A walnut meal fermented peptide, its preparation method and application

Abstract

The present invention relates to the technical field of functional peptides. The present invention discloses a walnut meal fermented peptide, a preparation method and an application thereof. By mixing defatted and dephenolized walnut meal with Ganoderma lucidum, a walnut peptide that is easy to preserve is obtained. The obtained walnut peptide includes oligopeptides shown in SEQ ID NO.1 - SEQ ID NO.10, and the oligopeptides have the effects of anti-fatigue and anti-aging; the walnut meal fermented peptide also has the effect of up-regulating the proportion of beneficial bacteria in the intestine. It can be widely applied to pharmaceutical preparations, foods, health products, feeds or cosmetics for delaying aging and regulating the intestinal flora. It has a wide range of applications, simple and easily available raw materials, and a simple preparation method, which is suitable for popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of functional peptides, and particularly relates to a walnut meal fermented peptide, a preparation method thereof, and an application thereof. Background Art

[0002] The statements in this section only provide background information related to the disclosure of the present application and may not constitute prior art.

[0003] Exercise-induced fatigue is a physiological phenomenon that occurs when the human body's mental and physical activities continue to a certain stage, manifested as the inability of the body's physiological processes to maintain their functions at a specific level or to maintain a predetermined exercise intensity. Excessive exercise-induced fatigue may cause sports injuries and affect human body functions. To relieve exercise-induced fatigue, active peptides can be supplemented to enhance the nutrients in the body and regulate the homeostasis of the body's internal environment. As a plant-derived active peptide, walnut peptide has a better absorption mechanism than individual proteins and amino acids and has functions such as regulating enzymes in organisms and controlling the transport and absorption of minerals in organisms.

[0004] Aging is a normal change in the human life cycle. Aging will lead to a decline in the body's functions (such as antioxidant stress, learning and memory, and metabolic capacity, etc.) and increase the risk of the body getting sick. Common animal aging models include natural aging models, chemical intervention models, physical intervention models, and gene knockout / knock-in models. The chemical intervention model often uses D-galactose to construct an aging model. The D-galactose-induced aging model has the advantages of simple operation, few side effects, and a high survival rate during the experimental period. Compared with the natural aging model, the modeling time is short, which can lead to a decline in the body's antioxidant capacity and cause oxidative stress and cognitive impairment in the brain tissue, etc., and it is an ideal model for anti-aging research. Exercise, nutritional supplementation, genetic, and drug interventions, etc. have the effect of delaying aging. Cellular aging can be delayed and aging-related diseases can be prevented by inhibiting oxidative stress, inflammation, telomere shortening, and DNA damage.

[0005] Walnut kernels (Juglans regia L.) are resources that can be used both as medicine and food and have high nutritional value. As an important cash crop, China leads the world in walnut production. Walnut meal is a by-product after walnut oil extraction and is rich in nutrients such as proteins and vitamins. Walnut meal can be used to prepare bioactive peptides, and walnut meal bioactive peptides have the effects of antioxidant, anti-fatigue, improving memory, and regulating intestinal flora, etc.

[0006] Most of the existing methods for preparing walnut peptides use enzymatic methods, alkali solution acid precipitation methods, etc., and can prepare walnut polypeptides with antioxidant activity and antibacterial activity. For example, papain and alkaline protease are used to enzymatically hydrolyze walnut meal, and enzymatic hydrolysis products with different antioxidant activities can be prepared under different enzymatic hydrolysis conditions. However, the cost of enzymatic hydrolysis raw materials is high, and the process control is complex. Walnut peptides can be prepared by a simpler process through strain fermentation; different raw materials, different strains, and different fermentation processes result in different fermentation products. For example, under liquid fermentation conditions using Lactobacillus plantarum, the highest polypeptide yield can reach 0.263 g / g, and polypeptides with a molecular weight less than 3 kDa have strong antioxidant activity. The solid-state fermentation method often uses Bacillus subtilis and Aspergillus niger to ferment walnut meal to prepare walnut polypeptides.

[0007] The prior art has prepared a walnut meal fermentate with high immunological activity by fermenting walnut meal with Ganoderma lucidum. The active peptides included are GAATK, LMVIL, or VIPPR. The fermentation raw materials of this prior art are different from those of the present application. It uses walnut meal without pre-dephenolization and defatting treatment for fermentation, and the differences in fermentation raw materials result in significant differences in fermentation efficiency and products (Wu Wanxing. Research on the preparation of antioxidant active peptides from solid-state fermented walnut meal [D]: Kunming University of Science and Technology, 2014.). There is no overlap between the sequences screened in the present application and the sequences of this prior art, and this prior art does not indicate the possible existence of the peptides in the present application and the functions of the peptides in the present application.

[0008] At present, there is no report on the research of using Ganoderma lucidum strains to ferment walnut meal to prepare anti-fatigue and anti-aging peptides in China. Developing active peptides with anti-fatigue and anti-aging effects and regulating intestinal flora from walnut meal has important theoretical significance and application value. Summary of the Invention

[0009] The object of the present invention is to provide a walnut meal fermented peptide, its preparation method and application, aiming at the lack of methods for preparing anti-fatigue and anti-aging peptides by fermenting walnut meal with strains. Ganoderma lucidum is used to ferment defatted and dephenolized walnut meal, and a walnut meal fermented peptide including active oligopeptides shown in SEQ ID NO.1 - SEQ ID NO.10 is prepared after fermentation. It has anti-fatigue and anti-aging effects and the effect of regulating intestinal flora, and can be applied to cosmetics, foods, health products, and drugs to exert anti-aging effects.

[0010] The technical solution of the present invention is as follows:

[0011] On the one hand, the present invention provides a walnut meal fermented peptide, including oligopeptides shown in SEQ ID NO.1 - SEQ ID NO.10.

[0012] Another aspect of the present invention provides a method for preparing a walnut meal fermented peptide as described above, comprising the following steps:

[0013] Step S1: defatting and dephenolizing: crushing the walnut meal, adding ethanol solution, heating and refluxing extraction, extracting twice, filtering, and drying to obtain defatted and dephenolized walnut meal;

[0014] Step S2: fermentation: crush the defatted and dephenolized walnut meal, sieve it, add grade tertiary water, prepare fermentation medium, sterilize and cool it, add red ganoderma suspension, mix well and ferment;

[0015] Step S3: Inactivation: After the fermentation is completed, the filtrate is inactivated, centrifuged, and filtered. The peptide sequences contained in the filtrate are then analyzed based on LC-MS / MS. Peptides below the heptapeptide are selected and screened according to the intensity peptide abundance > 150,000 and the peptide level > 0.5 to obtain walnut meal fermentation peptides with anti-fatigue and anti-aging activities and intestinal flora regulation effects.

[0016] According to a preferred embodiment, the ethanol solution is a 50%-70% ethanol solution, and the solid-liquid ratio of the walnut meal to the ethanol solution is 1:40-1:60 (g:mL).

[0017] According to a preferred embodiment, the fermentation conditions in step S2 are: the sieve aperture of defatted and dephenolized walnut meal is 20-120 mesh, the defatted and dephenolized walnut meal powder: tertiary water-to-liquid ratio is 1:3-1:7 (g:mL), and the fermentation time is 3-7d.

[0018] According to a preferred embodiment, in step S2, the sieve aperture of the defatted and dephenolized walnut meal is 120 mesh, the defatted and dephenolized walnut meal powder: tertiary water-to-liquid ratio is 1:5, the fermentation time is 6 days, and the fermentation temperature is 28°C.

[0019] On the other hand, the present application provides the use of the walnut meal fermented peptide as described above as a functional food additive.

[0020] On the other hand, the present application provides the use of the walnut meal fermented peptide as described above in anti-aging cosmetics.

[0021] On the other hand, the present application provides the use of the walnut meal fermented peptide as described above in the preparation of an anti-aging composition.

[0022] According to a preferred embodiment, the composition is a medicine, food, health product or feed.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. A walnut meal fermented peptide and its preparation method, which uses the by-product walnut meal after walnut processing as the raw material, effectively utilizes walnut resources and avoids waste; after fermentation, multiple oligopeptides with anti-fatigue and anti-aging effects are successfully produced, which can play the role of anti-fatigue and anti-aging;

[0025] 2. A walnut meal fermented peptide and its preparation method, the process of fermenting with Ganoderma rubra is simple, has lower cost and a simpler and easier-to-control fermentation process compared with enzymatic treatment, and active peptide segments that cannot be produced by enzymatic hydrolysis are produced. This walnut meal fermented peptide can play the role of anti-fatigue and anti-aging, and at the same time has the effect of reducing the relative abundance of unfavorable bacteria in the intestine;

[0026] 3. A walnut meal fermented peptide, its preparation method and application, the walnut meal fermented peptide with anti-fatigue and anti-aging effects and the effect of reducing the relative abundance of unfavorable intestinal bacteria can be applied to foods, health products, drugs, cosmetics, etc. to play the anti-aging effect, or be used in foods, health products, drugs to regulate the balance of intestinal flora and maintain intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shows the effects of different fermentation conditions on the peptide content in walnut meal fermented peptide; on the left is the effect of sieve pore size; in the middle is the effect of solid-liquid ratio; on the right is the effect of fermentation time;

[0028] Figure 2 Is the heat map of the docking results of the target target and the oligopeptide molecules in the walnut meal fermented peptide;

[0029] Figure 3 The effects of walnut meal fermented peptide on the load-bearing swimming time, serum urea nitrogen (BUN) and liver glycogen level of fatigued mice;

[0030] Figure 4 Shows the effects of walnut meal fermented peptide on the intestinal flora phylum (left) and genus-level species composition (right) of fatigued mice;

[0031] Figure 5 Shows the effects of walnut meal fermented peptide on the serum SOD activity, MDA content, GSH activity and brain IL-6, Aβ1-42, Ach content and AchE activity of aging mice;

[0032] Figure 6 Shows the effects of walnut meal fermented peptide on the intestinal flora phylum (left) and genus-level species composition (right) of aging mice. DETAILED DESCRIPTION OF THE INVENTION

[0033] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. To better illustrate the present invention, numerous specific details are given in the following specific implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Without special instructions, the units used in this specification are all international standard units, and the numerical values and numerical value ranges appearing in the present invention should be understood to include the systematic errors inevitable in industrial production.

[0035] The characteristics and performance of the present invention will be further described in detail below in combination with the embodiments.

[0036] The present invention uses walnut meal as the raw material, and after degreasing and dephenolizing, it is fermented by Ganoderma lucidum to prepare a peptide with the functions of anti-fatigue, anti-aging, and regulating intestinal flora. Through single-factor experiments and uniform design experiments, the preparation process of Ganoderma lucidum walnut meal fermented peptide with the functions of anti-fatigue, anti-aging, and regulating intestinal flora is optimized. The amino acid composition and the sequence of anti-fatigue and anti-aging active peptide segments in the walnut fermented peptide prepared under the optimal conditions are analyzed. Then, through animal experiments, the behavioral indexes of mice, antioxidant indexes such as serum SOD and MDA, brain AchE activity, brain Ach content, and intestinal flora distribution are analyzed, in order to provide a research basis and evidence for the health attributes and potential functional values of walnuts.

[0037] Ganoderma lucidum was purchased from the Guangdong Provincial Microbial Culture Collection Center, and the culture collection number is: GDMCC 5.250.

[0038] Example 1

[0039] A preparation method of a walnut meal fermentation product with the functions of anti-aging and regulating intestinal flora, the steps are as follows:

[0040] Step S1: Degreasing and dephenolizing: Crush the walnut meal, add 50% ethanol solution, heat under reflux for 1 h according to the material-liquid ratio of 1:50, extract twice, filter, and dry to obtain degreased and dephenolized walnut meal.

[0041] Step S2: Fermentation: The defatted and dephenolized walnut meal is crushed and sieved. Take the defatted and dephenolized walnut meal in a conical flask, add tertiary water according to a fixed solid-liquid ratio to prepare a fermentation medium, sterilize it at 121 °C under high pressure for 15 min, take it out to the ultra-clean workbench, and after the medium cools to an appropriate temperature, add the activated Ganoderma lucidum spore suspension. After mixing evenly, ferment at 28 °C.

[0042] Step S3: Inactivation: After fermentation, inactivate it at 94 °C for 15 min. After centrifuging at 3000 r / min for 15 min, filter it and take the filtrate to obtain walnut meal fermented peptides.

[0043] Design a single-factor experiment to optimize the fermentation process. The fermentation conditions in Step S2 are as follows: the sieve pore size of the defatted and dephenolized walnut meal is 20 mesh - 120 mesh, the solid-liquid ratio of the defatted and dephenolized walnut meal powder to tertiary water is 1:3 - 1:7, and the fermentation time is 3 - 7 d. Measure the polypeptide content of the walnut meal fermented peptides with different fermentation conditions. The specific results of the single-factor experiment optimization process are shown in Figure 1 Based on the optimization results of the single-factor experiment, design U6(6 3 ) for investigation. The factor levels are shown in Table 1. After optimization, the equation is shown as:

[0044]

[0045] Among them, Y is the polypeptide content (mg / mL); X1 is the solid-liquid ratio; X2 is the fermentation time; X3 is the sieve pore size.

[0046] Perform a significance test on the equation. The multiple correlation coefficient R = 0.999, the determination coefficient R 2 = 0.998, the F value = 195.90, P = 0.0535, indicating that the regression equation has significant significance. The results show that the optimal optimization process is as follows: in Step S2, the sieve pore size of the defatted and dephenolized walnut meal is 120 mesh, the solid-liquid ratio of the defatted and dephenolized walnut meal powder to tertiary water is 1:5, and the fermentation time is 6 d. Carry out 3 parallel verification experiments according to the optimal process parameters. The polypeptide contents obtained three times are 19.38 mg / mL, 18.88 mg / mL, and 19.89 mg / mL respectively, which are close to the predicted value of 18.12 mg / mL, indicating that this process is stable.

[0047] Table 1 Uniform design factor level table

[0048]

[0049] Example 2

[0050] Analysis results of the peptide sequence of walnut meal fermented peptides:

[0051] After freeze-drying the walnut meal fermentation peptides prepared under the optimal process of Example 1, the walnut meal fermentation peptides prepared under the optimal conditions in Example 1 were analyzed by gas chromatography-mass spectrometry (LC-MS / MS) and size exclusion chromatography (SEC). The molecular weight distribution of the walnut meal fermentation peptides is shown in Table 2. Based on the analysis of the peptide sequences contained in the HLZ walnut meal fermentation peptides by LC-MS / MS, peptides with less than heptapeptides were selected and screened according to the peptide segment abundance of Intensity > 150,000 and the peptide segment grade > 0.5. Ten short peptides were obtained (see Table 3). ToxinPred was used to predict the toxicity of the screened short peptides. The structure of the peptide sequence was calculated using the polypeptide structure calculator (https: / / www.allpeptide.com / jiegoutu.html), and the structure was optimized by removing water and residues using the experimental Spider (Pymol) software. Target proteins related to fatigue and aging were searched through the Gene Card website. According to the functional roles of the target proteins, 5 target targets related to fatigue and aging were screened from several receptors with relatively high correlation coefficients. The target structure was confirmed using the Uniprot website and the PDB website, and the structure was optimized using the software Chem3D 22.0.0. Molecular docking was performed referring to the method of Yu et al. The peptide sequence and the target target were molecularly docked using the Autodock software to confirm the intermolecular interaction. All amino acid residues were kept rigid, and the ligand was treated flexibly. The docking box (40A×40A×40A) covered as much protein surface as possible during the docking process. The docking model with the lowest ligand binding energy in the protein binding pocket was selected as the best model. The Vina score was used as a prediction of the binding affinity between the peptide and the target protein (calculated in kJ / mol). The results (see Figure 2 and Table 3) showed that through molecular docking, the binding energy heat map of the aging and fatigue-related targets and the 10 short peptides was obtained (see Figure 2 ). When the binding energy < -5.0 kJ / mol, it indicated a relatively stable binding between the active ingredient and the core target. Figure 2 In the figure, blue represents the magnitude of the binding energy. The darker the color, the more stable the binding between the target and the walnut short peptide. From Figure 2 it can be seen that there are 3 aging and fatigue-related proteins, namely apolipoprotein E (APOE), brain-derived neurotrophic factor (BDNF), and interleukin 6 (IL6), which have relatively strong binding energies with the obtained walnut short peptides in sequence. Among the walnut short peptides, NFYL is the strongest, followed by DIIAFP, SGSNI, and DNGDNV (see Figure 2). BDNF is a neuroprotective factor that can promote the repair of nerve cell damage after ischemia, protect the brain nerves, avoid brain damage after exercise fatigue, affect the activation of age-related microglia, and delay aging. APOE is related to the expression of astrocytes in the brain and can affect neurodegenerative diseases related to memory and cognition. APOE4 can even trigger a severe inflammatory response. Chronic inflammatory markers such as IL-6 can cause fatigue in the body, and the content of IL-6 in the body also increases with age.

[0052] Table 2 Molecular weight distribution of walnut meal fermented peptides

[0053]

[0054] Table 3 Summary of 10 oligopeptides in walnut meal fermented peptides

[0055]

[0056] Example 3:

[0057] Verification of the anti-fatigue effect of Ganoderma lucidum walnut meal fermented peptide is as follows:

[0058] Grouping of mice: 30 SPF-grade male Kunming mice were randomly divided into 3 groups, with 10 mice in each group, namely the fatigue model group, the American ginseng group (positive control group), and the Ganoderma lucidum walnut meal fermented peptide group (experimental group). The mice in the experimental group were given the walnut meal fermented peptide prepared by the optimal process of Example 1 at a corresponding dose, calculated as 2 mg / (g·d) according to the polypeptide content. The positive control group was given American ginseng at 0.6 mg / (g·d), and the fatigue model group was intragastrically administered 0.9% normal saline. Each group of animals was intragastrically administered at a volume of 10 ml / (kg·bw·d) for 6 consecutive weeks. During the feeding period, the mice drank water and ate normally. Observe the mental state, activity and hair condition of the mice, and measure the body weight of each group of mice once a week and record it.

[0059] 30 minutes after the last administration of the test sample, the mice with a 5% body weight lead sheet loaded at the tail root were placed in a swimming tank for swimming. The swimming tank was 30×70×40 cm, the water depth was greater than 30 cm, and the water temperature was 25℃±1.0℃. Record the time from the start of swimming until the mouse's head is submerged in the water for 7 seconds without surfacing, that is, the weight-bearing swimming time of the mouse.

[0060] After the weight-bearing swimming of the mice ended, they were immediately anesthetized, the eyeballs were taken for blood sampling, and then they were decapitated and sacrificed. After the blood samples were left standing at room temperature for 30 minutes, they were centrifuged (3000 r / min, 10 minutes) to obtain the supernatant, which was stored in a -80℃ refrigerator for later use; the liver and other organs were taken and stored frozen for later use; the content of serum urea nitrogen (BUN) was measured using a kit, the glycogen content of the mouse liver was measured, and the effect of Ganoderma lucidum walnut meal fermented peptide on the intestinal flora was measured from the colon contents.

[0061] The results of measuring the swimming endurance time with load, serum urea nitrogen (BUN), and hepatic glycogen content in mice are shown in Figure 3 . The results showed that compared with the model group, the swimming endurance time with load in the American ginseng group and the Ganoderma lucidum-fermented walnut meal peptide group was significantly increased. Compared with the model group, the BUN content in mice of the American ginseng group and the Ganoderma lucidum-fermented walnut meal peptide group was significantly decreased, and the hepatic glycogen content was significantly increased. Based on the comprehensive experimental results, it is indicated that the walnut meal peptide prepared under the optimal conditions of Example 1 has high anti-fatigue activity.

[0062] Example 4:

[0063] Verification of the effect of Ganoderma lucidum-fermented walnut meal peptide on regulating the intestinal flora of fatigued mice was carried out as follows:

[0064] Colon contents of the three groups of mice in Example 3 were taken, and the intestinal flora of the fatigue model group (model group), American ginseng group (AG), and Ganoderma lucidum-fermented walnut meal peptide group (WL) were measured. By analyzing the species composition at the phylum and genus levels of the flora, the results are shown in Figure 4 . At the phylum level, compared with the model group, the fermented walnut meal peptide could significantly reverse the change in the relative abundance of Campylobacterota in the intestine of mice fatigued by swimming with load ( Helicobacter <0.05), and increase the relative abundances of Bacteroidota and Proteobacteria. At the genus level, compared with the model group, the relative abundances of Campylobacterota in the American ginseng group and the fermented walnut meal peptide group were significantly decreased ( P <0.05). Helicobacter P <0.05).

[0065] Example 5:

[0066] Verification of the anti-aging effect of Ganoderma lucidum-fermented walnut meal peptide was carried out as follows:

[0067] Grouping of mice: 30 SPF-grade male Kunming mice were randomly divided into 3 groups, with 10 mice in each group, namely the aging model group, the normal group, and the Ganoderma lucidum-fermented walnut meal peptide group (experimental group). The Ganoderma lucidum-fermented walnut meal peptide group was given the walnut meal peptide prepared by the optimal process of Example 1 at the corresponding dose, which was 2 mg / (g·d) calculated by polypeptide content. The aging model group was intragastrically administered 0.9% normal saline, and the normal group was intragastrically administered 0.9% normal saline. During the administration period, except for the normal group, the aging model group and the Ganoderma lucidum-fermented walnut meal peptide group were injected with D-galactose at 0.36 mg / (g·d) to induce aging. Each group of animals was intragastrically administered at a volume of 10 ml / (kg·bw·d), and the administration continued for 8 weeks. During the feeding period, the animals drank water and ate normally, and the body weight, mental state, activity, and hair condition of the mice were observed, etc.

[0068] ​After the last administration to the mice, they were fasted for 12 h with free access to water, anesthetized, and sacrificed by decapitation after blood collection by eye socket puncture. The blood samples were allowed to stand at room temperature for 30 min and then centrifuged (3000 r / min, 10 min) to obtain the supernatant, which was stored in a -80 °C refrigerator for later use. Using a kit, the activities of serum superoxide dismutase (SOD), the contents of malondialdehyde (MDA), reduced glutathione (GSH), the activities of brain acetylcholinesterase (AchE), the contents of acetylcholine (Ach), β-amyloid (1-42) (Aβ1-42), and interleukin 6 (IL-6) were measured according to the operating instructions of the kit.

[0069] The results of the activities of SOD, the contents of MDA and GSH in mouse serum, and the activities of AchE, the contents of Ach, Aβ1-42, and IL-6 in the brain are shown in Figure 5 . The results showed that, compared with the normal group, the activities of serum SOD and the contents of GSH in the aging model group ( P <0.0001) were significantly decreased, the content of MDA was significantly increased ( P <0.001), the content of brain IL-6 ( P <0.0001), Aβ1-42 ( P <0.0001), and the content of AchE ( P <0.05) were all significantly increased, and the content of Ach was significantly decreased ( P <0.001), indicating that the aging model was successfully established. Compared with the aging model group, the activities of serum SOD ( P <0.001) and the contents of GSH ( P <0.0001) in the Ganoderma lucidum walnut meal fermented peptide group were both significantly increased, the content of MDA ( P <0.0001) was significantly decreased, the content of brain IL-6 ( P <0.001), the content of AchE ( P <0.0001), and Aβ1-42 ( P <0.05) were all significantly decreased, and the content of brain Ach ( P <0.01) was significantly increased. Based on the comprehensive experimental results, it was indicated that the Ganoderma lucidum walnut meal fermented peptide prepared under the optimal conditions of Example 1 had anti-aging activity.

[0070] Example 6:

[0071] To verify the effect of walnut meal fermented peptide on regulating the intestinal flora of aging mice, the steps were as follows:

[0072] The colonic contents of the three groups of mice in Example 5 were taken, and the intestinal flora of the mice in the aging model group, normal group, and Ganoderma lucidum walnut meal fermented peptide group were measured. By analyzing the species composition at the phylum and genus levels of the flora, the results are shown in Figure 6At the phylum level, the Ganoderma lucidum walnut meal fermented peptide can affect the structure of the colonic microbiota in D-galactose-induced senescent mice and increase the relative abundances of Actinobacteria and Verrucomicrobia in the colon of mice. Administering D-galactose can significantly reduce the relative abundance of Firmicutes Lactobacillus and significantly increase the relative abundance of Actinobacteria Enterorhabdus in the colonic contents of mice. Administering the Ganoderma lucidum walnut meal fermented peptide can reverse this change to a certain extent.

[0073] The above-described embodiments merely represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. Application of walnut meal fermented peptide in anti-aging cosmetics, characterized in that, The walnut meal fermented peptide is prepared by the following preparation method, comprising the following steps: Step S1: defatting and dephenolizing: crushing the walnut meal, adding ethanol solution, heating and refluxing extraction, extracting twice, filtering, and drying to obtain defatted and dephenolized walnut meal; Step S2: fermentation: crush the defatted and dephenolized walnut meal, sieve it, add grade tertiary water, prepare fermentation medium, sterilize and cool it, add red ganoderma suspension, mix well and ferment; Step S3: inactivation: inactivation after fermentation is completed, centrifugation, suction filtration and filtrate collection to obtain walnut meal fermentation peptides; the walnut meal fermentation peptides include oligopeptides as shown in SEQ ID NO.1-SEQ ID NO.10; The ethanol solution in step S1 is a 50%-70% ethanol solution, and the solid-liquid ratio of the walnut meal to the ethanol solution is 1:40-1:60 (g:mL).

2. The application according to claim 1, wherein The fermentation conditions in step S2 are as follows: the sieve aperture of the defatted and dephenolized walnut meal is 20-120 meshes, the defatted and dephenolized walnut meal powder: the tertiary water-to-liquid ratio is 1:3-1:7 (g:mL), and the fermentation time is 3-7 days.

3. The application according to claim 2, wherein In the step S2, the sieve aperture of the defatted and dephenolized walnut meal is 120 meshes, the defatted and dephenolized walnut meal powder: the third-grade water-to-liquid ratio is 1:5, the fermentation time is 6 days, and the fermentation temperature is 28°C.

4. Use of walnut meal fermented peptide in the preparation of an anti-aging pharmaceutical composition, characterized in that, The walnut meal fermented peptide is prepared by the following preparation method, comprising the following steps: Step S1: defatting and dephenolizing: crushing the walnut meal, adding ethanol solution, heating and refluxing extraction, extracting twice, filtering, and drying to obtain defatted and dephenolized walnut meal; Step S2: fermentation: crush the defatted and dephenolized walnut meal, sieve it, add grade tertiary water, prepare fermentation medium, sterilize and cool it, add red ganoderma suspension, mix well and ferment; Step S3: inactivation: inactivation after fermentation is completed, centrifugation, suction filtration and filtrate collection to obtain walnut meal fermentation peptides; the walnut meal fermentation peptides include oligopeptides as shown in SEQ ID NO.1-SEQ ID NO.10; The ethanol solution in step S1 is a 50%-70% ethanol solution, and the solid-liquid ratio of the walnut meal to the ethanol solution is 1:40-1:60 (g:mL).

5. The application according to claim 4, characterized in that, The fermentation conditions in step S2 are as follows: the sieve aperture of the defatted and dephenolized walnut meal is 20-120 meshes, the defatted and dephenolized walnut meal powder: the tertiary water-to-liquid ratio is 1:3-1:7 (g:mL), and the fermentation time is 3-7 days.

6. The application according to claim 5, wherein In the step S2, the sieve aperture of the defatted and dephenolized walnut meal is 120 meshes, the defatted and dephenolized walnut meal powder: the third-grade water-to-liquid ratio is 1:5, the fermentation time is 6 days, and the fermentation temperature is 28°C.

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