Walnut meal fermentation peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of functional peptides, and discloses a walnut meal fermentation peptide and a preparation method and application thereof.The walnut meal fermentation peptide easy to preserve is obtained through mixed fermentation of degreased and dephenolized walnut meal and ganoderma lucidum, the obtained walnut peptide comprises oligopeptides shown in SEQ ID NO.1-SEQ ID NO.10, and the oligopeptides have the anti-fatigue and anti-aging effects; the walnut meal fermentation peptide also has the effect of increasing the proportion of beneficial bacteria in intestinal tracts. The composition can be widely applied to pharmaceutical preparations, foods, health-care products, feeds or cosmetics for delaying senescence and regulating intestinal flora, and is wide in application range, simple and easily available in raw materials, simple in preparation method and suitable for popularization.

Description

Technical Field

[0001] The invention relates to the technical field of functional peptides, and in particular to a walnut meal fermentation peptide and a preparation method and application thereof. Background Art

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

[0003] Sports fatigue is a physiological phenomenon that occurs when the human body's mental and physical activities continue to reach a certain stage. It is manifested as the body's physiological process cannot maintain its function at a specific level or cannot maintain the predetermined exercise intensity. Excessive sports fatigue may cause sports injuries and affect human functions. In order to relieve sports fatigue, active peptides can be supplemented to enhance the body's nutrients and regulate the body's internal environment. As a plant-derived active peptide, walnut peptide has a better absorption mechanism than individual proteins and amino acids. It has the functions of regulating enzymes in the body and regulating the transportation and absorption of minerals in the body.

[0004] Aging is a normal change in the human life cycle. Aging can lead to a decline in body functions (such as anti-oxidative stress, learning and memory, and metabolic capacity) and increase the risk of disease. Commonly used animal aging models are natural aging models, chemical intervention models, physical intervention models, and gene knockout / knock-in models. Chemical intervention models often use D-galactose to construct aging models. The D-galactose-induced aging model has the advantages of simple operation, few side effects, and 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 brain tissue. It is an ideal model for anti-aging research. Exercise, nutritional supplements, genetic and drug interventions have the effect of delaying aging. Cell aging can be delayed and aging-related diseases can be prevented by inhibiting oxidative stress, inflammation, telomere shortening, and DNA damage.

[0005] Walnut kernel (Juglans regia L.) is a medicinal and edible resource with high nutritional value. As an important economic crop, China's production of walnuts leads the world. Walnut meal is a by-product of walnut oil extraction, rich in nutrients such as protein and vitamins. Walnut meal can be used to prepare bioactive peptides, which have antioxidant, anti-fatigue, memory improvement and intestinal flora regulation effects.

[0006] The preparation of walnut peptides currently mostly adopts enzymatic methods, alkaline dissolution and acid precipitation methods, etc., which can prepare walnut peptides with antioxidant and antibacterial activities. For example, walnut meal is hydrolyzed by papain and alkaline protease, and enzymatic products with different antioxidant activities can be prepared under different enzymatic conditions. However, the cost of enzymatic raw materials is high and the process control is complex. Walnut peptides can be prepared by a simpler process through bacterial fermentation; different raw materials, different strains and different fermentation processes produce different fermentation products. For example, under liquid fermentation conditions using Lactobacillus plantarum, the peptide yield can reach up to 0.263 g / g, and peptides with a molecular weight of less than 3 kDa have strong antioxidant activity. Solid-state fermentation often uses Bacillus subtilis and Aspergillus niger to ferment walnut meal to prepare walnut peptides.

[0007] The prior art prepares a walnut meal fermentation product with high immune activity by fermenting walnut meal with red ganoderma, including active peptides such as GAATK, LMVIL or VIPPR. The prior art is different from the fermentation raw materials of the present application. It uses walnut meal that has not been pre-dephenolized and defatted for fermentation. The difference in fermentation raw materials leads to large differences in fermentation efficiency and products (Wu Wanxing. Research on the preparation of antioxidant active peptides by solid-state fermentation of walnut meal [D]: Kunming University of Science and Technology, 2014.). The sequences screened by the present application do not overlap with the sequences of the prior art, and the prior art does not point out 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 red Ganoderma lucidum to ferment walnut meal to prepare anti-fatigue and anti-aging peptides. Using walnut meal to develop active peptides with anti-fatigue and anti-aging effects and regulating intestinal flora has important theoretical significance and application value. Summary of the invention

[0009] The purpose of the present invention is to provide a walnut meal fermented peptide and a preparation method and application thereof in view of the problem that there is currently a lack of methods for preparing anti-fatigue and anti-aging peptides by fermenting walnut meal with bacteria, and to use the walnut meal fermented peptide. The defatted and dephenolized walnut meal is fermented by red ganoderma lucidum to prepare walnut meal fermented peptides including active oligopeptides shown as SEQ ID NO.1-SEQ ID NO.10 after fermentation. The walnut meal fermented peptides have anti-fatigue and anti-aging effects and the function of regulating intestinal flora, and can be applied to cosmetics, foods, health products and medicines to exert anti-aging effects.

[0010] The technical solution of the present invention is as follows: In one aspect, the present invention provides a walnut meal fermentation peptide, including the oligopeptides shown as SEQ ID NO.1-SEQ ID NO.10.

[0011] Another aspect of the present invention provides a method for preparing a walnut meal fermented peptide as described above, 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: 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.

[0012] 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).

[0013] 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.

[0014] 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.

[0015] On the other hand, the present application provides the use of the walnut meal fermented peptide as described above as a functional food additive.

[0016] On the other hand, the present application provides the use of the walnut meal fermented peptide as described above in anti-aging cosmetics.

[0017] 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.

[0018] According to a preferred embodiment, the composition is a medicine, food, health product or feed.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. A walnut meal fermented peptide and a preparation method thereof, which uses walnut meal, a byproduct of walnut processing, as a raw material, effectively utilizing walnut resources and avoiding waste; after fermentation, multiple oligopeptides with anti-fatigue and anti-aging effects are successfully produced, which can play an anti-fatigue and anti-aging role; 2. A walnut meal fermented peptide and a preparation method thereof. The fermentation process using red ganoderma lucidum is simple, and compared with enzyme treatment, it has lower cost and a simpler and easier-to-control fermentation process, and produces active peptide segments that cannot be produced by enzymatic hydrolysis. The walnut meal fermented peptide can play an anti-fatigue and anti-aging role, and also has the effect of reducing the relative abundance of unfavorable flora in the intestine; 3. A walnut meal fermented peptide and its preparation method and application. The fermented walnut meal fermented peptide has anti-fatigue and anti-aging effects and can reduce the relative abundance of adverse intestinal flora. It can be used in food, health products, medicines, cosmetics, etc. to exert anti-aging effects, or can be used in food, health products, and medicines to regulate the balance of intestinal flora and maintain intestinal health. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effects of different fermentation conditions on the peptide content of walnut meal fermented peptides; the left one is the effect of sieve aperture; the middle one is the effect of material-liquid ratio; the right one is the effect of fermentation time; Figure 2 This is the heat map of the docking results between the target and the oligopeptide molecules in walnut meal fermentation peptides; Figure 3 Effects of walnut meal fermented peptides on weight-bearing swimming time, serum urea nitrogen (BUN) and liver glycogen levels in fatigue mice; Figure 4 Effects of walnut meal fermented peptides on the phylum (left) and genus-level species composition (right) of the intestinal flora in fatigue mice; Figure 5 To study the effects of walnut meal fermented peptides on serum SOD activity, MDA content, GSH activity and brain IL-6, Aβ1-42, Ach content and AchE activity in aging mice; Figure 6 Effects of walnut meal fermented peptides on the phylum (left) and genus-level species composition (right) of the intestinal flora in aged mice. DETAILED DESCRIPTION

[0021] 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, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0023] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0024] The present invention uses walnut meal as raw material, and after defatting and dephenolization, a peptide with anti-fatigue, anti-aging and intestinal flora regulating effects is prepared by fermentation of red ganoderma lucidum. The preparation process of the red ganoderma lucidum walnut meal fermented peptide with anti-fatigue, anti-aging and intestinal flora regulating effects is optimized through single factor experiments and uniform design experiments, the amino acid composition of the walnut fermented peptide prepared under the optimal conditions and the sequence of the peptide segment with anti-fatigue and anti-aging activity are analyzed, and then the behavioral indicators of mice, serum SOD and MDA and other antioxidant indicators, brain AchE activity, brain Ach content and intestinal flora distribution are analyzed through animal experiments, in order to provide a research basis and basis for the health attributes and potential functional value of walnuts.

[0025] Red Ganoderma lucidum was purchased from Guangdong Microbiological Culture Collection Center with the culture collection number of GDMCC 5.250.

[0026] Example 1 A method for preparing a walnut meal fermentation product having anti-aging and intestinal flora regulating effects, comprising the following steps: Step S1: defatting and dephenolizing: walnut meal is crushed, 50% ethanol solution is added, and the mixture is heated under reflux and extracted for 1 h at a solid-liquid ratio of 1:50, extracted twice, filtered, and dried to obtain defatted and dephenolized walnut meal.

[0027] Step S2: Fermentation: Grind the defatted and dephenolized walnut meal and sieve it, put the defatted and dephenolized walnut meal into a conical flask, add grade tertiary water according to a fixed solid-liquid ratio, prepare the fermentation medium, sterilize it under high pressure at 121°C for 15 min, take it out to the clean bench, wait for the medium to cool to an appropriate temperature, add the activated red ganoderma suspension, mix well, and ferment at 28°C.

[0028] Step S3: Inactivation: After fermentation, inactivate at 94°C for 15 min, centrifuge at 3000 r / min for 15 min, and filter the filtrate to obtain walnut meal fermentation peptide.

[0029] A single factor experiment was designed to optimize the fermentation process. The fermentation conditions in step S2 were: the sieve aperture of defatted and dephenolized walnut meal was 20-120 mesh, the defatted and dephenolized walnut meal powder: the three-level water-to-liquid ratio was 1:3-1:7, and the fermentation time was 3-7 days. The polypeptide content of walnut meal fermentation peptides under different fermentation conditions was determined. 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, we designed U6 (6 3 ) was investigated, and the factor levels are shown in Table 1. The optimized display equation is:

[0030] Among them, Y is the polypeptide content (mg / mL); X1 is the solid-liquid ratio; X2 is the fermentation time; X3 is the sieve aperture.

[0031] The significance test of the equation was carried out, and the multiple correlation coefficient R = 0.999, the determination coefficient R 2 =0.998, F value = 195.90, P =0.0535, indicating that the regression equation is significant. The results show that the best optimized process is: in step S2, the sieve aperture of defatted and dephenolized walnut meal is 120 mesh, the defatted and dephenolized walnut meal powder: the third-level water-liquid ratio is 1:5, and the fermentation time is 6 d. Three parallel verification experiments were carried out according to the optimal process parameters, and the polypeptide contents were obtained three times, 19.38 mg / mL, 18.88 mg / mL and 19.89 mg / mL, respectively, which is close to the predicted value of 18.12 mg / mL, indicating that this process is stable.

[0032] Table 1 Uniform design factor level table

[0033] Example 2 Peptide sequence analysis results of walnut meal fermentation peptides: The walnut meal fermentation peptide prepared by the optimal process of Example 1 was freeze-dried, and the walnut meal fermentation peptide prepared by the optimal conditions in Example 1 was analyzed by gas chromatography-mass spectrometry (LC-MS / MS) and size exclusion chromatography (SEC). The molecular weight distribution of the walnut meal fermentation peptide is shown in Table 2. Based on the LC-MS / MS analysis of the peptide sequence contained in the HLZ walnut meal fermentation peptide, the peptides below the heptapeptide were selected and screened according to the intensity peptide abundance>150000 and the peptide level>0.5, and 10 short peptides were screened (see Table 3). ToxinPred was used to predict the toxicity of the screened short peptides. The structure of the generated peptide sequence was calculated using the polypeptide structure calculator (https: / / www.allpeptide.com / jiegoutu.html), and the experimental Spider (Pymol) software was used to remove water and residues to optimize the structure. The target proteins related to fatigue and aging were searched through the Gene Card website. According to the functional role of the target protein, 5 target sites related to fatigue and aging were screened from several receptors with high correlation coefficients. The target structure was confirmed using the Uniprot website and the PDB website. The structure was optimized using the software Chem3D 22.0.0. Molecular docking was performed with reference to the method of Yu et al. The peptide sequence and the target site were molecularly docked using the Autodock software to confirm the interaction between the molecules. All amino acid residues remained rigid, the ligand was handled flexibly, and 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 the prediction of the affinity of the peptide binding to the target protein (calculated in kJ / mol). The results (see Figure 2 and Table 3) show that after molecular docking, the binding energy heat map of aging and fatigue related targets and 10 short peptides was obtained (see Figure 2 ). When the binding energy is <−5.0 kJ / mol, it indicates that there is a relatively stable binding between the active ingredient and the core target. Figure 2 The medium blue color indicates the size of the binding energy. The darker the color, the more stable the binding between the target and the walnut peptide. Figure 2 It can be seen that three aging and fatigue-related proteins, apolipoprotein E (APOE), brain-derived neurotrophic factor (BDNF), and interleukin 6 (IL6), have strong binding energy with the obtained walnut short peptides, and NFYL is the strongest walnut short peptide, 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 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, which can affect neurodegenerative diseases related to memory and cognition. APOE4 can even cause severe inflammatory reactions. Chronic inflammatory markers such as IL-6 can cause fatigue in the body. With age, the content of IL-6 in the body will also increase.

[0034] Table 2 Molecular weight distribution of walnut meal fermented peptides

[0035] Table 3 Summary of 10 oligopeptides from walnut meal fermentation peptides

[0036] Embodiment 3: The anti-fatigue effect of red Ganoderma lucidum walnut meal fermented peptide was verified by the following steps: Mouse grouping: 30 SPF male Kunming mice were randomly divided into 3 groups, 10 mice / group, namely fatigue model group, American ginseng group (positive control group), and red Ganoderma lucidum walnut meal fermented peptide group (experimental group). The experimental group was given a corresponding dose of walnut meal fermented peptide prepared by the optimal process of Example 1, calculated as 2 mg / (g·d) according to the polypeptide content, the positive control group was given American ginseng 0.6 mg / (g·d), and the fatigue model group was gavaged with 0.9% normal saline. Each group of animals was gavaged at a gavage volume of 10 ml / (kg·bw·d) for 6 weeks. During the feeding period, the mice drank water and ate normally, and the mental state, activity and hair condition of the mice were observed. The weight of the mice in each group was measured and recorded once a week.

[0037] 30 minutes after the last administration of the test sample, mice with a lead load of 5% of their body weight at the base of their tails were placed in a swimming box with a size of 30×70×40 cm, a water depth of more than 30 cm, and a water temperature of 25℃±1.0℃. The time from the start of swimming to the time when the head of the mouse was submerged in the water for 7 seconds without surfacing was recorded, which was the weighted swimming time of the mouse.

[0038] After the mice finished weight-bearing swimming, they were immediately anesthetized, their eyeballs were removed, and blood was collected. Then, the mice were killed by cervical dislocation. The blood samples were allowed to stand at room temperature for 30 min, then centrifuged (3000 r / min, 10 min) to obtain the supernatant, which was stored in a -80°C refrigerator for later use. The liver and other organs were removed and frozen for later use. The serum urea nitrogen (BUN) content was determined using a kit, and the liver of mice was removed to determine the glycogen content. The colon contents were taken to determine the effects of red Ganoderma lucidum walnut meal fermented peptides on intestinal flora.

[0039] The results of the mouse swimming weight-bearing time, serum urea nitrogen (BUN) and liver glycogen content are shown in Figure 3. The results showed that compared with the model group, the weight-bearing swimming time of the American ginseng group and the red ganoderma lucidum walnut meal fermented peptide group was significantly increased. Compared with the model group, the BUN content of mice in the American ginseng group and the red ganoderma lucidum walnut meal fermented peptide group was significantly decreased, and the glycogen content was significantly increased. The comprehensive experimental results indicate that the walnut meal fermented peptide prepared under the optimal conditions of Example 1 has high anti-fatigue activity.

[0040] Embodiment 4: The efficacy of red Ganoderma lucidum walnut meal fermented peptide in regulating the intestinal flora of fatigue mice was verified by the following steps: The colon contents of the three groups of mice in Example 3 were taken to measure the intestinal flora of the fatigue model group (model group), American ginseng group (AG) and red ganoderma lucidum walnut meal fermented peptide group (WL). The species composition of the flora at the genus level was analyzed. The results are shown in Figure 4 At the phylum level, compared with the model group, walnut meal fermented peptides could significantly reverse the growth of Campylobacter in the intestine of mice with fatigue induced by weighted swimming. Helicobacter The relative abundance changes of P <0.05), increased the relative abundance of Bacteroidetes and Proteobacteria. At the genus level, compared with the model group, the Campylobacter phylum in the American ginseng group and the walnut meal fermented peptide group Helicobacter The relative abundance of P <0.05).

[0041] Embodiment 5: The anti-aging efficacy of red Ganoderma lucidum walnut meal fermented peptides was verified by the following steps: Mouse grouping: 30 SPF male Kunming mice were randomly divided into 3 groups, 10 mice / group, namely, aging model group, normal group, and red Ganoderma lucidum walnut meal fermented peptide group (experimental group). The red Ganoderma lucidum walnut meal fermented peptide group was given the red Ganoderma lucidum walnut meal fermented peptide group with the corresponding dose of walnut meal fermented peptide prepared by the optimal process of Example 1, calculated as 2 mg / (g·d) according to the polypeptide content. The aging model group was gavaged with 0.9% saline, and the normal group was gavaged with 0.9% saline. During the administration period, except for the normal group, the aging model group and the red Ganoderma lucidum walnut meal fermented peptide group were injected with D-galactose at 0.36 mg / (g·d) to induce aging. All animals in each group were gavaged at a gavage volume of 10 ml / (kg·bw·d) for 8 weeks. During the feeding period, they drank water and ate normally, and the weight, mental state, activity and hair condition of the mice were observed.

[0042] After the last administration, mice were fasted for 12 h but not water. After anesthesia, the eyeballs were removed and blood was collected, and then the mice were killed by dislocation of the neck. The blood samples were allowed to stand at room temperature for 30 min, then centrifuged (3000 r / min, 10 min) to obtain the supernatant, which was stored in a refrigerator at -80°C for later use. The serum superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reduced glutathione (GSH) content, brain acetylcholinesterase (AchE) activity, acetylcholine (Ach) content, β-amyloid protein (1-42) (Aβ1-42) content, and interleukin 6 (IL-6) content were determined using the kit according to the kit's operating instructions.

[0043] The results of serum SOD activity, MDA content, GSH content and brain AchE activity, Ach content, Aβ1-42 content and IL-6 content of mice are shown in Figure 5 The results showed that compared with the normal group, the serum SOD activity and GSH content in the aging model group ( P <0.0001) was significantly decreased, and MDA content was significantly increased ( P <0.001), brain IL-6 content ( P <0.0001), Aβ1-42 ( P <0.0001) and AchE content ( P <0.05) were significantly increased, and Ach content was significantly decreased ( P <0.001, and the aging model was successfully established. Compared with the aging model group, the serum SOD activity of the red Ganoderma lucidum walnut meal fermented peptide group ( P <0.001) and GSH content ( P <0.0001) were significantly increased, MDA content ( P <0.0001) and the content of brain IL-6 ( P <0.001), AchE content ( P <0.0001) and Aβ1-42 ( P <0.05) were significantly decreased, and the brain Ach content ( P <0.01) was significantly increased. The comprehensive experimental results show that the red Ganoderma lucidum walnut meal fermented peptide prepared under the optimal conditions of Example 1 has anti-aging activity.

[0044] Embodiment 6: The efficacy of walnut meal fermented peptide in regulating the intestinal flora of aging mice is verified by the following steps: The colon contents of the three groups of mice in Example 5 were taken to measure the intestinal flora of the mice in the aging model group, the normal group and the red Ganoderma lucidum walnut meal fermented peptide group. The species composition of the flora was analyzed at the genus level. The results are shown in Figure 6At the phylum level, red Ganoderma lucidum walnut meal fermented peptides can affect the structure of the colonic flora of D-galactose-induced aging mice and increase the relative abundance of Actinobacteria and Verrucomicrobia in the colon of mice. Administration of D-galactose can increase the relative abundance of Firmicutes in the colonic contents of mice. Lactobacillus The relative abundance of Actinobacteria decreased significantly Enterorhabdus The relative abundance of Ganoderma lucidum increased significantly, and administration of walnut meal fermented peptides could reverse this change to a certain extent.

[0045] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A walnut meal fermented peptide, characterized in that: Includes oligopeptides shown as SEQ ID NO.1-SEQ ID NO.

10.

2. The method for preparing a walnut meal fermented peptide according to claim 1, characterized in that: The steps include: 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: 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.

3. The method for preparing walnut meal fermented peptide according to claim 2, characterized in that: 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).

4. The method for preparing walnut meal fermented peptide according to claim 2, 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.

5. The method for preparing walnut meal fermented peptide according to claim 4, characterized in that: 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 tertiary water-to-liquid ratio is 1:5, the fermentation time is 6 days, and the fermentation temperature is 28°C.

6. Use of the walnut meal fermented peptide as claimed in claim 1 as a functional food additive.

7. Use of the walnut meal fermented peptide as claimed in claim 1 in anti-aging cosmetics.

8. Use of the walnut meal fermented peptide as claimed in claim 1 in preparing an anti-aging composition.

9. The use according to claim 8, characterized in that: The composition is medicine, food, health product or feed.

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