Glycine soja seed extract and preparation method and application thereof

Abstract

The application discloses turtle peptides delaying aging of Caenorhabditis elegans and a preparation method and application thereof, relates to the technical field of active peptides, and aims to find products capable of reducing the incidence of aging-related diseases and slowing down the aging process. The prior art does not research active ingredients from the aspect of peptide sequences at present, while the application researches the anti-aging activity of active peptides of turtles, and discloses that the sequences of the turtle peptides with the anti-aging function and the anti-aging activity can be DIDLSQPQIGVV, KQRGDLSRELEEISERL, DNIEDEPKLIGYF, GDTDGDGKIGVDEFQAL, NKEQGTFEDFVEGL, TEGKNPPLFDIPIAPVDAF and KRNHLRVVDSL respectively.

Description

Technical Field

[0001] This application relates to the field of bioactive peptide technology, specifically to a turtle peptide that delays the aging of Caenorhabditis elegans, its preparation method, and its application. Background Technology

[0002] Aging is a biological process characterized by the decline of physiological functions; it is universal and irreversible. Anti-aging drugs have been a focus of attention since ancient times. The aim of anti-aging activities is to increase lifespan and quality of life. Anti-aging activities primarily involve treating age-related diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), cardiovascular disease, cancer, and chronic obstructive pulmonary disease (COPD). These diseases are triggered by multiple factors involving numerous molecular pathways, including telomere shortening, the NF-κB pathway, the adiponectin receptor pathway, insulin and IGF signaling pathways, AMPK, mTOR, and mitochondrial dysfunction. Natural products are considered effective molecules for slowing the aging process by influencing metabolic pathways, thereby ensuring extended lifespan.

[0003] In 2019, the global population aged 65 and over was 703 million. The United Nations (UN) predicts that by 2050, one in six people will be over 65, and the number of people aged 80 and over will triple. Population aging is increasingly becoming a significant social issue in many countries, including China, Japan, the UK, and Germany, and has attracted international attention in many fields. Finding products that can reduce the incidence of age-related diseases and slow down the aging process is currently a hot research topic. Summary of the Invention

[0004] The main purpose of this application is to provide a turtle peptide that delays the aging of Caenorhabditis elegans, as well as its preparation method and application, with the aim of finding products that can reduce the incidence of aging-related diseases and slow down the aging process.

[0005] To achieve the above objectives, the technical solutions adopted in the embodiments of this application are as follows: In a first aspect, embodiments of this application provide a turtle peptide that delays aging of Caenorhabditis elegans, the amino acid sequence of which is shown in SEQ ID No. 1 to SEQ ID No. 7.

[0006] Secondly, embodiments of this application provide a method for preparing turtle peptides that delay aging of Caenorhabditis elegans as described above, comprising the following steps: The turtle meat was dried, pulverized, and degreased in sequence, then dissolved in water, and subsequently subjected to enzymatic hydrolysis and centrifugation to obtain the supernatant. The supernatant was freeze-dried to obtain turtle peptide powder.

[0007] As some optional embodiments of this application, the drying process refers to steaming the turtle meat in boiling water for 4 hours, removing the bones, and then drying it in a 70°C oven.

[0008] As some optional embodiments of this application, the pulverization process refers to pulverizing the dried turtle meat to a suitable particle size and passing it through a 20-mesh sieve.

[0009] As some optional embodiments of this application, the deoiling process refers to using a continuous phase change device to deoil the pulverized turtle meat to obtain turtle meat powder; the desorption temperature during the deoiling process is 70°C and the extraction temperature is 50°C.

[0010] As some optional embodiments of this application, when dissolved in water, the volume ratio of the turtle meat powder to water is 1:9, and the dissolution temperature is 55°C.

[0011] As some optional embodiments of this application, the enzymatic hydrolysis treatment refers to adding alkaline protease for enzymatic hydrolysis, with the hydrolysis reaction temperature at 55°C, the hydrolysis reaction time at 4.5 h, and the pH at 9. After alkaline protease hydrolysis is complete, adjust the temperature to 85℃ and keep it at that temperature for 25 minutes to inactivate the enzyme.

[0012] As some optional embodiments of this application, the amount of alkaline protease added is 0.25% of the mass of the turtle meat powder; The pH was adjusted using 1 mol / L NaOH.

[0013] As some optional embodiments of this application, the centrifugation treatment refers to centrifuging at 4000 rpm for 10 minutes after cooling to room temperature to obtain the turtle meat enzymatic hydrolysate supernatant.

[0014] Thirdly, embodiments of this application provide an application of turtle peptides, which delay the aging of Caenorhabditis elegans as described above, for the preparation of beverages, wherein the beverages are solid beverages, sports drinks, and concentrated beverages.

[0015] Currently, in the field of active ingredient research, most existing technologies focus on efficacy verification at the whole extract or cellular level, without delving into the intrinsic relationship between peptide sequences and activity. This is undoubtedly a significant research gap in the field. This application takes a different approach, focusing on turtle active peptides and conducting a systematic and in-depth study of their anti-aging activity. Through advanced separation techniques and precise analytical methods, the core sequences of turtle peptides with significant anti-aging functions were successfully deciphered, as shown in any one of SEQ ID No. 1 to SEQ ID No. 7. These unique amino acid sequences are like the "golden keys" to unlocking the anti-aging code, containing powerful bioactive potential.

[0016] Furthermore, the anti-aging peptides prepared in this application demonstrate extremely high application value. Their excellent stability and compatibility allow them to be adapted to diverse product forms. Attached Figure Description

[0017] Figure 1 This is the total ion chromatogram of the turtle peptide involved in the embodiments of this application; Figure 2 This is a secondary mass spectrum of the polypeptide involved in the embodiments of this application; Figure 3 This is a secondary mass spectrum of the polypeptide SEQ ID No. 2 involved in the embodiments of this application; Figure 4 This is a secondary mass spectrum of the polypeptide involved in the embodiments of this application; Figure 5 This is a secondary mass spectrum of the polypeptide SEQ ID No. 4 involved in the embodiments of this application; Figure 6 This is a secondary mass spectrum of the polypeptide SEQ ID No. 5 involved in the embodiments of this application; Figure 7 This is a secondary mass spectrum of the polypeptide SEQ ID No. 6 involved in the embodiments of this application; Figure 8 This is a secondary mass spectrum of the polypeptide involved in the embodiments of this application; Figure 9 The figures show the lifetime test results of sample plates with different concentrations and the control group involved in the embodiments of this application; Figure 10 The figures show the results of heat stress experiments on sample plates with different concentrations and control groups involved in the embodiments of this application; Figure 11 The figures show the experimental results of egg production in sample plates with different concentrations and control groups involved in the embodiments of this application; Figure 12 The figures show the results of sinusoidal motion experiments on sample plates of different concentrations and control groups involved in the embodiments of this application; Figure 13 The figures show the results of head-swing experiments with sample plates of different concentrations and control groups involved in the embodiments of this application. Detailed Implementation

[0018] It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0019] As described above, this application has studied the anti-aging activity of turtle bioactive peptides and disclosed that the anti-aging peptides contain sequences with anti-aging activity as shown in SEQ ID No. 1 to SEQ ID No. 7.

[0020] Specifically, this application provides a turtle peptide that delays the aging of Caenorhabditis elegans, the amino acid sequence of which is shown in SEQ ID No. 1 to SEQ ID No. 7.

[0021] Among them, SEQ ID No. 1 to SEQ ID No. 7 are shown below: SEQ ID No. 1: DIDLSQPQIGVV; SEQ ID No.2: KQRGDLSRELEEISERL; SEQ ID No.3: DNIEDEPKLIGYF; SEQ ID No.4: GDTDGDGKIGVDEFQAL; SEQ ID No.5:NKEQGTFEDFVEGL; SEQ ID No.6: TEGKNPPLFDIPIAPVDAF; SEQ ID No. 7: KRNHLRVVDSL.

[0022] The above-mentioned turtle peptides were prepared using the following steps: Step S10: After drying, crushing and degreasing the turtle meat in sequence, dissolve it in water and then perform enzymatic hydrolysis and centrifugation in sequence to obtain the supernatant.

[0023] Specifically, the drying process refers to steaming the turtle meat in boiling water for 4 hours, removing the bones, and then drying it in a 70°C oven. The pulverization process refers to pulverizing the dried turtle meat to a suitable particle size and passing it through a 20-mesh sieve.

[0024] The oil removal process refers to using a continuous phase change device to remove oil from the pulverized turtle meat to obtain turtle meat powder; the desorption temperature during the oil removal process is 70℃, and the extraction temperature is 50℃. When dissolved in water, the volume ratio of the turtle meat powder to water is 1:9, and the dissolution temperature is 55°C. The enzymatic hydrolysis treatment refers to the addition of alkaline protease for enzymatic hydrolysis at a temperature of 55°C for 4.5 h and a pH of 9. After the alkaline protease hydrolysis is completed, the temperature is adjusted to 85°C and kept at that temperature for 25 min to inactivate the enzyme. The amount of alkaline protease added is 0.25% of the mass of the turtle meat powder. The pH is adjusted using 1 mol / L NaOH. The centrifugation process refers to centrifuging at 4000 rpm for 10 minutes after cooling to room temperature to obtain the enzymatic hydrolysate of turtle meat.

[0025] Step S20: Freeze-dry the supernatant to obtain turtle peptide powder.

[0026] To verify the anti-aging properties of the aforementioned turtle peptides, this application provides the following examples and experimental cases: Example 1: (1) After the turtle is slaughtered, the blood is removed, the turtle meat and shell are separated, and then it is steamed and sterilized (steamed in boiling water for 4 hours), and then the bones and meat are separated. (2) Place the turtle meat in a 70℃ oven to dry it; (3) Grind the dried turtle meat into powder using a grinder; (4) Use a continuous phase change device to perform oil removal treatment (desorption temperature 70℃, extraction temperature 50℃) to obtain turtle meat powder; (5) Add water with a mass of 9 times that of turtle meat powder and stir to dissolve at 55°C; (6) Add alkaline protease for enzymatic hydrolysis. The amount of enzyme added is 0.25% of the mass of turtle peptide powder. The reaction temperature is 55℃, the reaction time is 4.5 h, and the pH is 9 (the pH is adjusted with 1 mol / L NaOH). After alkaline protease hydrolysis is completed, the temperature is adjusted to 85℃ and kept at that temperature for 25 minutes to inactivate the enzyme. (7) After cooling to room temperature, centrifuge at 4000 rpm for 10 min to obtain the supernatant of turtle meat enzymatic hydrolysis.

[0027] (8) Freeze-dry the supernatant to obtain turtle peptide powder.

[0028] Experimental Example 1: Structural analysis was performed on the turtle peptide powder prepared in this example, namely: The structures of turtle peptides were analyzed using Orbitrap-MS / MS combined with proteomics techniques. The structural results were compared with three turtle-related protein libraries in NCBI: Testudines, Mauremys reevesii, and Testudinidae. Analysis revealed that four of the highly matched peptides had been previously reported, while seven peptides were primarily derived from muscle contraction-related proteins Myosin and Calsequestrin (details are shown in Table 1). Their functions are closely related to muscle movement, energy metabolism, and environmental adaptation in turtles (such as Mauremys reevesii and Chelonia mydas).

[0029] Table 1. Results of peptides related to muscle contraction in turtle peptides.

[0030] The total ion chromatogram of turtle peptide is shown below. Figure 1As shown, the secondary mass spectra of the peptides corresponding to SEQ ID No. 1 to SEQ ID No. 7 are as follows: Figures 2-8 As shown.

[0031] Experimental Example 2: 1) Preparation of Nematode Growth Culture Medium First, accurately weigh 6.8 g of agar powder, 1 g of tryptone, 1.2 g of sodium chloride, and 0.08 g of streptomycin, and dissolve them in 400 mL of ultrapure water to prepare the basal culture medium. The prepared basal culture medium, along with a pre-prepared 1 mol / L CaCl2, MgSO4, and K2HPO4-KH2PO4 buffer solution, was autoclaved (121 ℃, 20 min). After sterilization, the culture medium was allowed to cool naturally to 85±5 ℃ in a clean environment. Then, aseptic techniques were performed in a biosafety cabinet: 0.4 mL of sterile CaCl2 solution, 0.4 mL of sterile MgSO4 solution, 10 mL of phosphate buffer (pH 6.0), and 0.4 mL of cholesterol ethanol solution (5 mg / mL) sterilized through a 0.22 μm filter were added sequentially. The molten culture medium was thoroughly mixed and dispensed into pre-sterilized 60 mm Petri dishes. After the culture medium solidified at room temperature, it was transferred to a 4 ℃ refrigerator for storage. Throughout the entire operation, aseptic techniques must be followed, and all added reagents must be sterilized beforehand.

[0032] 2) Nematode synchronization Young adult nematodes were washed into sterile EP tubes with M9 buffer solution. A suitable amount of lysis buffer was added to lyse the nematodes. The tubes were centrifuged at 3000 rpm for 1 min in a low-speed centrifuge. The supernatant was discarded. The nematodes were washed twice with M9 solution. After centrifugation and discarding the supernatant, the nematodes at the bottom of the EP tube were pipetted and dropped into the sterile area of ​​the NGM. After about 48 hours, the fertilized eggs in the lysed nematodes basically developed into L4 stage larvae, and the synchronization was completed for lifespan experiments.

[0033] 3) Lifespan determination The experiment was divided into a control group and an experimental group. Synchronized L4 nematodes were transferred to the culture medium for each group. The survival time of the nematodes was calculated from the time of transfer, with the day of transfer recorded as day 0 of the lifespan experiment. Each day, they were transferred to a new culture dish, and the number of dead nematodes, surviving nematodes, dead nematodes, and the number of nematodes removed from the experiment were recorded. Criteria for nematode death: no movement or swallowing action, and no reaction after gentle touch. Removal criteria: ① escaping to the plate wall or lid and drying out; ② eggs hatching into cyst-like nematodes within the body; ③ burrowing into the agar. Accidentally dead, escaped, and cyst-like nematodes were removed. Criteria for nematode death: no reaction when gently touched with a platinum wire; this indicates death.

[0034] Nematodes are a highly efficient and powerful animal model due to their short lifespan (approximately 3-4 weeks, rapid growth), high reproductive capacity, transparent bodies, clear background, and the availability of a large and comprehensive database. Nematode lifespan experiments are one of the core models in the field of aging research, not only assessing lifespan but also revealing the molecular mechanisms of aging. Results include... Figure 9 As shown, the median lifespan of *C. elegans* in the control group was 20.5 days, the average lifespan was 18.78 days, and the longest lifespan was 30 days. The median lifespan of the low-concentration turtle peptide treatment group (100 μg / mL), the medium-concentration turtle peptide treatment group (200 μg / mL), and the high-concentration turtle peptide treatment group (500 μg / mL) all increased by 7.32%, and the average lifespan increased by 12.62%, 11.34%, and 15.97%, respectively.

[0035] In conclusion, turtle peptides exhibit good anti-aging capabilities in *C. elegans* and can significantly extend the lifespan of the worm.

[0036] 4) Heat stress experiment After 72 hours of synchronization, the nematodes were transferred to plates uniformly coated with 0.03% H2O2 (50 nematodes per plate, 3 replicates per group) and recorded as 0 h. The number of dead nematodes on each plate was recorded every 1 h until all nematodes were dead.

[0037] Heat stress experiments are primarily used to verify an organism's stress response and tolerance limits under high-temperature environments. Their core assessment focuses on the organism's ability to maintain protein homeostasis, the activation efficiency of heat shock pathways, and overall cellular protection mechanisms. Results are as follows... Figure 10 As shown, the median lifespan of nematodes in the control group was 6 h, and the average lifespan was 5.36 h. The average lifespan of the low-concentration turtle peptide treatment group (100 μg / mL), the medium-concentration turtle peptide treatment group (200 μg / mL), and the high-concentration turtle peptide treatment group (500 μg / mL) increased by 10.45%, 29.85%, and 15.67%, respectively. This indicates that turtle peptide can improve the survival and repair capabilities of nematodes under heat stress.

[0038] 5) Measurement of egg production The study was divided into a control group and an experimental group. Ten nematodes in the L4 stage were selected from each group and placed in their respective culture media, with one nematode per plate. Every 24 hours, the nematodes were transferred to new plates. After 4-5 transfers, the nematodes had essentially stopped laying eggs. All plates were then incubated in an incubator. After the progeny nematodes hatched and grew, their numbers were counted before they entered the oviposition period (incubated at 20°C for 24 hours). The total number of eggs laid by each female nematode in 6-7 plates was the total number of eggs laid by that nematode.

[0039] Some studies have indicated that *C. elegans* possesses a "compensatory mechanism," potentially extending its lifespan by sacrificing its reproductive capacity. Figure 11 It can be seen that the high-concentration sample plate had the highest number of nematode eggs, while there was no significant difference in the number of eggs laid between the medium- and low-concentration sample plates and the control group. In conclusion, turtle peptide can improve the lifespan of *C. elegans* without reducing its reproductive capacity.

[0040] 6) Caenorhabditis elegans movement index experiment (sine motion and head swing frequency experiment) The movement indicators of *C. elegans* are mainly judged based on the sinusoidal body movement within 1 minute and the head oscillation frequency within 30 seconds. A sinusoidal movement is counted as one complete 180° sinusoidal curve cycle during the worm's bending movement, and a head oscillation is counted as one back-and-forth swing of the worm's body near the pharynx.

[0041] Sine wave motion is a wave-like movement produced by nematodes through alternating contractions of their body wall muscles, regulated by central pattern generators and motor neurons. Figure 12 It can be seen that the nematodes in the high-concentration sample plate had the most sinusoidal movements, and the number of movements decreased with decreasing concentration, but there was still a significant difference compared with the control group. In conclusion, turtle peptide can effectively improve the motility of nematodes.

[0042] Head swaying is a head-shaking behavior in nematodes, dependent on the coordination of sensory neurons, interneurons, and muscles. The nematode's avoidance behavior towards harmful substances can be quantified by changes in head swaying frequency. Figure 13 The results showed that the nematodes in the high-concentration sample plate exhibited the most head swings, with the number of head swings decreasing progressively with decreasing concentration. There was no significant difference between the low-concentration group and the control group. In conclusion, this indicates that the turtle peptide sample is harmless and can improve the motility of nematodes to a certain extent.

[0043] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A turtle peptide composition for delaying aging of *C. elegans*, characterized in that, The turtle peptide composition comprises peptides with amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No. 7; The preparation method of the turtle peptide composition includes the following steps: The turtle meat was dried, pulverized, and degreased in sequence, then dissolved in water, and subsequently subjected to enzymatic hydrolysis and centrifugation to obtain the supernatant. The supernatant was freeze-dried to obtain turtle peptide powder; The enzymatic hydrolysis treatment refers to adding alkaline protease for enzymatic hydrolysis, with the hydrolysis reaction temperature at 55℃, the hydrolysis reaction time at 4.5 h, and the pH at 9. After alkaline protease hydrolysis is complete, adjust the temperature to 85℃ and keep it at that temperature for 25 minutes to inactivate the enzyme.

2. The turtle peptide composition for delaying aging of *C. elegans* according to claim 1, characterized in that, The drying process refers to steaming the turtle meat in boiling water for 4 hours, removing the bones, and then drying it in a 70°C oven.

3. The turtle peptide composition for delaying aging of *C. elegans* according to claim 1, characterized in that, The pulverization process refers to pulverizing the dried turtle meat to a suitable particle size and passing it through a 20-mesh sieve.

4. The turtle peptide composition for delaying aging of *C. elegans* according to claim 1, characterized in that, The deoiling process refers to using a continuous phase change device to deoil the pulverized turtle meat to obtain turtle meat powder; the desorption temperature during the deoiling process is 70℃ and the extraction temperature is 50℃.

5. The turtle peptide composition for delaying aging of *C. elegans* according to claim 1, characterized in that, When dissolved in water, the volume ratio of the turtle meat powder to water is 1:9, and the solvent temperature is 55°C.

6. The turtle peptide composition for delaying aging of *C. elegans* according to claim 5, characterized in that, The amount of alkaline protease added is 0.25% of the mass of the turtle meat powder; The pH was adjusted using 1 mol / L NaOH.

7. The turtle peptide composition for delaying aging of *C. elegans* according to claim 6, characterized in that, The centrifugation process refers to centrifuging at 4000 rpm for 10 minutes after cooling to room temperature to obtain the enzymatic hydrolysate of turtle meat.

8. The application of the turtle peptide composition for delaying aging of *C. elegans* as described in claim 1, characterized in that, Used for preparing beverages, wherein the beverages are solid beverages, sports drinks and concentrated beverages.

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