:50, 1:60, 1:70, and 1:80, respectively, and heated at 110°C for 15 min to prepare crude powder. The remaining operations were the same as step 1. The total phenol content results are shown in Table 3. Figure 1 C; 4. Based on the single-factor experimental results obtained in steps 1-3, a three-factor three-level experiment was designed using Design-Expert 13 software, with extraction temperature, extraction time, and solid-liquid ratio as independent variables and the total phenolic content of the deep-grained walnut seed coat as the response value. A total of 17 extractions were performed; the results are shown in Table 1. Figure 1 DF, the optimal process for subcritical water extraction of polyphenol extracts from deep-grained walnut seed coat was obtained as follows: temperature 112℃, time 12 min, and solid-liquid ratio 1:48.

[0014] 5. The crude extract prepared under the optimal process conditions obtained in step 4 was redissolved in distilled water to obtain a 2 mg / mL crude extract solution, and the pH of the solution was adjusted to 2; the pretreated NKA-9 macroporous resin was wet-loaded into a 16×60 cm chromatography column, and 500 mL of the 2 mg / mL, pH=2 crude extract solution was added to the chromatography column at a flow rate of 2 mL / min. After adsorption was completed, it was rinsed with distilled water for 12 h, and then eluted with 45% ethanol solution at a flow rate of 2 mL / min. The liquid in each tube was collected to determine the total phenol content, and an elution curve was drawn. The eluates with higher polyphenol content were combined, and vacuum rotary evaporation was performed at 45°C. After freeze-drying, a deep-grained walnut inner seed coat polyphenol extract (WPP) was obtained.

[0015] Example 2: Detection of total phenol content of polyphenol extracts from deep-grained walnut endocarp The polyphenol content of the crude extract in step 4 and the deep-grained walnut inner seed coat polyphenol extract WPP in step 5 of Example 1 was determined using the Folin & Ciocalteu method. 0.5 mL of a 0.02 mg / mL sample solution was vortexed with 2.5 mL of 10% Folin & Ciocalteu's Phenol Reagent and reacted for 5 minutes. Then, 2 mL of a 7.5% Na2CO3 solution was added and the mixture was reacted in the dark for 1 hour. The absorbance was measured at 765 nm, using gallic acid as a standard. The total phenol content (TPC) was expressed as gallic acid equivalent (mg GAE / g). See the results Figure 2 ,From the figure, it can be seen that the total phenol content of WPP is 585.30±2.54 mg GAE / g.

[0016] Example 3: Determination of the DPPH free radical scavenging ability of the deep-grained walnut endocarp polyphenol extract in Example 1 (DPPH method) 2.0 mL of 0.1 mmol / L DPPH working solution was mixed with 0.02 mg / mL crude extract solution and 0.5 mL of 0.02 mg / mL deep-grained walnut inner seed coat polyphenol extract solution, respectively. The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm. Trolox was used as a calibrant, and the DPPH radical scavenging capacity was expressed as Trolox equivalent (mmol TE / g). See the results Figure 3 ,It can be seen from the figure that the DPPH free radical capacity of WPP is 6.83±0.17 mmol TE / g.

[0017] Example 4: Determination of the ABTS free radical scavenging ability of the deep-grained walnut endocarp polyphenol extract in Example 1 (ABTS method) Adjust the absorbance of the ABTS+ working solution to 0.70 at 734 nm and store at 30°C until use. Mix 0.5 mL of the crude extract (0.02 mg / mL) and 0.02 mg / mL of the deep-grained walnut inner seed coat polyphenol extract with 4 mL of the ABTS+ working solution, incubate in a 30°C water bath for 6 minutes, and measure the absorbance at 734 nm. Using Trolox as a standard, the ABTS free radical scavenging capacity is expressed as Trolox equivalents (mmol TE / g). See the results Figure 4 ,It can be seen from the figure that the ABTS radical scavenging ability of WPP is 9.89±0.11 mmol TE / g.

[0018] Example 5: Determination of the FRAP Iron Ion Reducing Capacity of the Deep-grained Walnut Inner Seed Coat Polyphenol Extract in Example 1 (FRAP Method) Store the FRAP working solution at 37°C until use. Prepare the extract sample to be tested into a 2.0 mg / mL stock solution and dilute to a 0.02 mg / mL sample solution. Mix 150 μL of the 0.02 mg / mL sample solution with 4.5 mL of the FRAP working solution, incubate in a 37°C water bath for 10 minutes, and measure the absorbance at 593 nm. Using FeSO₄•7H₂O as the standard, the FRAP iron ion reducing capacity is expressed as FeSO₄•7H₂O equivalents (mmol FeSO₄•7H₂O / g). See the results Figure 5 ,It can be seen from the figure that the FRAP iron ion reducing ability of WPP is 22.54±0.27 mmolFeSO4•7H2O / g.

[0019] Example 6: In vitro simulated digestion of WPP and detection of polyphenol components in digestion products 1. In vitro simulated digestion 0.5 g of deep-grained walnut seed coat polyphenol extract (WPP), 35 mL of ultrapure water, and 15 mL of artificial gastric juice were mixed and placed in an anaerobic bottle. The pH was adjusted to 2 with 1 M HCl, and the mixture was sealed and incubated in a dark water bath at 100 rpm and 37°C under 10% O2 for 1 h to complete simulated gastric digestion. 10 mL of the gastric digestion mixture was frozen in a -80°C refrigerator to terminate the reaction. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and freeze-dried to obtain the gastric digestion product (GD), which was then stored at -20°C for later use. The remaining 40 mL of gastric digestion mixture was mixed with 20 mL of artificial small intestinal fluid, and the pH was adjusted to 7 with 6 M NaOH. The mixture was sealed under 8% O2 and incubated in a dark water bath at 100 rpm and 37°C for 3 h to complete the simulated small intestinal digestion. 10 mL of the small intestinal digestion mixture was placed in a -80°C refrigerator to terminate the reaction. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected and freeze-dried to obtain the small intestinal digestion stage product (ID), which was then stored at -20°C for later use.

[0020] 2. Using UPLC-Q-Orbitrap-MS 2 Analysis of polyphenol extracts from the inner seed coat of deep-grained walnut and its in vitro simulated digestion products HPLC conditions: Analyses were performed using a Poroshell 120EC-C18 column (2.1 mm × 100 mm, 1.9 μm) at 35°C, with an injection volume of 2 μL and a flow rate of 0.2 mL / min. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), with a gradient elution program: 0–1 min, 5% B; 1–5 min, 15% B; 5–10 min, 38% B; 10–15 min, 65% B; 15–18 min, 80% B; 18–20 min, 100% B; 20–22 min, 100% B.

[0021] MS analysis was performed in negative ion mode using an electrospray ionization (ESI) source. Mass spectrometry conditions included a spray voltage of 3.5 kV and a capillary temperature of 320°C. The full mass scan range was m / z 100–1000, with a resolution of 70,000 for full MS and 35,000 for MS2 measurements.

[0022] Data analysis and processing were performed using XCalibur software. Qualitative analysis was performed using MS and MS / MS comparisons based on known standards, publicly available data, and online databases such as MassBank and HMDB. The polyphenol composition of the WPP simulated in vitro digestion product was analyzed, and 18 compounds were identified. The identification results are shown in Table 1. The main components of the deep-grained walnut inner seed coat polyphenol extract (WPP) are HHDP-glucose, trigalloyl-HHDP-glucose, galloyl-HHDP-glucose, elagic acid pentoside, elagic acid hexoside, and monogalloyl-glucose. After simulated gastrointestinal digestion, the contents of most polyphenolic components decreased significantly. In particular, the contents of trigalloyl-HHDP-glucose, galloyl-HHDP-glucose, and bis-HHDP-glucose decreased by 86%, 57.65%, and 54.27%, respectively.

[0023] Table 1 .

[0024] Example 7: In vitro simulated fermentation of WPP and microbial diversity of in vitro simulated fermentation products 1. In vitro simulated fermentation The fermentation medium was prepared by mixing 2 g of peptone, 2 g of yeast extract, 2 g of NaHCO3, 2 mL of Tween-80, 0.1 g of NaCl, 0.04 g of KH2PO4, 0.04 g of K2HPO4, 0.01 g of MgSO4∙7H2O, 0.01 g of CaCl2∙6H2O, 0.02 g of hemin, 0.5 g of bile salts, 0.5 g of cysteine hydrochloride, 1 mg of resazurin, 10 μL of vitamin K1, and 1 L of ultrapure water. The mixture was homogeneously mixed and then sterilized by autoclaving at 121°C for 30 min.

[0025] The fecal culture used for fermentation was obtained from fecal samples collected from three healthy volunteers without metabolic or gastrointestinal diseases and who had not taken antibiotics for at least the past month. Immediately after fecal collection, the fecal sample was mixed with phosphate buffer (pH 7.0, 0.1 M) at a ratio of 1:9 (g:mL) in an anaerobic incubator. The mixture was then filtered through four layers of sterile gauze to prepare the fermentation broth, which was used within 1 hour. In a sterile anaerobic incubator, 0.4 g WPP, 18 mL of bacterial fermentation broth, and 22 mL of fermentation medium were added to an anaerobic bottle. A blank control group (without WPP) was set up and placed in a constant temperature incubator at 37°C, 120 rpm, and dark conditions for fermentation for 48 h. Samples were taken at 0, 6, 12, 24, and 48 h of fermentation. 1 mL of fermentation broth was taken for pH determination, 1 mL for short-chain fatty acids determination, 1 mL for microbial diversity detection, and the remaining 5 mL for polyphenol content, activity, and components.

[0026] 2. Microbial diversity analysis Total genomic DNA from the microbial community was extracted from fermentation samples according to the instructions of the EZNA® soil DNA kit (Omega Bio-tek, Norcross, GA, US). The quality of the extracted genomic DNA was assessed by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanoDrop 2000 (Thermo Scientific, USA). The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the upstream primer 27F (5'-AGRGTTYGATYMTGGGCTCAG-3') and the downstream primer 1492R (5'-RGYTACCTTGTTACGACTT-3') containing a barcode sequence. The PCR product was recovered on a 2% agarose gel and purified using a PCR Clean-Up Kit (Yuhua, China). The recovered product was quantified using a Qubit 4.0 (Thermo Fisher Scientific, USA). The purified PCR products were used to construct a library using the NEXTFLEX Rapid DNA-Seq Kit: (1) linker ligation; (2) using magnetic beads to screen and remove linker self-ligation fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using the Illumina Nextseq2000 platform (Shanghai Meiji Biotechnology Co., Ltd.). Using UPARSE v7.1 software (http: / / drive5.com / uparse / ), the quality control spliced sequences were clustered into operational taxonomic units (OTUs) based on a similarity of 97% and chimeras were removed. The RDP classifier [6] (http: / / rdp.cme.msu.edu / , version 2.11) was used to compare the Silva 16S rRNA gene database (v138) for OTU species taxonomy annotation, with a confidence threshold of 70%, and the community composition of each sample was statistically analyzed at different species classification levels. Figure 6 As can be seen from the results, there are significant differences in microbial diversity and structure between the control (Ctrl) group and the WPP group, indicating that WPP has a significant effect on the intestinal microbial community. Figure 7It can be seen that at the phylum level, the dominant intestinal microorganisms in all groups are Bacillota, Bacteroidota, and Actinomycetota, which is consistent with the typical characteristics of human intestinal microbial communities. After WPP fermentation, the abundance of Bacillota slowly decreased, while the abundance of Bacteroidota increased, resulting in a decrease in the ratio of Bacillota to Bacteroidota, which indicates that WPP has the potential to alleviate intestinal microbial imbalance. Figure 8 It can be seen that at the genus level, WPP fermentation significantly increased Megamonas (reaching a peak of 42.03% after 24 h) and Faecalibacterium The abundance of Ruminococcus 、 Dorea 、 Mediterraneibacter and Blautia Similarly, in Figure 9 Species level analysis of the bacterial community showed Megamonas rupellensis and Faecalibacterium prausnitzii The abundance of Blautia wexlerae 、 Dorea longicatena 、 Ruminococcus sp. ZG-924, Mediterraneibacter faecis 、 Ruminococcus sp. AF45-4BH and Dorea formicigenerans Overall, after in vitro simulated fermentation, WPP can be Megamonas rupellensis and Faecalibacterium prausnitzii abundance, while reducing Blautia wexlerae 、 Dorea longicatena 、 Ruminococcus sp. ZG-924, Mediterraneibacter faecis 、 Ruminococcus sp. AF45-4BH and Dorea formicigenerans to regulate the abundance of intestinal microbiota.

[0027] 3. Identification of polyphenolic components in the in vitro simulated digestion and fermentation products was performed using the same method as in Step 2 of Example 6. Seventeen compounds were identified, and the results are shown in Table 2. As can be seen from the table, the levels of most WPP components continued to decrease after colonic fermentation, likely due to their utilization by the microbial community. However, some components initially increased and then decreased. The content of Trigalloyl-HHDP-glucose increased from 20.54 μg / mL to 50.91 μg / mL after 6 h of fermentation, then decreased to 19.91 μg / mL after 48 h of fermentation. The content of Bis-HHDP-glucose increased from 14.39 μg / mL to 23.09 μg / mL after 12 h of fermentation, then decreased to 19.11 μg / mL after 48 h of fermentation.

[0028] Table 2 ; 4. Detection of short-chain fatty acids and indole derivatives Fermentation product samples were centrifuged for 5 minutes (4°C, 10,000 rpm). 0.4 mL of the supernatant was mixed with 0.2 mL of 50% H₂SO₄ and 0.4 mL of ethyl acetate and placed on ice for 10 minutes. The mixture was centrifuged at 10,000 rpm for 5 minutes. The upper organic layer was aspirated into a 2 mL centrifuge tube, and residual water was removed by adding anhydrous calcium chloride. The sample was then filtered through a 0.22 μm filter before analysis. Short-chain fatty acids were determined using a Shimadzu GCMS-QP2010 gas chromatograph-mass spectrometer using an HP-FFAP column (0.25 μm, 30 m × 0.32 mm). The column oven temperature was set initially at 100°C, then increased to 140°C at a rate of 6°C / min, and then to 200°C at a rate of 15°C / min for 3 minutes. Other operating instrument parameters were as follows: carrier gas was He, sample injection volume was 1 μL, split ratio was 1:50, and external standards (acetic acid, propionic acid, butyric acid, and isovaleric acid) were used for identification and quantification; See the results Figure 10 、 11 The figure shows that after in vitro simulated fermentation of deep-grained walnut seed coat polyphenols, the total SCFA content in the WPP group was significantly higher than that in the control group during fermentation, reaching a peak at 12 hours. Specifically, the WPP group promoted the production of short-chain fatty acids (acetic acid, propionic acid, butyric acid, and isovaleric acid) as metabolites of the bacterial community.

[0029] The results of the bacterial metabolite indole derivative test are shown in Figure 12 As can be seen from the figure, after in vitro simulated fermentation, the polyphenols in the seed coat of deep-grained walnut significantly promoted the production of bacterial metabolites indole-3-pyruvic acid and indole-3-propionic acid.

[0030] The results of WPP metabolizing urolithin after interaction with bacterial flora are shown in Figure 13 From the figure, it can be seen that the content of urolithin gradually increases with the extension of fermentation time, especially urolithin A, urolithin C, urolithin M5 and urolithin M6, which accumulate significantly during the fermentation process, among which the increase of urolithin M5 is the most obvious.

[0031] In summary, the method for extracting deep-grained walnut endocarp polyphenols of the present invention expands the activity of deep-grained walnut endocarp polyphenols and provides a new way for them to serve as prebiotics to prevent intestinal microbial disorders.

Claims

1. A method for preparing a deep-grained walnut endocarp extract, characterized in that: The crushed deep-grained walnut endocarp and distilled water are added to a high-pressure reactor, and the mixture is stirred and extracted at 100-120°C and 1-3 MPa for 10-20 min. The extract is then collected, concentrated by rotary evaporation, and freeze-dried to obtain a crude extract. The crude extract is dissolved in water, the pH is adjusted to 2-3, and the crude extract solution is purified using NKA-9 macroporous resin. The extract is eluted with a 45-65% by volume ethanol solution, and the eluate is collected, concentrated, and dried to obtain the deep-grained walnut endocarp extract.

2. Use of the deep-grained walnut endocarp extract prepared by the preparation method of the deep-grained walnut endocarp extract according to claim 1 in the preparation of a preparation for regulating intestinal microbial diversity and promoting microbial metabolism.

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