A postbiotic and composition for improving the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin and its application in anti-aging

Abstract

The present invention belongs to the technical field of microbial preparations, and particularly relates to a postbiotic and composition that can improve the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin, and their application in anti-aging. The postbiotic prepared by fermenting Lactobacillus rhamnosus used in the present invention, through synergistic action with astaxanthin, not only improves the bioavailability of astaxanthin, but also enhances their synergistic effect in antioxidant and anti-inflammatory aspects. In addition, the synergistic effect of astaxanthin and the postbiotic not only targets the skin, but also has a protective effect on internal organs. In addition to skin anti-aging, it also includes anti-aging of the ovaries and uterus. The above technical solution provides a new solution for the comprehensive health management of women, and thus has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial preparations, and particularly relates to a postbiotic and composition for improving the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin, and its application in anti-aging. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Astaxanthin, also known as astaxanthin and lobster shell pigment, is a carotenoid and the highest-level product of carotenoid synthesis. It is dark pink and is the strongest antioxidant found in nature. Astaxanthin is widely present in the biological world, especially in relatively high concentrations in shrimp, crabs, fish, algal bodies, yeast and the feathers of birds, and is one of the main carotenoids in marine organisms. It has anti-aging, anti-inflammatory, immunomodulatory, anti-cancer, lipid-lowering and anti-diabetic effects. In addition, astaxanthin can cross the blood-brain barrier and improve nerve function.

[0004] With the increasing pursuit of health and beauty by people, anti-aging for women has become a hot topic. Due to its super antioxidant properties, astaxanthin has become a star ingredient in the anti-aging field in recent years. By scavenging free radicals, inhibiting oxidative stress and enhancing the activity of antioxidant enzymes in cells, it can effectively slow down skin aging, inhibit wrinkle formation and enhance skin barrier function. At the same time, astaxanthin also has anti-inflammatory effects, which helps to relieve skin inflammatory reactions caused by ultraviolet rays, so it is widely used in sunscreen and repair products.

[0005] However, despite the excellent anti-aging effects of astaxanthin, the bottleneck of bioavailability limits its application effect. There are two forms of astaxanthin, one is the free state, and the other is the esterified state (monoester type and diester type). Esterified astaxanthin has stronger stability than free astaxanthin. As a lipophilic molecule, its absorption rate in the gastrointestinal tract after oral administration is limited. Especially in the case of poor water solubility, the absorption rate of astaxanthin is slow, and it is easily metabolized and excreted from the body. This results in that even if a large amount of astaxanthin is ingested, the actual amount entering the blood and tissues is relatively small, weakening the actual role of astaxanthin in anti-aging and other aspects. Therefore, improving the utilization rate of astaxanthin still has great research prospects in making full use of its physiological functions.

[0006] In addition, most of the current anti-aging products on the market mainly target a certain aspect, and there are few reports on products with comprehensive anti-aging effects, especially in multiple aspects such as skin, ovaries and uterus. Summary of the Invention

[0007] Based on the deficiencies of the above-mentioned prior art, the present invention provides a postbiotic and composition for improving the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin, and its application in anti-aging. Through experiments, it has been proved that the postbiotic product prepared from Lactobacillus rhamnosus of the present invention can effectively improve the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin. By combining the two, a comprehensive anti-aging effect is shown, especially for the skin, ovaries and uterus of women. Based on the above research results, the present invention is completed.

[0008] To achieve the above technical objectives, the present invention relates to the following technical solutions:

[0009] In the first aspect of the present invention, there is provided the use of a postbiotic in the preparation of a product for improving the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin;

[0010] Among them, the postbiotic contains inactivated Lactobacillus rhamnosus ncxt-1;

[0011] The Lactobacillus rhamnosus ncxt-1 is preserved in the China Center for Type Culture Collection (address: Wuchang Luojia Mountain, Wuhan University, Hubei Province), and the preservation date is November 6, 2024, and its biological preservation number is CCTCC NO: M 20242441.

[0012] In the second aspect of the present invention, there is provided a postbiotic astaxanthin composition, which contains a postbiotic and astaxanthin;

[0013] The mass ratio of the postbiotic to astaxanthin is 5-50:1, further 10-30:1, such as 10:1, 20:1 or 30:1.

[0014] Among them, the postbiotic contains inactivated Lactobacillus rhamnosus ncxt-1.

[0015] In the third aspect of the present invention, there is provided a preparation method of the above postbiotic astaxanthin composition, and the preparation method includes the step of mixing the postbiotic and astaxanthin.

[0016] The astaxanthin can be obtained through commercial channels, and no specific limitation is imposed on its source. As is well known to those skilled in the art, astaxanthin is a keto - type carotenoid with the chemical name 3,3′ - dihydroxy - 4,4′ - diketo - β,β′ - carotene. Specifically, it is a red solid powder and widely exists in the biological world. It has a relatively high content in shrimp, crab, fish, algal bodies, yeast, and the feathers of birds, and is one of the main carotenoids in marine organisms. In addition to being obtained by biological extraction methods, astaxanthin can also be obtained by chemical synthesis methods, etc., without specific limitation here. For example, the astaxanthin can be astaxanthin derived from Haematococcus pluvialis, or it can be a product rich in astaxanthin, such as Antarctic krill oil.

[0017] In the fourth aspect of the present invention, there is provided the application of the above - mentioned postbiotic astaxanthin composition in the preparation of anti - aging products.

[0018] Through research, the present invention has found that postbiotics can effectively improve the bioavailability of astaxanthin. When the two are used in combination, they can produce synergistic antioxidant and anti - inflammatory effects, and thus can be used as anti - aging products, especially as anti - aging products for women. The present invention has confirmed through experiments that this product has good anti - aging effects on the skin, ovaries, and uterus.

[0019] Among them, the product is a medicine.

[0020] The beneficial technical effects of the above - mentioned one or more technical solutions:

[0021] The above - mentioned technical solution uses postbiotics prepared by Lactobacillus rhamnosus fermentation, which acts synergistically with astaxanthin. It not only improves the bioavailability of astaxanthin but also enhances their synergistic effects in antioxidant and anti - inflammatory aspects. In addition, the synergistic effect of astaxanthin and postbiotics not only targets the skin but also has a protective effect on internal organs. In addition to skin anti - aging, it also includes anti - aging of the ovaries and uterus. The above - mentioned technical solution provides a new solution for the comprehensive health management of women, and thus has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0023] Figure 1 It shows the change in the content of astaxanthin in the serum of rats in the effect verification of the present invention.

[0024] Figure 2 It shows the change in the content of astaxanthin in the intestinal villi of rats in the effect verification of the present invention.

[0025] Figure 3 It shows the change in the content of astaxanthin in the intestinal contents of rats in the effect verification of the present invention.

[0026] Figure 4 This is the inhibition rate curve of CI-TNF-α of the astaxanthin and postbiotics composition in the effect verification of the present invention.

[0027] Figure 5 This is the improvement rate curve of CI-SOD of the astaxanthin and postbiotics composition in the effect verification of the present invention.

[0028] Figure 6 This is the expression of related genes in the skin tissues of rats in each group in the effect verification of the present invention.

[0029] Figure 7 This is the synthesis of elastin and collagen in the skin tissues of rats in each group in the effect verification of the present invention.

[0030] Figure 8 This is the hormone levels of rats in each group in the effect verification of the present invention.

[0031] Figure 9 This is the HE section diagram of the ovaries of rats in each group in the effect verification of the present invention. Detailed Description of the Invention

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] In a typical specific embodiment of the present invention, there is provided the use of postbiotics in the preparation of a product for improving the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin;

[0035] Among them, the postbiotics comprise inactivated Lactobacillus rhamnosus ncxt-1.

[0036] The Lactobacillus rhamnosus ncxt-1 is deposited at the China Center for Type Culture Collection (address: Wuchang Luojiashan, Wuhan University, Hubei Province), and the deposit date is November 6, 2024, and its biological deposit number is CCTCC NO: M 20242441.

[0037] In another specific embodiment of the present invention, a postbiotic astaxanthin composition is provided, which comprises postbiotics and astaxanthin;

[0038] The mass ratio of the postbiotics to astaxanthin is 5 - 50:1, further 10 - 30:1, such as 10:1, 20:1 or 30:1.

[0039] Among them, the postbiotics comprise inactivated Lactobacillus rhamnosus ncxt - 1.

[0040] Furthermore, the postbiotics can be prepared by the following method:

[0041] S1. Strain activation: Streak the Lactobacillus rhamnosus ncxt - 1 onto an activation medium to obtain a pure strain;

[0042] S2. Preparation of the first - stage seed liquid: Pick a single colony of Lactobacillus rhamnosus obtained in step S1 and place it in a first culture medium at 28 - 30 °C for static culture for 12 - 14 hours to obtain the first - stage seed liquid;

[0043] S3. Preparation of the second - stage seed liquid: Inoculate the above - cultured first - stage seed liquid into a second culture medium, with an inoculation amount of 1 - 5% (preferably 0.5%, v / v), and perform static culture at 28 - 35 °C (preferably 30 °C) for 12 - 14 hours to obtain the second - stage seed liquid;

[0044] S4. Liquid fermentation of Lactobacillus rhamnosus: Inoculate the above - obtained second - stage seed liquid into a third culture medium, with an inoculation amount of 2 - 8% (preferably 5%, v / v), at 25 - 30 °C (preferably 28 °C), 30 - 50 r / min (preferably 40 r / min), and culture for 3 - 4 hours; then increase the stirring speed to 60 - 80 r / min (preferably 70 r / min), raise the fermentation temperature to 28 - 35 °C (preferably 30 °C), keep the tank pressure at 0.05 - 0.09 MPa (preferably 0.08 Mpa), adjust the pH to weakly acidic (pH 6.5), and ferment for 2 - 3 hours; then keep the stirring speed and temperature unchanged, raise the tank pressure to 0.1 - 0.12 MPa (preferably 0.1 Mpa), maintain the pH at weakly acidic (pH 6.5), and continue to ferment for 4 - 5 hours; then keep the stirring speed and tank pressure unchanged, raise the temperature to 40 - 50 °C (preferably 45 °C), add glycerol accounting for 0.1 - 0.2% (preferably 0.1%) of the volume of the fermentation broth to the fermentation broth, and then perform high - temperature sterilization.

[0045] Among them, in the step S1, the composition of the activation medium is as follows: yeast peptone 0.2%-1.0%, beef extract 0.3-1.0%, glucose 1.0%-2%, yeast extract powder 0.05-0.2%, sodium acetate 0.05-0.2%, ammonium citrate 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, agar powder 0.15-0.2%, adjust the pH to weakly acidic (pH 6.5).

[0046] In the step S2, the composition of the first culture medium is as follows: glucose 1.0%-2%, yeast peptone 0.15-0.2%, beef extract 0.3-1.0%, yeast extract powder 0.1-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, ammonium citrate 0.05-0.2%, adjust the pH to weakly acidic (pH 6.5).

[0047] In the step S3, the composition of the second culture medium is as follows: glucose 1.0%-2%, yeast peptone 0.5-2%, beef extract 0.1-0.5%, yeast extract powder 0.1-0.5%, ammonium citrate 0.05-0.2%, Tween-80 0.1-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, adjust the pH to weakly acidic (pH 6.5).

[0048] In the step S4, the composition of the third culture medium is as follows: fructooligosaccharide 1.0%-2%, yeast peptone 0.5-2%, beef extract 0.1-0.5%, yeast extract powder 0.1-0.5%, ammonium citrate 0.05-0.2%, Tween-80 0.1-0.2%, sodium acetate 0.1-0.2%, potassium dihydrogen phosphate 0.1-0.2%, adjust the pH to weakly acidic (pH 6.5).

[0049] Further, in the step S4, the high-temperature sterilization can specifically be carried out by raising the temperature to 85°C and maintaining it for 30 minutes for sterilization; furthermore, after spray-drying the culture medium after high-temperature sterilization, a postbiotic solid preparation is obtained.

[0050] In another specific embodiment of the present invention, a preparation method of the above-mentioned postbiotic astaxanthin composition is provided, and the preparation method includes the step of mixing the postbiotic with astaxanthin.

[0051] Among them, the postbiotic is obtained by the above-mentioned preparation method based on Lactobacillus rhamnosus.

[0052] The astaxanthin can be obtained commercially, and its source is not specifically limited. It is well-known to those skilled in the art that astaxanthin is a keto-carotenoid with the chemical name 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene. Specifically, it is a red solid powder and widely exists in the biological world, especially in aquatic animals such as shrimps, crabs, fish, and the feathers of birds. In addition to being obtained by biological extraction methods, astaxanthin can also be synthesized by chemical synthesis methods. For example, the astaxanthin can be astaxanthin derived from Haematococcus pluvialis, or it can be a product rich in astaxanthin, such as Antarctic krill oil.

[0053] In another specific embodiment of the present invention, there is provided the use of the above-mentioned postbiotic astaxanthin composition in the preparation of anti-aging products.

[0054] Through research, the present invention has found that postbiotics can effectively improve the bioavailability of astaxanthin. When the two are used in combination, they can produce synergistic antioxidant and anti-inflammatory effects, and thus can be used as anti-aging products, especially as anti-aging products for women. The present invention has confirmed through experiments that this product has good anti-aging effects on the skin, ovaries, and uterus.

[0055] Among them, the product is a medicine.

[0056] The medicine of the present invention may also contain common carriers, excipients, diluents, etc. Moreover, according to the usual methods, it can be made into dosage forms such as powders, granules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injection solutions.

[0057] The following further explains the present invention through examples, but does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In each example,

[0058] The composition of the activation medium is as follows: per 1000 mL, it contains 10 g of yeast peptone, 5 g of beef extract, 10 g of glucose, 2 g of yeast extract powder, 1 g of sodium acetate, 1 g of ammonium citrate, 1 g of potassium dihydrogen phosphate, 20 g of agar powder, and the pH is adjusted to 6.5.

[0059] The composition of the first culture medium is as follows: per 1000 mL, it contains 10 g of glucose, 2 g of yeast peptone, 5 g of beef extract, 2 g of yeast extract powder, 2 g of sodium acetate, 2 g of potassium dihydrogen phosphate, 1 g of ammonium citrate, and the pH is adjusted to 6.5;

[0060] The composition of the second culture medium is as follows: per 1000 mL, it contains 10 g of glucose, 20 g of yeast peptone, 5 g of beef extract, 5 g of yeast extract powder, 1 g of ammonium citrate, 2 g of Tween-80, 2 g of sodium acetate, 2 g of potassium dihydrogen phosphate, and the pH is 6.5.

[0061] The composition of the third culture medium is as follows: per 1000 mL, it contains 10 g of fructooligosaccharide, 20 g of yeast peptone, 5 g of beef extract, 5 g of yeast extract powder, 1 g of ammonium citrate, 2 g of Tween-80, 2 g of sodium acetate, 2 g of potassium dihydrogen phosphate, and the pH is 6.5.

[0062] Example 1

[0063] A method for preparing postbiotics, comprising the following steps:

[0064] Streak lactobacillus rhamnosus ncxt-1 onto an activation medium to obtain a pure strain; pick a single colony of the obtained lactobacillus rhamnosus and place it in the first culture medium at 30 °C and statically culture for 14 hours to obtain a primary seed solution;

[0065] Inoculate the primary seed solution into the second culture medium at an inoculation amount of 1% and statically culture at 30 °C for 14 hours to obtain a secondary seed solution;

[0066] Inoculate lactobacillus rhamnosus into a fermenter containing sterilized third culture medium at an inoculation amount of 5%, at 28 °C, 40 r / min, and culture for 4 hours; after 4 hours, increase the stirring speed to 80 r / min, raise the fermentation temperature to 30 °C, keep the tank pressure at 0.08 MPa, adjust the pH to 6.5 using ammonia water, and ferment for 3 hours; after 7 hours, keep the stirring speed and temperature unchanged, raise the tank pressure to 0.1 MPa, adjust the pH to 6.5 using ammonia water, and ferment for 4 hours; after 11 hours, keep the stirring speed unchanged, raise the temperature to 45 °C, add glycerol at 0.1% of the volume of the fermentation broth to the fermenter, raise the temperature to 85 °C and keep it for 30 min, and then spray dry.

[0067] Example 2

[0068] A method for preparing a postbiotic astaxanthin composition, comprising the following steps:

[0069] Streak lactobacillus rhamnosus ncxt-1 onto an activation medium to obtain a pure strain; pick a single colony of the obtained lactobacillus rhamnosus and place it in the first culture medium at 30 °C and statically culture for 14 hours to obtain a primary seed solution;

[0070] Inoculate the primary seed solution into the second culture medium at an inoculation amount of 1% and statically culture at 30 °C for 14 hours to obtain a secondary seed solution;

[0071] Inoculate Lactobacillus rhamnosus into a fermenter containing sterilized third culture medium at an inoculation amount of 5%, culture at 28°C and 40 r / min for 4 hours; after 4 hours, increase the stirring speed to 80 r / min, raise the fermentation temperature to 30°C, keep the tank pressure at 0.08 MPa, adjust the pH to 6.5 with ammonia water, and ferment for 3 hours; after 7 hours, keep the stirring speed and temperature unchanged, raise the tank pressure to 0.1 MPa, adjust the pH to 6.5 with ammonia water, and ferment for 4 hours; after 11 hours, keep the stirring speed unchanged, raise the temperature to 45°C, add glycerol accounting for 0.1% of the fermentation broth volume to the fermenter, raise the temperature to 85°C and keep it for 30 min, and then spray-dry to obtain postbiotics.

[0072] Take 2000 g of postbiotics and mix it with 100 g of astaxanthin powder. In a three-dimensional mixer, mix at 100 r / min for 60 minutes to obtain a postbiotic-astaxanthin composition.

[0073] Effect verification

[0074] A composition for improving the bioavailability of astaxanthin, and its verification method is as follows:

[0075] Select 60 healthy Wistar male rats with uniform body weight. After 1 week of adaptive feeding, randomly divide them into an astaxanthin group and a postbiotic + astaxanthin group, with 30 rats in each group. Perform gavage treatment and feed 20.0 mg / kg of the postbiotics prepared in Example 1 and 1.0 mg / kg of astaxanthin. After gavage, at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 10 h, 12 h, 18 h, and 24 h, anesthetize and sacrifice the mice, take blood and intestines, separate the serum, strip and take out the small intestinal villi and intestinal contents, and collect the intestinal contents by rinsing with normal saline. After extraction, use high-performance liquid chromatography to detect the content of astaxanthin in the serum, small intestinal villi, and intestinal contents at different time periods. Compare the content of astaxanthin in the same time period of different groups.

[0076] The content of astaxanthin in the serum of different groups is as Figure 1 shown. The content of astaxanthin in the serum of the two groups of rats began to rise slowly after 0.5 h after gavage, reached the maximum value at 8 h, then began to decline, and returned to the initial level at 16 - 24 h. At 1 h, 2 h, 4 h, 8 h, 10 h, and 12 h, the content of astaxanthin in the serum of the rats in the postbiotic + astaxanthin group was greater than that in the group fed only astaxanthin. After 8 h of feeding, the content of astaxanthin in the postbiotic-astaxanthin group was 24.7 ± 0.5 μg / L, which was 12.35 times that in the group fed only astaxanthin (2.0 ± 0.3 μg / L). The content of astaxanthin in the small intestinal villi of different groups is as Figure 2As shown, the astaxanthin content in the small intestinal villi gradually increased, reached its peak at 4 h, and then gradually decreased, with no detection at 16 - 24 h. At time points such as 1 h, 2 h, 4 h, 8 h, 10 h, and 12 h, the serum astaxanthin content of rats in the postbiotics + astaxanthin group was greater than that of the group fed only astaxanthin. After 4 h of feeding, the astaxanthin content in the postbiotics - astaxanthin group was 810.0 ± 20.5 ng, which was 7.04 times that of the group fed only astaxanthin (115.0 ± 20.3 μg / L). The astaxanthin content in the intestinal contents of different groups was as Figure 3 shown. The astaxanthin content in the intestinal contents of the two groups of rats began to rise slowly after 0.5 h of gavage, reached its maximum at 4 h, then began to decline, and returned to the initial level at 16 - 24 h. At 1 h, 2 h, 4 h, 8 h, 10 h, and 12 h, the serum astaxanthin content of rats in the postbiotics + astaxanthin group was greater than that of the group fed only astaxanthin.

[0077] Furthermore, by calculating the area under the curve (AUC0 - t), the bioavailability of astaxanthin in the serum of different experimental groups was measured.

[0078] AUC0 - t = 0.25A + 0.5B + 0.75C + 1.5D + 3E + 3F + 2G + 3H + 6I + 4J (A, B, C, D, E, F, G, H, I, J represent the serum astaxanthin concentration levels at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 10 h, 12 h, 18 h, and 24 h respectively). The AUC0 - t value of rats in the astaxanthin group was (25.1 ± 1.3) μg·h / L; the AUC0 - t value of rats in the postbiotics + astaxanthin group was (158.5 ± 12.3) μg·h / L, which was 6.31 times that of the astaxanthin group.

[0079] For the composition that synergistically enhances anti - inflammatory and antioxidant abilities, the verification method is as follows:

[0080] Disperse RAW264.7 cells in RPMI1640 culture medium (containing 10% fetal bovine serum, 100 μmol / L penicillin, and streptomycin), and perform routine culture in a cell incubator. Passage the cells when they cover 70% - 80% of the culture dish. When the cell growth state is good, inoculate RAW264.7 cells into a 96 - well plate at a density of 1×10 4 cells per well, add 100 μL of culture medium to each well, and incubate at 37 °C for 24 h. According to the drug - adding requirements of each group of experiments, change the culture medium. After continuing to culture for 24 h, add 10 μL of CCK - 8 reagent to each well, incubate at 37 °C in the dark for 2 h, detect the OD value at 450 nm, and calculate the cell viability. Cell viability % = ((experimental group OD - blank OD) / (normal group OD - blank OD))×100%.

[0081] (1) Effects of lipopolysaccharide (LPS) at different concentrations on cell viability

[0082] The experiment was divided into a blank group, a normal group, and experimental groups with different concentrations of LPS. Blank group: no cells; Normal group: cells present, no LPS; Experimental groups with different concentrations of LPS were set up with 6 concentration gradient groups with final LPS concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, and 25 μg / mL, and each treatment was repeated in 5 wells. The CCK-8 method was used to detect the effect of LPS on the viability of RAW264.7 cells, and the experimental concentration of LPS was finally determined to be 1.0 μg / mL.

[0083] (2) Effects of astaxanthin at different concentrations on cell viability

[0084] The experiment was divided into a blank group, a normal group, and experimental groups with different concentrations of astaxanthin. Blank group: no cells; Normal group: cells present, no astaxanthin; Experimental groups with different concentrations of astaxanthin were set up with 6 concentration gradient groups with final astaxanthin concentrations of 0.625, 1.25, 2.5, 5, 8, and 10 μg / mL, and each treatment was repeated in 5 wells. The CCK-8 method was used to detect the effect of astaxanthin on the viability of RAW264.7 cells. The experimental results showed that when the astaxanthin concentration was ≤1.25 μg / mL, there was no significant difference in the viability of RAW264.7 cells compared with the normal group. When the astaxanthin concentration was 1.25 - 10 μg / mL, the viability of RAW264.7 cells showed a trend of first increasing and then decreasing; when the astaxanthin concentration reached 10 μg / mL, there was no obvious change in the viability of RAW264.7 cells compared with the control. Based on cell viability and drug cost considerations, the final mass concentration of astaxanthin was selected to be ≤10 μg / mL.

[0085] (3) Effects of postbiotics at different concentrations on cell viability

[0086] The experiment was divided into a blank group, a normal group, and experimental groups with different concentrations of postbiotics. Blank group: no cells; Normal group: cells present, no postbiotics; Experimental groups with different concentrations of postbiotics were set up with 6 concentration gradient groups with final postbiotic concentrations of 12.5, 25, 50, 100, 200, and 300 μg / mL, and each treatment was repeated in 5 wells. The CCK-8 method was used to detect the effect of postbiotics on the viability of RAW264.7 cells. The experimental results showed that when the postbiotic concentration was 12.5 - 300 μg / mL, as the postbiotic concentration increased, the viability of RAW264.7 cells showed a trend of first increasing and then decreasing. When the final mass concentration of postbiotics reached 300 μg / mL, there was no obvious change in the viability of RAW264.7 cells compared with the control. Therefore, when using postbiotics to intervene in the inflammatory response of RAW264.7 cells, the final mass concentration should be lower than the maximum non-toxic dose of 300 μg / mL.

[0087] (4) Detection of the inhibitory effect of the combination of three concentrations of astaxanthin and postbiotics on the inflammatory factor TNF-α by RT-qPCR and analysis of antioxidant capacity

[0088] RAW264.7 cells in good growth state were taken and seeded in 6-well plates at a density of 1×10 6 cells per well, and 2 mL of medium was added to each well and incubated at 37 °C for 24 h. The experiment was divided into a normal group, a model group, and an astaxanthin + postbiotic experimental group. Normal group: There are cells, no LPS, no astaxanthin + postbiotics; Astaxanthin + postbiotic experimental group, set the combination of astaxanthin + postbiotics as three final mass concentration combinations of 10 + 300, 10 + 200, 10 + 100 μg / mL (select appropriate final drug mass concentrations for compatibility according to the above-mentioned cell activity results). According to the reagents added in each group experiment, the medium was changed. After the drug acted for 2 h, LPS with a final concentration of 1 μg / mL was added, and the cells were collected after continuing to culture for 24 h; The RNA of cells in each group was extracted using a nucleic acid extraction kit, and the relative expression levels of TNF-α in each group were detected by RT-PCR, and the SOD level was detected by ELISA kit. Compared with the control group, the expression level of the inflammatory factor TNF-α in the model group increased significantly; Compared with the model group, the three (10 + 300, 10 + 200, 10 + 100 μg / mL, that is, the mass ratios are 1:30, 1:20, and 1:10) astaxanthin + postbiotic compositions could all down-regulate the expression of the inflammatory factor TNF-α after LPS stimulation; When the combination of astaxanthin + postbiotics was 10 + 200 μg / mL, the expression level of the TNF-α inflammatory factor in RAW264.7 cells was the lowest. When the mass ratio of astaxanthin + postbiotics was 1:20, the inhibitory effect on the TNF-α inflammatory factor in RAW264.7 cells was the best. Compared with the control group, the SOD activity in the model group decreased, and compared with the model group, the three (10 + 300, 10 + 200, 10 + 100 μg / mL, that is, the mass ratios are 1:30, 1:20, and 1:10) astaxanthin + postbiotic compositions could all stimulate the SOD activity; When the combination of astaxanthin + postbiotics was 10 + 200 μg / mL, the antioxidant capacity shown by RAW264.7 cells was the most obvious.

[0089] (5) Analysis of the inhibitory effect of astaxanthin, postbiotics and their combination on the expression of the inflammatory factor TNF-α and antioxidant capacity

[0090] RAW264.7 cells in good growth state were taken and seeded at 1×10 6The density of cells was used to seed RAW264.7 cells in a 6-well plate, with 2 mL of medium added to each well, and incubated at 37 °C for 24 h. The experiments were divided into a normal group, a model group, an astaxanthin group, a postbiotic group, and an astaxanthin + postbiotic group. Normal group: There were cells, no LPS, no astaxanthin, and no postbiotic; Model group: There were cells stimulated with LPS, no astaxanthin and no postbiotic; Astaxanthin group: The final mass concentration gradients were set as 10, 8, 6, 5, 1 μg / mL; The final mass concentration gradients of postbiotics were set as 200, 160, 120, 100, 20 μg / mL; Astaxanthin + postbiotic final mass concentration gradients were set as 10 + 200, 8 + 160, 6 + 120, 5 + 100, 1 + 20 μg / mL. According to the drug addition requirements of each group of experiments, the medium was replaced. After the drugs acted for 2 h, LPS with a final concentration of 1 μg / mL was added for stimulation, and the cells were harvested after continued culture for 24 h; Kits were used to extract the RNA of cells in each group, and RT-PCR was used to detect and calculate the mRNA expression levels and inhibition rates of TNF-α in each group. Inhibition rate = (1 - relative mRNA expression level of the drug addition group / relative mRNA expression level of the model group) × 100%. ELISA kits were used to detect the SOD level and the SOD increase rate. Increase rate = (relative mRNA expression level of the drug addition group - relative mRNA expression level of the model group) / relative mRNA expression level of the model group × 100%.

[0091] According to the results, the simulation equation for the inhibition rate of astaxanthin on TNF-α was: y = 2.1087x + 3.3478

[0092] (R 2 = 0.9929); The simulation equation for the inhibition rate of postbiotics on TNF-α was: y = 0.2033x + 3.4087

[0093] (R 2 = 0.9966); The simulation equation for the inhibition rate of the astaxanthin + postbiotic composition on TNF-α was:

[0094] y = 0.3157x + 2.2174 (R 2 = 0.9962). According to the Chou-Talalay equation CI = D1 / D 1x + D2 / D 2x , the combination index CI of astaxanthin and postbiotics was calculated. D1 and D2 were the respective concentrations of the astaxanthin + postbiotic composition when inhibiting the expression of the inflammatory factor TNF-α, D 1x 、D 2xThe concentrations of astaxanthin and postbiotics when used alone to achieve the above effects. Evaluate the synergistic inhibitory effect of the two (mass ratio 1:20) on the expression of inflammatory factor TNF-α. When CI < 1, the combined effect of astaxanthin and postbiotics is a synergistic effect; when CI = 1, the combined effect of astaxanthin and postbiotics is an additive effect; when CI > 1, the combined effect of astaxanthin and postbiotics is an antagonistic effect.

[0095] It can be seen from the experimental results that the inhibition rates of astaxanthin and postbiotics on the expression of inflammatory factor TNF-α both show a concentration-dependent increase. Taking an inhibition rate of 40 as an example, the concentration of astaxanthin used alone is 17.38 ± 2.31 μg / mL, and the concentration of postbiotics used alone is 179.98 ± 8.12 μg / mL. The dosages of the composition are: astaxanthin 5.69 ± 0.61 μg / mL and postbiotics 113.97 ± 5.22 μg / mL. Compared with the use of astaxanthin and postbiotics alone, when achieving a specific drug effect, the concentrations of the single drugs in the composition are significantly reduced. Evaluate the synergistic compatibility effect of astaxanthin and postbiotics under the compatibility of mass ratio 1:20 according to the median effect principle and the Chou-Talalay combined index method. Draw a curve graph of the inhibition rate and CI according to the calculation results, as Figure 4 shown, astaxanthin and postbiotics can play an obvious role in synergistically inhibiting the expression of inflammatory factor TNF-α under the compatibility of mass ratio 1:20.

[0096] According to the results, the simulation equation of the increase rate of SOD by astaxanthin is: y = 0.0357x - 0.0059 (R 2 = 0.993); the simulation equation of the increase rate of SOD by postbiotics is: y = 0.002x + 0.16 (R 2 = 1); the simulation equation of the increase rate of SOD by the astaxanthin + postbiotics composition is: y = 0.0046x + 0.0797 (R 2 = 0.9909). According to the Chou-Talalay equation CI = D1 / D 1x + D2 / D 2x , calculate the combined index CI of astaxanthin and postbiotics. D1 and D2 are the respective concentrations of astaxanthin and postbiotics in the astaxanthin + postbiotics composition when the SOD increases to a certain extent. D 1x and D 2x are the concentrations of astaxanthin and postbiotics when used alone to achieve the above effects. Evaluate the synergistic inhibitory effect of the two (mass ratio 1:20) on the increase of SOD. When CI < 1, the combined effect of astaxanthin and postbiotics is a synergistic effect; when CI = 1, the combined effect of astaxanthin and postbiotics is an additive effect; when CI > 1, the combined effect of astaxanthin and postbiotics is an antagonistic effect.

[0097] It can be seen from the experimental results that the increase in SOD by astaxanthin and postbiotics also shows a concentration-dependent increase. Taking a 40% increase rate as an example, the dosage of astaxanthin alone is 11.37±0.81 μg / mL, and the dosage of postbiotics alone is 120.00±3.32 μg / mL. The dosage of the composition is: astaxanthin 3.32±0.12 μg / mL, postbiotics 66.31±1.23 μg / mL. Compared with the single use of astaxanthin and postbiotics, when achieving a specific drug effect, the concentration of the single drug in the composition is significantly reduced. According to the median effect principle and the Chou-Talalay combination index method, the synergistic compatibility effect of astaxanthin and postbiotics at a mass ratio of 1:20 is evaluated. According to the calculation results, an inhibition rate-CI curve graph is drawn, as Figure 5 shown. Astaxanthin and postbiotics can play an obvious synergistic role in increasing SOD at a mass ratio of 1:20.

[0098] The application in the field of comprehensive anti-aging for women:

[0099] (1) Verification of skin anti-aging:

[0100] After one week of adaptive feeding, SD female rats were subcutaneously injected with D-galactose at 125 mg·kg -1 .d -1 , and continuously administered for 42 days to establish a rat aging model. After successful modeling, the rats were randomly divided into a model group and an astaxanthin+postbiotics group, with 10 rats in each group. The model group was fed normally, and the astaxanthin+postbiotics group was intragastrically administered at a dose of (1+20) mg / kg. Continuously treated for 30 days. After the medication, the rats were anesthetized and sacrificed. After shaving, the skin tissues of rats in each group were taken, and the contents of collagen and elastin in the skin tissues of each group were detected by Western-Bloting, and the changes in the expression levels of TGF-β1, smad3, colⅠ, and colⅢ genes in the skin tissues of each group were detected by RT-PCR method.

[0101] Collagen in the skin is mainly secreted and synthesized by fibroblasts. Therefore, regulating the collagen production of these cells is closely related to anti-aging. Research shows that the degradation and synthesis of collagen are affected by various skin cytokines. Among them, TGF-β1, as an important regulator of tissue fibrosis, participates in processes such as cell proliferation, growth, differentiation, remodeling, and wound healing by activating the downstream Smads signaling pathway. It plays a key role in organ fibrosis, extracellular matrix secretion, and collagenase activity regulation. The experimental results show that the mRNA expression levels of TGF-β1 and Smad3 in the dermal fibroblasts of rats treated with the composition are significantly higher than those in the model group. This indicates that the composition enhances the expression of TGF-β1 / Smad3 at the gene level, thereby further promoting collagen synthesis and achieving the anti-aging effect.

[0102] (2) Verification of anti-aging effects on the ovaries and uterus:

[0103] Ten-month-old female Sprague-Dawley (SD) rats and three-month-old female SD rats were selected and acclimated for 1 week under conditions of 12 h light / dark cycle, 40%-60% humidity, (23±3)°C temperature, regular diet, and free access to water to establish a control for middle-aged and young rats. The vaginal exfoliated cells of the rats were continuously monitored to determine the completion of the estrous cycle (5 days), and rats with estrus cycle disorders were selected as experimental subjects to serve as a natural aging rat model. Rats were randomly screened for observation of ovarian and uterine tissue sections. The number of primordial follicles, primary follicles, and secondary follicles in the ovaries of naturally aged rats was significantly reduced, the endometrium was thinned, epithelial cell hyperplasia occurred, the cells were low columnar, glandular and blood vessels were sparse, indicating successful establishment of the perimenopausal rat model. The successfully modeled rats were randomly divided into a model group and an astaxanthin + postbiotics group, with 10 rats in each group. The model group was fed normally, and the astaxanthin + postbiotics group was gavaged at a dose of (1 + 20) mg / kg. The treatment was continued for 60 days.

[0104] After the treatment, blood was collected from rats in each group, and the levels of five hormones, namely testosterone (T), estradiol (E2), anti-Müllerian hormone (AMH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH), were detected by ELISA. The rats were anesthetized and sacrificed, and ovarian tissues were taken for HE staining to observe follicle development.

[0105] The hormone content levels of rats in each group were as Figure 8 shown. After feeding with the composition, the serum E2 and AMH contents in each administration group increased, and the contents of T, FSH, and LH were lower compared with the model group. The hormone levels in each group were comparable to the control, indicating that the composition has a certain protective effect on the ovaries and can prevent the occurrence of premature ovarian failure.

[0106] Through the experiment, it was found that the number of follicles at all levels in the model group rats decreased, the number of granulosa cell layers in the follicles was small, and the number of atretic follicles increased. In the composition group, follicles at all levels developed well, approaching the blank group. The follicles were larger in volume, and it could be seen that primordial follicles, growing follicles, and mature follicles were actively growing, and the number of granulosa cell layers was large and well-developed.

[0107] (3) Small-scale human trial:

[0108] Thirty women aged 30 - 60 years volunteered to participate in the trial and were able to complete the questionnaire and records. They orally took 300 mg of the composition (product prepared in Example 2) on an empty stomach every morning for 4 weeks. Their original eating habits were not changed during the drinking period, and they had a normal diet. After the administration ended, a questionnaire survey was conducted.

[0109] After the end of the administration cycle, 100% of the users reported obvious physical sensations. By analyzing the questionnaire results of everyone, it was found that in general, after taking the product, the skin became smoother, the menstrual cycle became more regular, and the physical sensations during ovulation became more obvious compared to before taking the product.

[0110] Table 1 Statistical Table of the Comprehensive Anti-aging Population Trial of the Composition

[0111]

[0112]

[0113]

[0114] The above-mentioned effect verification experiments show that the composition preparation of the present invention has a relatively comprehensive anti-aging and repair function.

[0115] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of them. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. Although the specific implementation manners of the present invention have been described above, it does not limit the protection scope of the present invention. Those skilled in the art should understand that based on the technical solutions of the present invention, various modifications or deformations that can be made without creative labor by those skilled in the art are still within the protection scope of the present invention.

Claims

1. A postbiotic astaxanthin composition, characterized in that, It contains postbiotics and astaxanthin; The mass ratio of the postbiotics to the astaxanthin is 5 - 50:1; Among them, the postbiotic contains inactivated Lactobacillus rhamnosus ( lactobacillus rhamnosus ) ncxt-1; The Lactobacillus rhamnosus ( lactobacillus rhamnosus ) ncxt-1, which is deposited in the China Center for Type Culture Collection, with the deposit date of November 6, 2024, and its biological deposit number is CCTCC NO: M 20242441.

2. The postbiotic astaxanthin composition according to claim 1, characterized in that, The mass ratio of the postbiotics to the astaxanthin is 10 - 30:

1.

3. The postbiotic astaxanthin composition according to claim 1, wherein The astaxanthin includes products rich in astaxanthin.

4. The postbiotic astaxanthin composition according to claim 3, characterized in that, The astaxanthin includes astaxanthin derived from Haematococcus pluvialis.

5. The postbiogenic astaxanthin composition according to claim 3, wherein The astaxanthin is derived from Antarctic krill oil.

6. The postbiotic astaxanthin composition according to claim 1, wherein The postbiotics are prepared by the following method: S1. Strain activation: Streak the Lactobacillus rhamnosus ( lactobacillus rhamnosus ) ncxt-1 onto an activation medium to obtain a pure strain; S2. Preparation of primary seed liquid: Pick a single colony of Lactobacillus rhamnosus obtained in step S1 and place it in the first culture medium, and statically culture it at 28 - 30 °C for 12 - 14 hours to obtain the primary seed liquid; S3. Secondary seed liquid preparation: Inoculate the above-cultured primary seed liquid into the second culture medium, with an inoculation amount of 1-5%, v / v , and statically culture at 28-35 °C for 12-14 hours to obtain the secondary seed liquid; S4. Liquid fermentation of Lactobacillus rhamnosus: Inoculate the above secondary seed liquid into the third culture medium, with an inoculation amount of 2-8%, v / v , and culture at 25-30 °C and 30-50 r / min for 3-4 hours; then increase the stirring speed to 60-80 r / min, raise the fermentation temperature to 28-35 °C, keep the tank pressure at 0.05-0.09 MPa, adjust the pH to weakly acidic, and ferment for 2-3 hours; then keep the stirring speed and temperature unchanged, raise the tank pressure to 0.1-0.12 MPa, maintain the pH at weakly acidic, and continue to ferment for 4-5 hours; then keep the stirring speed and tank pressure unchanged, raise the temperature to 40-50 °C, add glycerol accounting for 0.1-0.2% of the volume of the fermentation broth to the fermentation broth, and then perform high-temperature sterilization.

7. The postbiotic astaxanthin composition according to claim 6, wherein, S3. Preparation of secondary seed liquid: Inoculate the above - cultured primary seed liquid into the second culture medium, with an inoculation amount of 1%, v / v, and statically culture it at 30 °C for 12 - 14 hours to obtain the secondary seed liquid.

8. The postbiotic astaxanthin composition according to claim 6, wherein S4. Liquid fermentation of Lactobacillus rhamnosus: Inoculate the above secondary seed liquid into the third culture medium with an inoculation amount of 5%. v / v , and culture at 28°C and 40 r / min for 3 - 4 hours; then increase the stirring speed to 70 r / min, raise the fermentation temperature to 30°C, keep the tank pressure at 0.08 Mpa, adjust the pH to 6.5, and ferment for 2 - 3 hours; then keep the stirring speed and temperature unchanged, increase the tank pressure to 0.1 Mpa, maintain the pH at 6.5, and continue to ferment for 4 - 5 hours; then keep the stirring speed and tank pressure unchanged, raise the temperature to 45°C, add glycerol accounting for 0.1% of the volume of the fermentation broth to the fermentation broth, and then perform high-temperature sterilization.

9. The postbiotic astaxanthin composition according to claim 6, wherein In the above - mentioned step S1, the composition of the activation medium is as follows: yeast peptone 0.2% - 1.0%, beef extract 0.3 - 1.0%, glucose 1.0% - 2%, yeast extract powder 0.05 - 0.2%, sodium acetate 0.05 - 0.2%, ammonium citrate 0.05 - 0.2%, potassium dihydrogen phosphate 0.05 - 0.2%, agar powder 0.15 - 0.2%, and adjust the pH to weakly acidic.

10. The postbiotic astaxanthin composition according to claim 9, characterized in that, In the above - mentioned step S1, the composition of the activation medium is as follows: yeast peptone 0.2% - 1.0%, beef extract 0.3 - 1.0%, glucose 1.0% - 2%, yeast extract powder 0.05 - 0.2%, sodium acetate 0.05 - 0.2%, ammonium citrate 0.05 - 0.2%, potassium dihydrogen phosphate 0.05 - 0.2%, agar powder 0.15 - 0.2%, and adjust the pH to pH 6.

5.

11. The postbiotic astaxanthin composition according to claim 6, wherein In the above - mentioned step S2, the composition of the first culture medium is as follows: glucose 1.0% - 2%, yeast peptone 0.15 - 0.2%, beef extract 0.3 - 1.0%, yeast extract powder 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, ammonium citrate 0.05 - 0.2%, and adjust the pH to weakly acidic.

12. The postbiotic astaxanthin composition according to claim 11, wherein In the above - mentioned step S2, the composition of the first culture medium is as follows: glucose 1.0% - 2%, yeast peptone 0.15 - 0.2%, beef extract 0.3 - 1.0%, yeast extract powder 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, ammonium citrate 0.05 - 0.2%, and adjust the pH to pH 6.

5.

13. The postbiotic astaxanthin composition according to claim 6, wherein In the above - mentioned step S3, the composition of the second culture medium is as follows: glucose 1.0% - 2%, yeast peptone 0.5 - 2%, beef extract 0.1 - 0.5%, yeast extract powder 0.1 - 0.5%, ammonium citrate 0.05 - 0.2%, Tween - 80 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, and adjust the pH to weakly acidic.

14. The postbiotic astaxanthin composition according to claim 13, wherein In the step S3, the composition of the second culture medium is as follows: glucose 1.0% - 2%, yeast peptone 0.5 - 2%, beef extract 0.1 - 0.5%, yeast extract powder 0.1 - 0.5%, ammonium citrate tribasic 0.05 - 0.2%, Tween - 80 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, adjust the pH to pH 6.

5.

15. The postbiotic astaxanthin composition according to claim 6, characterized in that, In the step S4, In the step S4, the composition of the third culture medium is as follows: fructooligosaccharide 1.0% - 2%, yeast peptone 0.5 - 2%, beef extract 0.1 - 0.5%, yeast extract powder 0.1 - 0.5%, ammonium citrate tribasic 0.05 - 0.2%, Tween - 80 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, adjust the pH to weakly acidic.

16. The postbiotic astaxanthin composition according to claim 15, wherein, In the step S4, In the step S4, the composition of the third culture medium is as follows: fructooligosaccharide 1.0% - 2%, yeast peptone 0.5 - 2%, beef extract 0.1 - 0.5%, yeast extract powder 0.1 - 0.5%, ammonium citrate tribasic 0.05 - 0.2%, Tween - 80 0.1 - 0.2%, sodium acetate 0.1 - 0.2%, potassium dihydrogen phosphate 0.1 - 0.2%, adjust the pH to pH 6.

5.

17. The postbiotic astaxanthin composition according to claim 6, wherein The high - temperature sterilization is specifically carried out by raising the temperature to 85 °C and maintaining it for 30 min for sterilization.

18. The postbiotic astaxanthin composition according to claim 17, wherein, After spray - drying the culture medium after high - temperature sterilization, the postbiotic solid preparation is obtained.

19. The preparation method of the postbiotic astaxanthin composition according to any one of claims 1-18, characterized in that, The preparation method includes the step of mixing the postbiotic with astaxanthin.

Images