An anti-oxidative stress preparation and its preparation method and application

Abstract

The present invention discloses an antioxidant stress preparation, a preparation method and an application thereof, and belongs to the field of food technology. The antioxidant stress food provided by the present invention is prepared by mixing and drying bovine milk exosomes, microalgae, probiotics and cationic polysaccharides that embed active antioxidants. The antioxidant stress food provided by the present invention has good biosafety. The food can not only delay aging caused by oxidative stress and alleviate neurodegenerative diseases, but also has the functions of lowering blood sugar and blood lipids. It can be used as a functional food or a functional food additive for the efficient prevention and treatment of neurodegenerative diseases and liver diseases related to oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the field of food technology and relates to an antioxidant stress preparation and a preparation method and application thereof. Background Art

[0002] Excessive free radicals or excessive consumption of oxidative substances can lead to oxidative stress, which can contribute to aging and cause neurodegenerative diseases, liver dysfunction, cardiovascular disease, inflammatory responses, and tumors. Currently, most treatments for oxidative stress-related diseases rely on single antioxidant ingredients. However, due to the poor water solubility, poor targeting, and low bioavailability of most antioxidants, these results in unsatisfactory antioxidant protection. In recent years, the combination of bidirectional communication between the intestine and major organs such as the brain, liver, and heart, mediated by intestinal probiotics, and anti-oxidative stress strategies has provided new approaches for the effective prevention and treatment of oxidative stress-related diseases. However, to date, no such functional foods have been developed. Summary of the Invention

[0003] The primary purpose of this invention is to delay aging and prevent and treat oxidative stress-related diseases, including neurodegenerative diseases and liver diseases such as hyperglycemia and hyperlipidemia, by synergistically combating oxidative stress through bidirectional communication between the intestines and major organs such as the brain, liver, and heart, mediated by intestinal probiotics. Based on this, the present invention proposes an antioxidant preparation, its preparation method, and its use.

[0004] In a first aspect, the present invention provides an anti-oxidative stress preparation, wherein the anti-oxidative stress food is composed of inner nanospheres and an outer three-dimensional framework;

[0005] The nanospheres are made of milk exosomes encapsulating schisandrin B to form active exosomes;

[0006] The three-dimensional framework is formed by the interaction and connection of lactic acid bacteria, spirulina and chitosan.

[0007] Furthermore, in the anti-oxidative stress preparation provided by the present invention, the particle size of active exosomes is 1449.64±102.35nm;

[0008] The three-dimensional framework is a three-dimensional structure with a pore size of 10 to 60 μm.

[0009] In a second aspect, the present invention provides a method for preparing an antioxidant stress preparation, comprising the following steps:

[0010] (1) Preparation of milk exosomes: fresh milk is centrifuged at low temperature to obtain skim milk; after adjusting the pH of the skim milk, it is centrifuged at low temperature to obtain a whey solution; the whey solution is filtered and centrifuged at low temperature to obtain a yellow colloidal precipitate; the yellow colloidal precipitate is washed with PBS solution and dispersed in PBS solution, and the obtained milk exosomes are stored for future use;

[0011] (2) Preparation of active exosomes: After dilution, the milk exosomes were mixed with an ethanol solution containing schisandrin B to remove the unencapsulated schisandrin B and obtain active exosomes;

[0012] (3) After the active exosomes, spirulina, and chitosan are completely mixed, lactic acid bacteria are added, mixed and reacted, and an antioxidant stress-resistant food is prepared.

[0013] Specifically, in step (1), the pH of skim milk is adjusted to 4.6 using 2M hydrochloric acid, and the storage temperature of milk exosomes is -20°C.

[0014] Specifically, the dilution of the milk exosomes in step (2) is as follows: 2 mL of the milk exosomes in step (1) is diluted to 10 mL; the volume of the ethanol solution containing schisandrin B is 10 mL, and the concentration of the ethanol solution containing schisandrin B is 5 mg·mL -1 ; The mixing reaction time is 24h.

[0015] Specifically, the volume of active exosomes in step (3) was 20 mL, and the concentration of active exosomes was 5 μg mL -1 The mass of spirulina is 45-90 mg; the mass of chitosan is 100 mg; the mass of lactic acid bacteria is 10-55 mg.

[0016] Preferably, the volume of active exosomes is 20 mL and the concentration of active exosomes is 5 μg·mL -1 The mass of Spirulina is 90 mg; the mass of chitosan is 100 mg; the mass of lactic acid bacteria is 10 mg.

[0017] Furthermore, in the preparation method of the antioxidant stress preparation provided by the present invention, the final concentration of lactic acid bacteria is 1×10 10 CFU·mL -1 .

[0018] In a third aspect, the antioxidant stress preparation of the present invention or the preparation method thereof is used in the preparation of food having the following effects, wherein the effects include: preventing or treating diseases caused by oxidative stress;

[0019] The disease comprises at least one of a neurodegenerative disease and a liver disease;

[0020] The neurodegenerative disease is Alzheimer's disease.

[0021] In a fourth aspect, the antioxidant stress preparation of the present invention or the preparation method thereof is used in the preparation of a food additive having the following effects, wherein the effects include: preventing or treating diseases caused by oxidative stress;

[0022] The disease comprises at least one of a neurodegenerative disease and a liver disease;

[0023] The neurodegenerative disease is Alzheimer's disease.

[0024] In a fifth aspect, the anti-oxidative stress preparation of the present invention or the preparation method thereof is used in the preparation of a medicament having the following effects, wherein the effects include: preventing or treating diseases caused by oxidative stress;

[0025] The disease comprises at least one of a neurodegenerative disease and a liver disease;

[0026] The neurodegenerative disease is Alzheimer's disease.

[0027] Furthermore, the present invention provides an antioxidant stress preparation that acts on Caenorhabditis elegans with Alzheimer's mutations to extend the lifespan of Caenorhabditis elegans, inhibit the production of lipofuscin in Caenorhabditis elegans, eliminate ROS in Caenorhabditis elegans, increase the activity of antioxidant enzymes in Caenorhabditis elegans, and alleviate neurotoxicity, indicating that the antioxidant stress preparation described in the present invention can be used to prevent or treat Alzheimer's disease.

[0028] Furthermore, the present invention provides an antioxidant stress preparation for use in C. elegans nematodes induced by a high-fat diet. The antioxidant stress preparation of the present invention has positive effects on extending lifespan, reducing fat accumulation, lowering non-esterified fatty acid levels, increasing antioxidant enzyme activity, and reducing malondialdehyde levels in C. elegans fed a high-fat diet, demonstrating that the antioxidant stress preparation of the present invention can be used to prevent or treat liver diseases.

[0029] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0030] This invention leverages the bidirectional communication between the intestines and major organs, such as the brain, liver, and heart, mediated by intestinal probiotics, and proposes a strategy for effectively preventing and treating oxidative stress-related neurodegenerative and liver diseases through synergistic anti-oxidative stress. Specifically, the invention combines antioxidant-loaded active bovine milk exosomes, microalgae, probiotics, and cationic polysaccharides to create an antioxidant-resistant preparation. The preparation extended the average lifespan of N2 C. elegans by 69.81%, the median lifespan by 66.67%, and the maximum lifespan by 45.45%. It also reduced the aging marker lipofuscin by 45.94%, significantly slowing the normal aging rhythm. Furthermore, the preparation significantly extended the average, median, and maximum lifespan of the Alzheimer's mutant N2 C. elegans (CL4176). While reducing lipofuscin levels, it also increased the activity of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). It also significantly reduced the content of malondialdehyde (MDA), a lipid peroxidation product, by 84.77%, indicating that the preparation can prevent and treat Alzheimer's disease by combating oxidative stress. Furthermore, under high glucose induction, the preparation significantly extended the lifespan of glucose-induced N2 nematodes while increasing antioxidant enzyme activity and reducing MDA levels. In addition, under the intervention of this preparation, not only the lifespan of the N2 type Caenorhabditis elegans high-glucose model was significantly prolonged, but the triglyceride (TG) and non-esterified fatty acid (NEFA) content in the glucose-induced N2 nematode body was also significantly reduced, indicating that this anti-oxidative stress preparation has significant blood sugar and lipid-lowering effects.

[0031] In summary, this preparation can be used as a food, food additive or drug to eliminate excess free radicals in the body, enhance the activity of antioxidant enzymes, and reduce the content of lipid peroxidation products through anti-oxidative stress, thereby achieving efficient prevention and treatment of neurodegenerative diseases and liver diseases related to oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the Zeta potential diagram of anti-oxidative stress functional foods.

[0033] Figure 2 Microscopic morphology of functional foods that protect against oxidative stress. (A) shows milk exosomes; (B) shows active exosomes; (C) shows spirulina; (D) shows lactic acid bacteria; (E) shows functional food 1; and (F) shows a mixture.

[0034] Figure 3 The following are the survival curves of C. elegans after different treatments. (A) is N2 type C. elegans; (B) is CL4176 type C. elegans.

[0035] Figure 4These are fluorescence images of lipofuscin in N2 Caenorhabditis elegans after 10 days of different treatments.

[0036] Among them, (A) is the lipofuscin content of the blank group; (B) is the lipofuscin content of the spirulina group; (C) is the lipofuscin content of the active exosome group; (D) is the lipofuscin content of the milk exosome group; (E) is the lipofuscin content of the schisandrin B group; (F) is the lipofuscin content of the functional food 1 group; (G) is the lipofuscin fluorescence intensity diagram in N2 type Caenorhabditis elegans.

[0037] Figure 5 These are fluorescence images of lipofuscin in CL4176 Caenorhabditis elegans after 10 days of different treatments. (A) shows the lipofuscin content in the blank group; (B) shows the lipofuscin content in the spirulina-treated group; (C) shows the lipofuscin content in the active exosome-treated group; (D) shows the lipofuscin content in the milk exosome-treated group; (E) shows the lipofuscin content in the schisandrin B-treated group; (F) shows the lipofuscin content in the functional food 1-treated group; and (G) shows the fluorescence intensity of lipofuscin in CL4176 Caenorhabditis elegans.

[0038] Figure 6 This is a fluorescence image of ROS in N2 type Caenorhabditis elegans after 10 days of different treatments.

[0039] Among them, (A) is the ROS fluorescence image of the blank group; (B) is the ROS fluorescence image of the Spirulina treatment; (C) is the ROS fluorescence image of the active exosome treatment; (D) is the ROS fluorescence image of the milk exosome treatment; (E) is the ROS fluorescence image of the Schisandrin B treatment; (F) is the ROS fluorescence image of the functional food 1 treatment; (G) is the ROS fluorescence intensity image in the N2 type Caenorhabditis elegans.

[0040] Figure 7 Fluorescence images of ROS in CL4176 Caenorhabditis elegans after 10 days of different treatments. (A) is the ROS fluorescence image of the blank group; (B) is the ROS fluorescence image of the Spirulina treatment; (C) is the ROS fluorescence image of the active exosome treatment; (D) is the ROS fluorescence image of the milk exosome treatment; (E) is the ROS fluorescence image of the Schisandrin B treatment; (F) is the ROS fluorescence image of the functional food 1 treatment; (G) is the ROS fluorescence intensity image of CL4176 Caenorhabditis elegans.

[0041] Figure 8Figure 1: Changes in antioxidant enzyme activities in C. elegans after different treatments. (A) SOD activity in N2 C. elegans; (B) CAT activity in N2 C. elegans; (C) GSH-Px activity in N2 C. elegans; (D) MDA content in N2 C. elegans; (E) SOD activity in CL4176 C. elegans; (F) CAT activity in CL4176 C. elegans; (G) GSH-Px activity in CL4176 C. elegans; and (H) MDA content in CL4176 C. elegans.

[0042] Figure 9 This is a survival curve of high-fat-induced N2-type Caenorhabditis elegans after different treatments.

[0043] Figure 10 Figure 3 is the triglyceride content in N2 nematodes induced by high fat in different treatments.

[0044] Figure 11 The non-esterified fatty acid content in N2 nematodes induced by high fat in different treatments.

[0045] Figure 12 Figure 4 shows the CAT enzyme activity in N2-type Caenorhabditis elegans induced by high-fat diet after different treatments.

[0046] Figure 13 The SOD enzyme activity in N2-type Caenorhabditis elegans induced by high-fat diet after different treatments.

[0047] Figure 14 The GSH-Px enzyme activity in N2-type Caenorhabditis elegans induced by high-fat diet after different treatments.

[0048] Figure 15 The MDA content in high-fat-induced N2-type Caenorhabditis elegans after different treatments. DETAILED DESCRIPTION

[0049] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0050] Example 1

[0051] This embodiment provides a method for preparing an antioxidant stress-relieving preparation.

[0052] 1. Preparation of active exosomes

[0053] Fresh milk stored at low temperature was divided into centrifuge tubes and centrifuged at low temperature to obtain skim milk. The pH of the skim milk was adjusted to about 4.6 with hydrochloric acid (2M). Centrifuged again at low temperature to obtain whey solution, which was filtered to remove residual protein. Centrifuged again at low temperature in an ultra-high-speed refrigerated centrifuge to obtain a yellow colloidal precipitate. After washing the precipitate three times with pre-cooled PBS solution, the exosomes were dispersed in PBS and stored in a -20°C refrigerator for later use. 2 mL of milk exosome solution was diluted to 10 mL and mixed with 10 mL and 5 mg mL -1 The ethanol solution of schisandrin B was mixed evenly and stirred at room temperature for 24 hours, and then dialyzed to remove unencapsulated schisandrin B, thereby obtaining milk exosomes encapsulating schisandrin B, referred to as active exosomes.

[0054] 2. Preparation of functional food 1 for anti-oxidative stress

[0055] 20 mL of active exosomes (5 μg mL -1 ), 90mg spirulina and 100mg chitosan were mixed thoroughly, and then 10mg lactic acid bacteria were added to make the lactic acid bacteria reach 1×10 10 CFU·mL -1 , mixed for 24 hours and then dried to obtain an antioxidant stress preparation, which was recorded as functional food 1.

[0056] 3. Preparation of the mixture

[0057] 20 mL of active exosomes (5 μg mL -1 ), 90mg spirulina and 100mg chitosan were mixed thoroughly, and then 10mg lactic acid bacteria were added to make the lactic acid bacteria reach 1×10 10 CFU·mL -1 , the samples that were directly dried without mixing for 24 h were regarded as physical mixtures (referred to as mixtures, as the control group).

[0058] 4. Preparation of functional food 1 for anti-oxidative stress

[0059] 20 mL of active exosomes (5 μg mL -1 ), 45 mg of spirulina and 100 mg of chitosan were mixed thoroughly, and then 55 mg of lactic acid bacteria was added to make the lactic acid bacteria reach 1×10 10 CFU·mL -1 , mixed for 24 hours and then dried to obtain an antioxidant stress preparation, which was recorded as functional food 2.

[0060] Example 2

[0061] This example provides the zeta potential of an anti-oxidative stress preparation.

[0062] The zeta potential of milk exosomes, active exosomes, chitosan, lactic acid bacteria and the functional food prepared in Example 1 was measured using a zeta potential meter.

[0063] like Figure 1 As shown, the Zeta potential of bovine milk exosomes, active exosomes, and lactic acid bacteria were 5.29±0.8mV, -26.93±0.39mV, and -10.72±0.53mV, respectively; the Zeta potential of chitosan was 20.25±1.33mV. After interacting with chitosan active exosomes and lactic acid bacteria to form functional foods 1 and 2, their Zeta potentials decreased to 5.42±0.18mV and 5.28±0.02mV, respectively, confirming that functional foods 1 and 2 were successfully prepared.

[0064] Example 3

[0065] This example provides a microscopic image of the anti-oxidative stress preparation.

[0066] A 10 μL dispersion of milk exosomes and active exosomes was dripped onto a copper grid, dried at room temperature, and then negatively stained with 2% phosphotungstic acid. After negative staining, the copper grid was gently rinsed 5–6 times in distilled water droplets and allowed to air dry at room temperature. The micromorphology of milk exosomes and active exosomes was observed using a transmission electron microscope at an accelerating voltage of 80 kV at room temperature. The micromorphology of spirulina, lactic acid bacteria, functional food 1, and the mixture was observed using a scanning electron microscope. The procedure was as follows: conductive adhesive was pre-applied to the sample stage. Appropriate amounts of dry spirulina powder, lactic acid bacteria, functional food 1, and the mixture were adhered to the conductive adhesive. Excess sample was removed using an ear bulb, and gold was sprayed onto the surface. The micromorphology of spirulina, lactic acid bacteria, functional food 1, and the mixture was observed using a scanning electron microscope.

[0067] like Figure 2 As shown in (A), milk exosomes are spherical structures with a particle size of 204.48±3.57nm. After embedding schisandrin B, a large number of nanoscale black dots appeared in the active exosomes (such as Figure 2 As shown in (B), this is formed after the hydrophobic Schisandrin B aggregates. Compared with the milk exosomes, the particle size of the active exosomes increased to 1449.64±102.35nm, which proves the successful loading of Schisandrin B. Figure 2 As shown in (C), Spirulina has a spiral structure with a diameter of 3 to 5 μm. Figure 2 (D) in the figure shows the microstructure of the lactic acid bacteria after freeze-drying. It can be clearly seen that the lactic acid bacteria presents a curled lamellar structure. This lays the structural foundation for the subsequent interaction with chitosan. Figure 2In (E), it can be found that functional food 1 presents a three-dimensional framework structure with a pore size of 10 to 60 μm composed of lamellar lactic acid bacteria, spirulina and chitosan, and nano-scale active exosomes (nanospheres) are embedded in the three-dimensional framework structure. When chitosan, schisandrin B, spirulina and active exosomes do not interact in the solution for 24 hours, they do not assemble to form a special structure. The above components are randomly distributed in the SEM field of view ( Figure 2 (F) in the.

[0068] Example 4

[0069] This example provides information on the lifespan extension of Caenorhabditis elegans after the treatment with an antioxidant stress preparation.

[0070] The C. elegans used in this example are divided into N2 and CL4176 strains, both fed with E. coli OP50. The C. elegans growth medium (NGM) was placed in a biochemical incubator at 20°C. After synchronization, the C. elegans were fully lysed. The C. elegans were centrifuged at 4000 rpm for 2 minutes and then washed twice with M9 buffer. The collected precipitate was the C. elegans eggs. Finally, the C. elegans eggs were transferred to NGM medium topped with OP50 and incubated at 20°C for 24 hours to obtain L1-stage C. elegans larvae, and approximately 48-72 hours to obtain L4-stage C. elegans adults.

[0071] L4 stage Caenorhabditis elegans were transferred to the culture medium containing functional food 1 (15 mg mL -1 )、Functional food 2(15mg·mL -1 ), Spirulina (4.8 mg mL -1 ), schisandrin B (2 μg·mL -1 ), active exosomes (0.1 mg mL -1 ), milk exosomes (30 μL), mixture (15 mg mL -1 ) in a 24-well plate, and 1 mL of PBS buffer, 10 μL of 5-fluorouracil (1.25 mg mL -1 ). This time was recorded as day 0 of the experiment, and the plates were rotated every two days. C. elegans were considered dead when they did not respond to the prod. The number of deaths was observed and recorded until all C. elegans tested had died. This was used to plot a C. elegans survival curve. The experiment was repeated three times, and the calculation formula was as follows:

[0072]

[0073] like Figure 3As shown in (A), the average lifespan of N2 type Caenorhabditis elegans exposed to schisandrin B, spirulina, milk exosomes, active exosomes, functional food 1, functional food 2 and the mixture was 10.13±0.89 days, 16.00±0.83 days, 13.27±0.68 days, 13.20±0.75 days, 17.40±0.48 days, 18.93±0.33 days and 15.20±0.64 days, respectively. In addition, compared with the blank group (untreated N2 C. elegans), the lifespan of N2 C. elegans was increased. The maximum lifespan of N2 C. elegans after treatment with schisandrin B, spirulina, milk exosomes, active exosomes, functional food 1, and functional food 2 was 24.00±1.37 days, 26.00±2.24 days, 24.00±1.67 days, 24.00±3.36 days, 32.00±2.84 days, and 32.00±2.11 days, respectively, which was 9.09%, 18.18%, 9.09%, 9.09%, 45.45%, and 45.45% longer than that of the blank group (Table 1). However, the mixture can only extend the lifespan of N2 type Caenorhabditis elegans to 24.00±2.24 days, but the extension is similar to that of bovine milk exosomes, indicating that when active exosomes, spirulina, chitosan, and lactic acid bacteria are simply mixed, their effect on extending the lifespan of N2 type Caenorhabditis elegans is not as good as functional food 1 and functional food 2 that form a three-dimensional framework structure through interaction.

[0074] Table 1 Lifespan and its change rate of N2 type Caenorhabditis elegans

[0075] sample Average lifespan (d) Median lifespan (d) Maximum lifespan (d) Rate of change (%) Significance analysis blank 10.60±0.64 12.00±0.65 22.00±1.98 - na Schisandrin B 10.13±0.89 14.00±1.29 24.00±1.37 9.09％↑ ns Spirulina 16.00±0.83 17.00±1.91 26.00±2.24 18.18％↑ 0.0009，*** milk exosomes 13.27±0.68 16.00±1.08 24.00±1.67 9.09％↑ 0.0077，** active exosomes 13.20±0.75 16.00±1.58 24.00±3.36 9.09％↑ 0.0103，* Functional Foods 1 17.40±0.48 20.00±0.84 32.00±2.84 45.45％↑ 0.0001，*** Functional Foods 2 18.93±0.33 20.00±1.25 32.00±2.11 45.45％↑ 0.0003，*** mixture 15.20±0.64 16.00±1.17 24.00±2.24 9.09％↑ 0.0043，**

[0076] like Figure 2As shown in (B), the average lifespan of CL4176 Caenorhabditis elegans exposed to schisandrin B, spirulina, milk exosomes, active exosomes, functional food 1, functional food 2 and the mixture was 12.07±2.87 days, 14.73±1.46 days, 10.93±2.62 days, 15.20±1.14 days, 17.40±1.27 days, 18.86±2.23 days and 15.00±1.22 days, respectively, and the average lifespan of Caenorhabditis elegans in the blank group was 9.73±0.33 days. The maximum lifespan of the above-mentioned groups of C. elegans were 22.00±1.29 days, 24.00±2.27 days, 22.00±1.64 days, 24.00±0.88 days, 30.00±3.79 days and 30.00±2.84 days, respectively, which were extended by 22.22%, 33.33%, 22.22%, 33.33%, 66.67% and 66.67% respectively compared with the blank group. The extension of the mixture was similar to that of bovine milk exosomes, which could only extend the lifespan of C. elegans to 22.00±3.21 days (Table 2), once again confirming the biosafety of functional foods and their very positive effect in extending the lifespan of C. elegans with Alzheimer's mutations. In particular, the lifespan of the CL4176 type Caenorhabditis elegans model can be restored to a level consistent with that of N2 after being treated with functional food 1 and functional food 2, confirming its great potential for extending the lifespan of Alzheimer's mutant Caenorhabditis elegans.

[0077] Table 2 Lifespan and its change rate of CL4176 Caenorhabditis elegans

[0078] sample Average lifespan (d) Median lifespan (d) Maximum lifespan (d) Rate of change (%) Significance analysis blank 9.73±0.33 12.00±1.77 18.00±1.45 - na Schisandrin B 12.07±2.87 14.00±0.87 22.00±1.29 22.22％↑ ns Spirulina 14.73±1.46 18.00±2.82 24.00±2.27 33.33％↑ 0.0044,** milk exosomes 10.93±2.62 13.00±3.41 22.00±1.64 22.22％↑ ns active exosomes 15.20±1.14 16.00±1.88 24.00±0.88 33.33％↑ 0.0013,** Functional Foods 1 17.40±1.27 20.00±2.79 30.00±3.79 66.67％↑ 0.0005,*** Functional Foods 2 18.86±2.23 20.00±1.74 30.00±2.84 66.67％↑ 0.0022,** mixture 15.00±1.22 16.00±2.12 22.00±3.21 22.22％↑ ns

[0079] Example 5

[0080] This example provides information on the lipofuscin content in Caenorhabditis elegans after treatment with an antioxidant stress preparation.

[0081] Synchronized C. elegans were cultured on NGM plates containing schisandrin B, spirulina, milk exosomes, and active exosome functional food 1 for 10 days. The cells were anesthetized with 10 mM tetramisole hydrochloride and then fixed. Twenty randomly selected C. elegans from each group were imaged under a fluorescence microscope at an excitation wavelength of 380 nm and an emission wavelength of 430 nm. The fluorescence intensity of lipofuscin in C. elegans was analyzed using ImageJ, which reflects the lipofuscin content in the worms.

[0082] As the body ages, the balance of cellular metabolism is disrupted, leading to a large accumulation of free radicals. These free radicals may react with unsaturated fatty acids to form lipofuscin, a pigmentation phenomenon associated with age. Simply put, the higher the lipofuscin fluorescence intensity, the more obvious the aging of the C. elegans nematode.

[0083] Figure 4 (A) is the lipofuscin fluorescence image of untreated N2 type Caenorhabditis elegans after 10 days (blank group). Figure 4 (B) is the lipofuscin fluorescence image of N2 type Caenorhabditis elegans treated with Spirulina after 10 days. Figure 4 (C) is the lipofuscin fluorescence image of N2 type Caenorhabditis elegans treated with active exosomes 10 days later. Figure 4 (D) is the lipofuscin fluorescence image of N2 type Caenorhabditis elegans treated with milk exosomes 10 days later. Figure 4 (E) is the lipofuscin fluorescence image of N2 type Caenorhabditis elegans treated with schisandrin B after 10 days. Figure 4 (F) in the middle is the lipofuscin fluorescence image of N2 type Caenorhabditis elegans treated with functional food 1 after 10 days. Figure 5 (A) is the lipofuscin fluorescence image of untreated CL4176 Caenorhabditis elegans after 10 days (blank group). Figure 5 (B) is the lipofuscin fluorescence image of CL4176 type Caenorhabditis elegans treated with Spirulina after 10 days. Figure 5 (C) is the lipofuscin fluorescence image of CL4176 Caenorhabditis elegans treated with active exosomes 10 days later. Figure 5 (D) is the lipofuscin fluorescence image of CL4176 Caenorhabditis elegans treated with milk exosomes 10 days later. Figure 5 (E) is the lipofuscin fluorescence image of CL4176 type Caenorhabditis elegans treated with schisandrin B after 10 days. Figure 5 (F) is the lipofuscin fluorescence image of CL4176 type Caenorhabditis elegans treated with functional food 1 after 10 days. Figure 4 and Figure 5 It was found that the overall lipofuscin level of N2 type Caenorhabditis elegans was lower than that of CL4176. Functional food 1 and its various components all have the effect of alleviating the aging of Caenorhabditis elegans.

[0084] Depend on Figure 5 (A-F) It can be seen that the lipofuscin content in C. elegans is in the following order from high to low: blank > milk exosomes > spirulina > active exosomes > schisandra chinensis B > functional food 1; Figure 4Figure (G) in Figure 1 and Figure (G) in Figure 5 show that the lipofuscin content in N2 type Caenorhabditis elegans and CL4176 Caenorhabditis elegans intervened by functional food 1 is the lowest, confirming that functional food can significantly inhibit the production of lipofuscin in Caenorhabditis elegans with Alzheimer's mutation, thereby delaying the aging rhythm of Caenorhabditis elegans.

[0085] Example 6

[0086] This example provides the endogenous ROS levels in Caenorhabditis elegans after the action of an antioxidant stress preparation on the nematode.

[0087] During the experiment, the C. elegans cells were first carefully cleaned to ensure a clean surface. Subsequently, the fluorescent labeling reagent DCFA-DH (50 μM) was evenly distributed into individual wells of a 24-well plate, with 1 mL of solution injected into each well. The treated C. elegans were transferred to individual wells of the 24-well plate, the wells sealed to prevent light interference, and dark-adapted in a 20°C incubator for 1 hour. To eliminate any residual fluorescent dye, the C. elegans were washed three times with M9 buffer after completing the above steps. The washed C. elegans were placed in a centrifuge tube, and 1 mL of 60 μM levamisole anesthetic was mixed with the C. elegans solution. The mixture containing the anesthetized C. elegans was then dripped onto a dedicated glass slide to form the desired sample. Finally, the samples were imaged using a laser confocal fluorescence microscope, and clear fluorescence images were captured at an excitation wavelength of 485 nm. Twenty C. elegans were randomly selected from each group and photographed, and relative fluorescence intensity was analyzed using ImageJ software.

[0088] During the natural aging process, the body continuously produces reactive oxygen species (ROS). These molecules have powerful oxidative effects and are considered a key factor in aging. In this example, endogenous ROS levels in C. elegans were monitored using the DCFH-DA fluorescent probe method. Fluorescence intensity is positively correlated with ROS content. Therefore, fluorescence intensity was used to assess ROS content in C. elegans, thereby evaluating the potential regulatory effects of antioxidants on its oxidative stress status. Figure 6 (A) is the ROS fluorescence image of untreated N2 type Caenorhabditis elegans after 10 days (blank group). Figure 6 (B) is the ROS fluorescence image of N2 type Caenorhabditis elegans treated with Spirulina after 10 days. Figure 6 (C) is the ROS fluorescence image of N2 type Caenorhabditis elegans treated with active exosomes after 10 days. Figure 6 (D) is the ROS fluorescence image of N2 type Caenorhabditis elegans treated with milk exosomes after 10 days. Figure 6(E) is the ROS fluorescence image of N2 type Caenorhabditis elegans treated with schisandrin B after 10 days. Figure 6 (F) is a ROS fluorescence image of N2 C. elegans treated with functional food 1 after 10 days. The ROS level in N2 C. elegans was quantified using ImageJ software. Functional food 1 and its components all had significant ROS scavenging effects, with functional food 1 having the most significant ROS scavenging effect (e.g. Figure 6 (G) in the , further confirming the great potential of functional foods in resisting oxidative stress.

[0089] Figure 7 (A) is the ROS fluorescence image of untreated CL4176 Caenorhabditis elegans after 10 days (blank group). Figure 7 (B) is the ROS fluorescence image of N2 type Caenorhabditis elegans treated with Spirulina after 10 days. Figure 7 (C) is the ROS fluorescence image of CL4176 type Caenorhabditis elegans treated with active exosomes after 10 days. Figure 7 (D) is the ROS fluorescence image of CL4176 type Caenorhabditis elegans treated with milk exosomes after 10 days. Figure 7 (E) is the ROS fluorescence image of CL4176 type Caenorhabditis elegans treated with schisandrin B after 10 days. Figure 7 (F) is the ROS fluorescence image of CL4176 C. elegans treated with functional food 1 for 10 days. The ROS content in CL4176 C. elegans is in the following order from high to low: blank group > Schisandrin B > exosomes > Spirulina > active exosomes > functional food 1. By comparison, the fluorescence intensity of CL4176 C. elegans in the functional food 1 group was the weakest ( Figure 7 (G) in the figure indicates that the ROS content in the CL4176 type Caenorhabditis elegans of this group is the lowest, which not only confirms the biosafety of functional food 1, but also further confirms its ability to scavenge ROS.

[0090] Example 7

[0091] This example provides information on changes in antioxidant enzyme activities in Caenorhabditis elegans after exposure to an antioxidant stress preparation.

[0092] Synchronized L1-stage Caenorhabditis elegans were exposed to the same conditions for the first time. The experiment ended 10 days later and the treated Caenorhabditis elegans were collected and washed three times in M9 buffer to ensure cleanliness. The treated Caenorhabditis elegans were then transferred to pre-sterilized centrifuge tubes. 2 mL of PBS buffer was added to each tube, along with two sterilized steel balls as grinding aids. Vortexing was performed three times for 30 seconds each, followed by a 20-second pause, to fully disrupt and evenly grind the Caenorhabditis elegans. This process was maintained at a low temperature to protect the protein structure. The tubes were centrifuged at 8,000 rpm for 10 minutes. After this process, the supernatant was transferred to a new centrifuge tube for subsequent total protein content determination. SOD, CAT, GSH-Px activities, and MDA content were calculated by measuring optical density. Each determination was repeated three times.

[0093] SOD can separate superoxide anions into H2O2 and O2, while CAT can decompose H2O2 into H2O and O2. GSH-Px, an important non-enzymatic antioxidant, exists within cells. GSH-Px catalyzes the production of reduced glutathione (GSH), which reduces intracellular H2O2. Simultaneously, GSH is oxidized to oxidized glutathione (GSSG), which, under GSH-Px catalysis, regenerates GSH. This reaction accumulates GSH, which scavenges oxygen ions and other free radicals in the body, delaying aging and effectively activating the nematode's antioxidant defense mechanisms.

[0094] Figure 8 (A) in the figure is the SOD activity in N2-type Caenorhabditis elegans. Figure 8 (B) in the figure shows the CAT activity in N2-type Caenorhabditis elegans. Figure 8 (C) in the figure is the GSH-Px activity in N2-type Caenorhabditis elegans. Figure 8 (E) in the figure is the SOD activity in CL4176 type Caenorhabditis elegans. Figure 8 (F) in the middle is the CAT activity in CL4176 type Caenorhabditis elegans. Figure 8The data (G) represents GSH-Px activity in CL4176 C. elegans, as well as the changes in antioxidant enzyme activity in N2 C. elegans in Table 3 and CL4176 C. elegans in Table 4. Functional Food 1 and its components increased antioxidant enzyme activity in both N2 and CL4176 C. elegans to varying degrees, with Functional Food 1 being the most significant. Specifically, MDA, one of the end products of cellular lipid peroxidation, is often used as an important indicator of lipid oxidation, reflecting the level of intracellular oxidative damage. Following treatment with Functional Food 1, MDA levels in N2 and CL4176 C. elegans were 1.84±0.46 nmol / mgprot and 4.52±0.70 nmol / mgprot, respectively.

[0095] Figure 8 (D) in the figure is the MDA content in N2 type Caenorhabditis elegans. Figure 8 As shown in (A-D) and Table 3, the SOD activity of N2 type Caenorhabditis elegans exposed to functional food 1 was significantly increased by 441.93%, the CAT activity was significantly increased by 261%, the GSH-Px activity was increased by 258.97%, and the MDA content was reduced by 88.25% compared with the blank group, indicating that functional food 1 can prolong the lifespan of normal Caenorhabditis elegans through anti-oxidation, confirming its prospect for development as functional food 1.

[0096] Figure 8 (H) in the figure is the MDA content in CL4176 C. elegans. Figure 8 As shown in (E~H) and Table 4, after exposure to functional food 1, the SOD enzyme activity of CL4176 Caenorhabditis elegans increased by 144.34%, the CAT enzyme activity increased by 72.30%, the GSH-Px activity increased by 612.39%, and the MDA content decreased by 84.63% compared with the blank group.

[0097] Table 3 Changes in antioxidant enzyme activities in N2 Caenorhabditis elegans

[0098]

[0099] Table 4 Changes in antioxidant enzyme activities in CL4176 Caenorhabditis elegans

[0100]

[0101] This functional food 1 increases antioxidant enzyme activity, reduces MDA content, resists oxidative stress, and delays body aging, thereby effectively activating the antioxidant defense mechanism in C. elegans, alleviating neurotoxicity, and thereby extending the lifespan of C. elegans. The above results also prove that the antioxidant stress food described in the present invention can be used for the prevention or treatment of Alzheimer's disease.

[0102] Example 8

[0103] This example provides information on the lifespan extension of high-fat Caenorhabditis elegans after different treatments.

[0104] Adult Caenorhabditis elegans at the L4 stage in Example 4 were cultured for 5 days on a high-fat (HF) culture medium containing Escherichia coli OP50 and 10 mM glucose to obtain high-fat Caenorhabditis elegans. The high-fat Caenorhabditis elegans were then transferred to a culture medium containing no antioxidants (blank group) and a culture medium containing functional food 1 (15 mg mL -1 ), Spirulina (4.8 mg mL -1 ), schisandrin B (2 μg·mL -1 ), active exosomes (0.1 mg mL -1 elegans were cultured in 24-well plates containing 2% (0.174%), 1% (0.176%), and milk exosomes (30 μL) for 48 h, and lifespan analysis was performed.

[0105] like Figure 9 As shown in the results, after high-fat diet induction, schisandrin B, spirulina, and active exosomes all extended the mean, median, and maximum lifespan of C. elegans. Functional Food 1 had the most significant effect on extending the lifespan of C. elegans N2 induced by a high-fat diet. Compared to the blank control group, the maximum lifespan of nematodes in the schisandrin B, spirulina, active exosomes, and functional food 1 groups was extended by 10%, 10%, 20%, 20%, and 60%, respectively (Table 5), indicating that Functional Food 1 has a beneficial effect on extending the lifespan of C. elegans fed a high-sugar diet.

[0106] Table 5 Lifespan and its change rate of N2 Caenorhabditis elegans after high-fat induction

[0107]

[0108]

[0109] Example 9

[0110] This example provides information on the activities of antioxidant enzymes in high-fat Caenorhabditis elegans after different treatments.

[0111] L4-stage adult C. elegans worms, treated differently as in Example 8, were washed three times with M9 buffer and then sonicated. Triglyceride (TG), non-esterified fatty acids (NEFA), CAT, SOD, GSH-Px, and MDA levels in each treatment group were measured using the corresponding kits.

[0112] Non-esterified fatty acids (NEFA) are products of neutral fat metabolism, and excess NEFA can produce free radicals. Free radicals can react with the lipid bilayer of the cell membrane and cause lipid peroxidation. This process damages the cell membrane, leading to cell dysfunction, which may in turn affect the health of liver cells. A large amount of evidence shows that excessive NEFA can lead to metabolic disorders such as obesity and hyperlipidemia. When fat intake exceeds the body's needs, excess NEFA accumulates in the liver and is converted into triglycerides (TG) for storage. If this state persists, it may lead to increased TG levels in the liver and the formation of fatty liver. Therefore, TG levels can be used as a marker of fat accumulation, and monitoring NEFA and TG levels is of certain significance for the prevention and treatment of liver disease.

[0113] Depend on Figure 10 It can be seen that the TG content of C. elegans treated with functional food 1 and fed a high-fat diet was reduced by 43.24% compared with that of C. elegans in the blank group, indicating that functional food 1 can improve the fat accumulation of TG in C. elegans. In addition, the NEFA levels of C. elegans under various treatment conditions were also evaluated. Figure 11 As shown, NEFA levels were significantly elevated in response to a high-fat diet. Feeding functional food 1 and its individual components effectively reduced NEFA levels, consistent with the TG results. Specifically, compared to C. elegans fed a high-fat diet (native), NEFA content in C. elegans decreased by 40% after functional food 1 intervention. Therefore, functional food 1 demonstrated a significant ability to mitigate fat accumulation and reduce NEFA levels in C. elegans fed a high-fat diet.

[0114] Figure 12 is the CAT level of high-fat Caenorhabditis elegans after different treatments, Figure 13 is the SOD level of high-fat Caenorhabditis elegans after different treatments, Figure 14The GSH-Px levels of high-fat Caenorhabditis elegans after different treatments. After high-fat culture, the activities of CAT, SOD and GSH-Px in Caenorhabditis elegans decreased. Through the intervention of functional food 1, the activities of CAT, SOD and GSH-Px antioxidant enzymes were all improved. After the intervention of functional food 1, the CAT, SOD and GSH-Px activities of high-fat diet Caenorhabditis elegans increased by 245.45%, 184.38% and 625%, respectively. These results indicate that the functional food 1 can increase the activity of antioxidant enzymes in Caenorhabditis elegans exposed to a high-fat diet. MDA is a key biomarker that can reflect the rate of organ lipid peroxidation and the intensity of peroxidative damage. As Figure 15 As shown in the results, after feeding functional food 1 to C. elegans on a high-fat diet, the MDA content of C. elegans decreased by 78.64%, verifying that functional food 1 can significantly reduce the MDA content of C. elegans under a high-fat environment.

[0115] The embodiments described above are some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. An anti-oxidative stress preparation, characterized in that It is composed of inner nanospheres and outer three-dimensional framework; The nanospheres are made of milk exosomes encapsulating schisandrin B to form active exosomes; The milk exosomes were diluted and mixed with an ethanol solution containing schisandrin B to remove unencapsulated schisandrin B to obtain the active exosomes; 2 mL of the milk exosomes were diluted to 10 mL; the volume of the ethanol solution containing schisandrin B was 10 mL, and the concentration of the ethanol solution containing schisandrin B was 5 mg·mL -1 ; The mixing reaction time is 24h; The three-dimensional framework is formed by the interaction and connection of lactic acid bacteria, spirulina and chitosan; After the active exosomes, spirulina, and chitosan are completely mixed, lactic acid bacteria are added, and the mixture is reacted for 24 hours and then dried to obtain the antioxidant stress preparation; the volume of the active exosomes is 20 mL, and the concentration of the active exosomes is 5 μg·mL -1 The mass of the spirulina is 45-90 mg; the mass of the chitosan is 100 mg; the mass of the lactic acid bacteria is 10-55 mg; The particle size of the active exosomes was 1449.64±102.35nm; The three-dimensional framework is a three-dimensional structure with a pore size of 10 to 60 μm.

2. The anti-oxidative stress preparation according to claim 1, characterized in that The preparation of bovine milk exosomes comprises: centrifuging fresh bovine milk at low temperature to obtain skim milk; adjusting the pH of the skim milk and then centrifuging at low temperature to obtain a whey solution; filtering the whey solution and centrifuging at low temperature to obtain a yellow colloidal precipitate; washing the yellow colloidal precipitate with a PBS solution and dispersing it in a PBS solution, and storing the obtained bovine milk exosomes for future use.

3. The anti-oxidative stress preparation according to claim 2, characterized in that The pH of the skim milk is adjusted to 4.6 with 2M hydrochloric acid; The storage temperature of the milk exosomes is -20°C.

4. The anti-oxidative stress preparation according to claim 1, characterized in that The mass of the spirulina is 90 mg; The mass of the chitosan is 100 mg; The mass of the lactic acid bacteria is 10 mg.

5. The anti-oxidative stress preparation according to claim 1, characterized in that The final concentration of lactic acid bacteria was 1×10 10 CFU·mL -1 .

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