Postbiotic and composition for improving anti-inflammatory capacity, anti-oxidation capacity and bioavailability of astaxanthin and application of postbiotic and composition in anti-aging

Abstract

The invention belongs to the technical field of microbial preparations, and particularly relates to a metagen and a composition for improving the anti-inflammatory capacity, the antioxidant capacity and the bioavailability of astaxanthin and an application of the metagen and the composition in anti-aging. According to the invention, the metabiotics prepared by fermentation of lactobacillus rhamnosus and the astaxanthin have a synergistic effect, so that the bioavailability of the astaxanthin is improved, and the synergistic effect of the astaxanthin in the aspects of oxidation resistance and inflammation resistance is enhanced. Besides, due to the synergistic effect of the astaxanthin and the metaplasts, the astaxanthin and the metaplasts have the protection effect on the skin and internal organs, and besides skin aging resistance, ovary aging resistance and uterus aging resistance, the technical scheme provides a new solution for comprehensive health management of women, and therefore the astaxanthin and the metaplasts have good practical application value.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled 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 shrimp, crab, fish, algae, yeast and bird feathers. It 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 also cross the blood-brain barrier and improve nerve function.

[0004] As people's pursuit of health and beauty grows, anti-aging for women has become a hot topic. Astaxanthin has become a star ingredient in the anti-aging field in recent years due to its strong antioxidant properties. By capturing 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 inflammation caused by ultraviolet rays, making it widely used in sunscreen and repair products.

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

[0006] In addition, most of the anti-aging products on the market are mainly targeted at a certain type, and there are few reports on comprehensive anti-aging products, especially anti-aging in multiple aspects such as skin, ovaries and uterus. Summary of the invention

[0007] Based on the above-mentioned deficiencies in the prior art, the present invention provides a postbiotic and composition that improves the anti-inflammatory, antioxidant and bioavailability of astaxanthin and its application in anti-aging. Experiments have shown that the postbiotic product prepared based on Lactobacillus rhamnosus in the present invention can effectively improve the anti-inflammatory, antioxidant and bioavailability of astaxanthin, and the two are combined to show a comprehensive anti-aging effect, especially for women's skin, ovaries and uterus. Based on the above research results, the present invention is completed.

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

[0009] The first aspect of the present invention provides the use of postbiotics in the preparation of products that improve the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin;

[0010] Wherein, the postbiotic comprises inactivated lactobacillus rhamnosus ncxt-1;

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

[0012] A second aspect of the present invention provides a postbiotic astaxanthin composition comprising postbiotics and astaxanthin;

[0013] 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.

[0014] Wherein, the postbiotics contain inactivated lactobacillus rhamnosus ncxt-1.

[0015] The third aspect of the present invention provides a method for preparing the above-mentioned postbiotic astaxanthin composition, which comprises the step of mixing the postbiotic with astaxanthin.

[0016] The astaxanthin can be obtained by commercial means, and its source is not specifically limited. It is well known to those skilled in the art that astaxanthin is a keto-carotenoid with a chemical name of 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene. It is specifically a red solid powder and is widely present in the biological world, especially in shrimp, crab, fish, algae, yeast and bird feathers. It is one of the main carotenoids in marine organisms. In addition to being obtained by biological extraction, astaxanthin can also be obtained by chemical synthesis and other methods, which are not specifically limited 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] A fourth aspect of the present invention provides the use of the above-mentioned postbiotic astaxanthin composition in the preparation of anti-aging products.

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

[0019] Wherein, the product can be food or medicine.

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] The above technical solution uses postbiotics prepared by fermentation of Lactobacillus rhamnosus, which synergize with astaxanthin, not only improving the bioavailability of astaxanthin, but also enhancing their synergistic effects in anti-oxidation and anti-inflammatory. 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 anti-aging of the skin, it also includes anti-aging of the ovaries and uterus. The above technical solution provides a new solution for the all-round health management of women, and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is the change of astaxanthin content in rat serum in verifying the effect of the present invention.

[0024] Figure 2 The figure shows the change of astaxanthin content in rat small intestinal villi in verifying the effect of the present invention.

[0025] Figure 3 This is the change of astaxanthin content in the intestinal contents of rats in verifying the effect of the present invention.

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

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

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

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

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

[0031] Fig. 9 These are HE sections of the ovaries of rats in each group used in the validation of the effects of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and are 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 meanings as those commonly understood by those skilled in the art 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 of the present invention. 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, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

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

[0035] Wherein, the postbiotics contain inactivated lactobacillus rhamnosus ncxt-1.

[0036] The lactobacillus rhamnosus ncxt-1 is deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the preservation date is November 6, 2024, and its biological preservation 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] Wherein, the postbiotics contain inactivated lactobacillus rhamnosus ncxt-1.

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

[0041] S1. Activation of bacterial strains: streaking the lactobacillus rhamnosus ncxt-1 into an activation medium to obtain a pure strain;

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

[0043] S3, preparation of secondary seed solution: inoculate the primary seed solution cultured above into the second culture solution, with an inoculation amount of 1-5% (preferably 0.5%, v / v), and culture at 28-35° C. (preferably 30° C.) for 12-14 hours to obtain the secondary seed solution;

[0044] S4, Lactobacillus rhamnosus liquid fermentation: the above-mentioned secondary seed liquid is inoculated into the third culture liquid, the inoculation amount is 2-8% (preferably 5%, v / v), and the culture is carried out at 25-30°C (preferably 28°C), 30-50r / min (preferably 40r / min) for 3-4 hours; then the stirring speed is increased to 60-80r / min (preferably 70r / min), the fermentation temperature is increased to 28-35°C (preferably 30°C), the tank pressure is maintained at 0.05-0.09MPa (preferably 0.08Mpa), and the pH is adjusted to weak acidity (pH 6.5), ferment for 2-3 hours; then keep the stirring speed and temperature unchanged, increase the tank pressure to 0.1-0.12MPa (preferably 0.1Mpa), maintain the pH at weak acidity (pH6.5), and continue fermenting for 4-5 hours; then control the stirring speed and tank pressure unchanged, increase 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 liquid to the fermentation liquid, and then sterilize at high temperature.

[0045] Wherein, in step S1, the composition of the activation culture 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%, triammonium citrate 0.05-0.2%, potassium dihydrogen phosphate 0.05-0.2%, agar powder 0.15-0.2%, and the pH is adjusted to weak acidity (pH 6.5).

[0046] In step S2, the first culture solution is composed of: 1.0%-2% glucose, 0.15-0.2% yeast peptone, 0.3-1.0% beef extract, 0.1-0.2% yeast extract powder, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, 0.05-0.2% triammonium citrate, and the pH is adjusted to weak acidity (pH 6.5).

[0047] In step S3, the second culture solution is composed of: 1.0%-2% glucose, 0.5-2% yeast peptone, 0.1-0.5% beef extract, 0.1-0.5% yeast extract powder, 0.05-0.2% triammonium citrate, 0.1-0.2% Tween-80, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, and the pH is adjusted to weak acidity (pH 6.5).

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

[0049] Furthermore, in step S4, the high temperature sterilization may be performed by raising the temperature to 85° C. and maintaining the temperature for 30 minutes for sterilization; further, the culture solution after high temperature sterilization is spray-dried to obtain the postbiotic solid preparation.

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

[0051] Wherein, the postbiotics are obtained based on Lactobacillus rhamnosus using the above-mentioned preparation method.

[0052] The astaxanthin can be obtained by commercial means, and its source is not specifically limited. It is well known to those skilled in the art that astaxanthin is a ketocarotenoid with a chemical name of 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene. It is specifically a red solid powder and is widely present in the biological world, especially in the feathers of aquatic animals such as shrimp, crab, fish and birds. In addition to being obtained by biological extraction, astaxanthin can also be synthesized by chemical synthesis. 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] The present invention has found through research that postbiotics can effectively improve the bioavailability of astaxanthin. The combination of the two can produce synergistic antioxidant and anti-inflammatory effects, and can be used as an anti-aging product, especially as an anti-aging product for women. The present invention has confirmed through experiments that this product has good anti-aging effects on the skin, ovaries and uterus.

[0055] Wherein, the product can be food or medicine.

[0056] The term "food" used in the present invention can be in any form that can be eaten, including ordinary food and special food. The special food includes health food and special medical formula food; and ordinary food is relative to special food, which is food suitable for everyone.

[0057] The medicine of the present invention may also contain commonly used carriers, carriers, excipients and diluents, etc. Moreover, according to the usual method, it can be made into oral preparations, external preparations, suppositories and sterile injection solutions in the form of powders, granules, suspensions, emulsions, syrups, sprays, etc.

[0058] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In each example,

[0059] The composition of the activation culture medium is as follows: per 1000 mL, it contains 10 g yeast peptone, 5 g beef extract, 10 g glucose, 2 g yeast extract powder, 1 g sodium acetate, 1 g triammonium citrate, 1 g potassium dihydrogen phosphate, 20 g agar powder, and the pH is adjusted to 6.5.

[0060] The composition of the first culture solution is as follows: per 1000 mL, it contains 10 g glucose, 2 g yeast peptone, 5 g beef extract, 2 g yeast extract powder, 2 g sodium acetate, 2 g potassium dihydrogen phosphate, 1 g triammonium citrate, and the pH is adjusted to 6.5;

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

[0062] The composition of the third culture solution is as follows: per 1000 mL, it contains: 10 g oligofructose, 20 g yeast peptone, 5 g beef extract, 5 g yeast extract powder, 1 g triammonium citrate, 2 g Tween-80, 2 g sodium acetate, 2 g potassium dihydrogen phosphate, pH 6.5.

[0063] Example 1

[0064] A method for preparing postbiotics comprises the following steps:

[0065] Streaking Lactobacillus rhamnosus ncxt-1 into an activation medium to obtain a pure strain; picking a single colony of Lactobacillus rhamnosus obtained and placing it in a first culture solution for static culture at 30° C. for 14 hours to obtain a primary seed solution;

[0066] The first-level seed solution was inoculated into the second culture solution at an inoculation rate of 1%, and then cultured at 30°C for 14 hours to obtain the second-level seed solution;

[0067] According to the inoculation amount of 5%, Lactobacillus rhamnosus was inoculated into a fermentation tank containing the sterilized third culture solution, and cultured at 28°C, 40r / min for 4 hours; after 4 hours, the stirring speed was increased to 80r / min, the fermentation temperature was increased to 30°C, the tank pressure was maintained at 0.08MPa, the pH was adjusted to 6.5 with ammonia water, and fermented for 3 hours; after 7 hours, the stirring speed and temperature remained unchanged, the tank pressure was increased to 0.1MPa, the pH was adjusted to 6.5 with ammonia water, and fermented for 4 hours; after 11 hours, the stirring speed was unchanged, the temperature was increased to 45°C, 0.1% glycerol by volume of the fermentation solution was added to the fermentation tank, the temperature was increased to 85°C, maintained for 30 minutes, and then spray-dried.

[0068] Example 2

[0069] A method for preparing a postbiotic astaxanthin composition comprises the following steps:

[0070] Streaking Lactobacillus rhamnosus ncxt-1 into an activation medium to obtain a pure strain; picking a single colony of Lactobacillus rhamnosus obtained and placing it in a first culture solution for static culture at 30° C. for 14 hours to obtain a primary seed solution;

[0071] The first-level seed solution is inoculated into the second culture solution at an inoculation rate of 1%, and then cultured at 30°C for 14 hours to obtain the second-level seed solution;

[0072] According to the inoculation amount of 5%, Lactobacillus rhamnosus was inoculated into a fermentation tank containing the sterilized third culture solution, and cultured at 28°C, 40r / min for 4 hours; after 4 hours, the stirring speed was increased to 80r / min, the fermentation temperature was increased to 30°C, the tank pressure was maintained at 0.08MPa, the pH was adjusted to 6.5 with ammonia water, and fermented for 3 hours; after 7 hours, the stirring speed and temperature remained unchanged, the tank pressure was increased to 0.1MPa, the pH was adjusted to 6.5 with ammonia water, and fermented for 4 hours; after 11 hours, the stirring speed was unchanged, the temperature was increased to 45°C, 0.1% glycerol by volume of the fermentation solution was added to the fermentation tank, the temperature was increased to 85°C and maintained for 30 minutes, and then spray-dried to obtain postbiotics.

[0073] 2000 g of postbiotics were mixed with 100 g of astaxanthin powder, and mixed in a three-dimensional mixer at 100 r / min for 60 minutes to obtain a postbiotic astaxanthin composition.

[0074] Effect verification

[0075] The composition for improving the bioavailability of astaxanthin is verified by:

[0076] 60 healthy Wistar male rats with uniform body weight were selected. After adaptive feeding for 1 week, they were randomly divided into 2 groups of astaxanthin group and postbiotics + astaxanthin, 30 in each group, and gavage treatment was performed to feed Example 1 to obtain postbiotics 20.0 mg / kg and astaxanthin 1.0 mg / kg. After gavage at 0h, 0.5h, 1h, 2h, 4h, 8h, 10h, 12h, 18h and 24h, mice were anesthetized and killed, blood and intestines were taken, serum was separated, small intestinal villi and intestinal contents were stripped and taken out, and intestinal contents were collected by washing with normal saline. After extraction, the content of astaxanthin in serum, small intestinal villi and intestinal contents in different time periods was detected by high performance liquid chromatography. The content of astaxanthin in the same time period of different groups was compared.

[0077] The content of astaxanthin in serum of different groups Figure 1 As shown, the astaxanthin content in the serum of the two groups of rats began to rise slowly 0.5h after gavage, reached the maximum value at 8h, then began to decline, and returned to the initial level at 16-24h. At 1h, 2h, 4h, 8h, 10h, and 12h, the serum astaxanthin content of rats in the postbiotic + astaxanthin group was greater than that in the group fed with astaxanthin alone. After 8h of feeding, the astaxanthin content in the postbiotic astaxanthin group was 24.7±0.5μg / L, which was 12.35 times that of the group fed with astaxanthin alone (2.0±0.3μg / L). The content of astaxanthin in the small intestinal villi of different groups is shown in Figure 2As shown, the astaxanthin content in the small intestinal villi gradually increased, reaching a peak at 4h, and then gradually decreased until it was undetectable at 16-24h. At 1h, 2h, 4h, 8h, 10h, 12h and other time points, the serum astaxanthin content of rats in the postbiotic + astaxanthin group was greater than that in the group fed with astaxanthin alone. After 4h of feeding, the astaxanthin content in the postbiotic astaxanthin group was 810.0±20.5ng, which was 7.04 times that of the group fed with astaxanthin alone (115.0±20.3μg / L). The content of astaxanthin in the intestinal contents of different groups is shown in Figure 3 As shown in the figure, the astaxanthin content in the intestinal contents of the two groups of rats began to rise slowly 0.5h after gavage, reached the maximum value at 4h, then began to decline, and returned to the initial level at 16-24h. At 1h, 2h, 4h, 8h, 10h, and 12h, the serum astaxanthin content of rats in the postbiotics + astaxanthin group was greater than that in the astaxanthin-only group.

[0078] The bioavailability of astaxanthin in serum of different experimental groups was further measured by calculating the area under the curve (AUC0-t).

[0079] 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 0h, 0.5h, 1h, 2h, 4h, 8h, 10h, 12h, 18h and 24h, 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 postbiotic+astaxanthin group was (158.5±12.3)μg·h / L, which was 6.31 times that of the astaxanthin group.

[0080] The verification method of the composition for synergistically improving anti-inflammatory and antioxidant abilities is as follows:

[0081] RAW264.7 cells were dispersed in RPMI1640 culture medium (containing 10% fetal bovine serum, 100 μmol / L penicillin and streptomycin) and cultured in a cell culture incubator. When the cells covered 70%-80% of the culture dish, they were subcultured. When the cells were growing well, 1×10 4 The density of cells was 100 μL, and RAW264.7 cells were inoculated in 96-well plates. Each well was inoculated with 100 μL of culture medium and incubated at 37°C for 24 hours. According to the drug addition requirements of each group of experiments, the culture medium was replaced. After continuing to culture for 24 hours, 10 μL of CCK-8 reagent was added to each well, and incubated at 37°C in the dark for 2 hours. The OD value was detected at 450 nm to calculate the cell viability. Cell viability % = (experimental group OD - blank OD / normal group - blank OD) × 100%.

[0082] (1) Effects of different concentrations of lipopolysaccharide (LPS) on cell activity

[0083] The experiment was divided into blank group, normal group, and different concentrations of LPS experimental groups. Blank group: no cells; normal group: cells, no LPS; different concentrations of LPS experimental groups, set the final concentration of LPS to 0.78125, 1.5625, 3.125, 6.25, 12.5 and 25μg / mL 6 concentration gradient groups, each group was treated with 5 replicates. The CCK-8 method was used to detect the effect of LPS on the activity of RAW264.7 cells, and the final experimental concentration of LPS was determined to be 1.0μg / mL.

[0084] (2) Effects of different concentrations of astaxanthin on cell activity

[0085] The experiment was divided into a blank group, a normal group, and different concentrations of astaxanthin experimental groups. Blank group: no cells; normal group: cells, no astaxanthin; different concentrations of astaxanthin experimental groups, set the final concentration of astaxanthin to 0.625, 1.25, 2.5, 5, 8, 10μg / mL 6 concentration gradient groups, each group was treated with 5 replicates. The CCK-8 method was used to detect the effect of astaxanthin on the activity of RAW264.7 cells. The experimental results showed that when the astaxanthin concentration was ≤1.25μg / mL, the activity of RAW264.7 cells was not significantly different from that of the normal group. When the astaxanthin concentration was 1.25-10μg / mL, the activity of RAW264.7 cells showed a trend of first increasing and then decreasing; when the astaxanthin concentration reached 10μg / mL, the activity of RAW264.7 cells was compared with the control, there was no significant change. Based on cell activity and drug cost considerations, the final mass concentration of astaxanthin was selected to be ≤10μg / mL.

[0086] (3) Effects of different concentrations of postbiotics on cell activity

[0087] The experiment was divided into a blank group, a normal group, and different concentrations of postbiotic experimental groups. Blank group: no cells; normal group: cells, no postbiotics; different concentrations of postbiotic experimental groups, set the final concentration of postbiotics to 12.5, 25, 50, 100, 200, 300 μg / mL 6 concentration gradient groups, each group was treated with 5 replicates. The CCK-8 method was used to detect the effect of postbiotics on the activity of RAW264.7 cells. The experimental results showed that when the concentration of postbiotics was 12.5-300 μg / mL, with the increase of the concentration of postbiotics, the activity of RAW264.7 cells showed a trend of first increasing and then decreasing. When the final mass concentration of postbiotics reached 300 μg / mL, the activity of RAW264.7 cells did not change significantly compared with the control. Therefore, when postbiotics are used 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.

[0088] (4) RT-qPCR method to detect the inhibitory effect of astaxanthin and postbiotics at three concentrations on the inflammatory factor TNF-α and analyze the antioxidant capacity

[0089] RAW264.7 cells with good growth status were taken and 1×10 6 The density of cells was 100 cells / well, and RAW264.7 cells were inoculated in 6-well plates. 2 mL of culture 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: cells, no LPS, no astaxanthin + postbiotics; astaxanthin + postbiotic experimental group, astaxanthin + postbiotics were set to three mass final concentration combinations of 10 + 300, 10 + 200, and 10 + 100 μg / mL (refer to the cell activity results in the above steps to select the appropriate drug mass final concentration for matching), according to each group of experiments, reagents were added, culture medium was replaced, LPS was added at a final concentration of 1 μg / mL after 2 hours of drug action, and cells were collected after continued culture for 24 hours; RNA of cells in each group was extracted using a nucleic acid extraction kit, and the relative expression of TNF-α in each group was calculated by RT-PCR, and SOD levels were detected by ELISA kit. Compared with the control group, the expression of inflammatory factor TNF-α in the model group increased significantly; compared with the model group, three (10+300, 10+200, 10+100μg / mL, i.e., mass ratios of 1:30, 1:20 and 1:10) astaxanthin + postbiotic combinations can downregulate the expression of inflammatory factor TNF-α after LPS stimulation; when the astaxanthin + postbiotic combination is 10+200μg / mL, the expression of inflammatory factor TNF-α in RAW264.7 cells is the lowest. When the mass ratio of astaxanthin + postbiotics is 1:20, the inhibitory effect of inflammatory factor TNF-α in RAW264.7 cells is the best. Compared with the control group, the SOD activity of the model group was decreased. Compared with the model group, three combinations of astaxanthin + postbiotics (10+300, 10+200, 10+100 μg / mL, i.e., mass ratios of 1:30, 1:20 and 1:10) could stimulate SOD activity; when the astaxanthin + postbiotic combination was 10+200 μg / mL, the antioxidant capacity of RAW264.7 cells was most obvious.

[0090] (5) Analysis of the inhibitory effect of astaxanthin, postbiotics and their combination on the expression of inflammatory factor TNF-α and their antioxidant capacity

[0091] RAW264.7 cells with good growth status were taken and 1×10 6The RAW264.7 cells were seeded in a 6-well plate at a density of 10 cells / well, 2 mL of culture medium was added to each well, and incubated at 37°C for 24 h. The experiment was divided into normal group, model group, astaxanthin group, postbiotic group, and astaxanthin group + postbiotic group. Normal group: cells, no LPS, no astaxanthin, postbiotics; model group: cells and LPS stimulation, no astaxanthin and postbiotics, astaxanthin group: final mass concentration gradient set to 10, 8, 6, 5, 1μg / mL; postbiotic final mass concentration gradient set to 200, 160, 120, 100, 20μg / mL; astaxanthin group + postbiotic final mass concentration gradient set to 10+200, 8+160, 6+120, 5+100, 1+20μg / mL, according to the experimental drug addition needs of each group, the culture medium was replaced, and LPS stimulation with a final concentration of 1μg / mL was added after 2h of drug action, and the cells were collected after 24h of continuous culture; RNA of each group of cells was extracted using a kit, and RT-PCR was used to calculate the mRNA expression and inhibition rate of TNF-α in each group. Inhibition rate = (1-relative expression of mRNA in the drug addition group / relative expression of mRNA in the model group) × 100%. The ELISA kit was used to detect the SOD level and SOD improvement rate, where improvement rate = (relative mRNA expression level in the drug-added group - relative mRNA expression level in the model group) / relative mRNA expression level in the model group × 100%.

[0092] According to the results, the simulation equation of astaxanthin inhibition rate on TNF-α is: y = 2.1087x + 3.3478 (R 2 =0.9929); the simulation equation of the inhibition rate of postbiotics on TNF-α is: y = 0.2033x + 3.4087 (R 2 =0.9966); The simulation equation of the inhibition rate of TNF-α by the combination of astaxanthin + postbiotics is: y = 0.3157x + 2.2174 (R 2 =0.9962). According to the Chou-Talalay equation, CI = D 1 / D 1x +D 2 / D 2x , calculate the combined index of astaxanthin and postbiotics CI, D 1 , D 2 is the concentration of astaxanthin and postbiotic composition at the inhibition rate of inflammatory factor TNF-α expression, D 1x , D 2x The concentrations of astaxanthin and postbiotics when used alone to achieve the above effects were evaluated. The synergistic inhibitory effect of the two (mass ratio of 1:20) on the expression of inflammatory factor TNF-α was evaluated. When CI < 1, the combined effect of astaxanthin and postbiotics was a synergistic effect; when CI = 1, the combined effect of astaxanthin and postbiotics was an additive effect; when CI > 1, the combined effect of astaxanthin and postbiotics was an antagonistic effect.

[0093] The experimental results show that the inhibition rates of astaxanthin and postbiotics on the expression of inflammatory factor TNF-α both show a concentration-dependent increase. Taking the inhibition rate of 40 as an example, the inhibition rate of astaxanthin alone is 17.38±2.31μg / mL, the inhibition rate of postbiotics alone is 179.98±8.12μg / mL, and the dosage of the combination is: astaxanthin 5.69±0.61μg / mL, postbiotics 113.97±5.22μg / mL. Compared with the use of astaxanthin and postbiotics alone, when a specific drug effect is achieved, the concentration of a single drug in the combination is significantly reduced. The synergistic effect of astaxanthin and postbiotics at a mass ratio of 1:20 was evaluated according to the median effect principle and the Chou-Talalay combined index method. The inhibition rate and CI curve were plotted based on the calculation results, as shown in the figure. Figure 4 As shown in the results, astaxanthin and postbiotics can play a significant synergistic inhibitory role in the expression of inflammatory factor TNF-α when combined in a mass ratio of 1:20.

[0094] According to the results, the simulation equation of astaxanthin's effect on SOD improvement is: y = 0.0357x - 0.0059 (R 2 =0.993); the simulation equation of the postbiotics on the SOD improvement rate is: y = 0.002x + 0.16 (R 2 =1); the simulation equation of the astaxanthin + postbiotic combination on the SOD improvement rate is: y = 0.0046x + 0.0797 (R 2 =0.9909). According to the Chou-Talalay equation, CI = D 1 / D 1x +D 2 / D 2x , calculate the combined index of astaxanthin and postbiotics CI, D 1 , D 2 is the concentration of astaxanthin and postbiotic combination when SOD is increased to a certain extent, D 1x , D 2x The concentrations of astaxanthin and postbiotics when used alone to achieve the above effects were evaluated. The synergistic inhibitory effect of the two (mass ratio of 1:20) on the increase of SOD was evaluated. 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] The experimental results show that the improvement of SOD by astaxanthin and postbiotics also shows a concentration-dependent increase. Taking the improvement rate of 40 as an example, the use of astaxanthin alone is 11.37±0.81μg / mL, the use of postbiotics alone is 120.00±3.32μg / mL, and the dosage of the combination is: astaxanthin 3.32±0.12μg / mL, postbiotics 66.31±1.23μg / mL. Compared with the use of astaxanthin and postbiotics alone, when a specific drug effect is achieved, the concentration of a single drug in the combination is significantly reduced. The synergistic effect of astaxanthin and postbiotics at a mass ratio of 1:20 was evaluated according to the median effect principle and the Chou-Talalay combined index method. The inhibition rate and CI curve were plotted based on the calculation results, as shown in the figure. Figure 5 As shown in the figure, astaxanthin and postbiotics can play a significant synergistic role in improving SOD when combined in a mass ratio of 1:20.

[0096] The application in the field of comprehensive anti-aging for women:

[0097] (1) Verification of skin anti-aging:

[0098] After one week of adaptive feeding, female SD rats were subcutaneously injected with D-galactose 125 mg kg -1 .d -1 , and the drug was administered continuously for 42 days to establish a rat aging model. After the model was successfully established, the rats were randomly divided into a model group and an astaxanthin + postbiotic group, with 10 rats in each group. The model group was fed normally, and the astaxanthin + postbiotic group was gavage treated at a dose of (1+20) mg / kg. The treatment was continued for 30 days. After the end of the medication, the rats were anesthetized and killed, and the skin tissues of the rats in each group were obtained after shaving. Western-Bloting was used to detect the content of collagen and elastin in the skin tissues of each group, and RT-PCR was used to detect the changes in the expression of TGF-β1, smad3, colⅠ and colⅢ genes in the skin tissues of each group.

[0099] 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. Studies have shown that the degradation and synthesis of collagen are affected by a variety of skin cytokines. Among them, TGF-β1, as an important regulator of tissue fibrosis, participates in 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 the regulation of collagenase activity. The experimental results showed that the mRNA expression of TGF-β1 and Smad3 in rat dermal fibroblasts treated with the composition was significantly increased compared with the model group. This shows that the composition enhances the expression of TGF-β1 / Smad3 at the genetic level, thereby further promoting collagen synthesis and achieving anti-aging effects.

[0100] (2) Verification of ovarian and uterine anti-aging:

[0101] Female SD rats aged 10 months and female SD rats aged 3 months were selected and adapted for 1 week in an environment with alternating light / darkness for 12 hours, humidity of 40% to 60%, temperature of (23±3)℃, regular diet and free drinking water, and used as controls for young and middle-aged rats. Vaginal exfoliated cells of rats were continuously monitored to determine the completion of the estrous cycle (5d), and those with estrous disorder were selected as experimental subjects as the natural aging rat model. Rats were randomly selected for observation of ovarian tissue 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, the epithelial cells proliferated, the cells were low columnar, the glands were scarce, and the blood vessels were scarce, indicating that the rat perimenopausal model was successfully established. The rats with successful modeling were randomly divided into a model group and an astaxanthin + epibiotic group, with 10 rats in each group. The model group was fed normally, and the astaxanthin + epibiotic group was gavage treated at a dose of (1+20) mg / kg. The treatment was continued for 60 days.

[0102] After the medication, blood was collected from each group of rats, and ELISA was used to detect the levels of testosterone T, estrogen E2, anti-Mullerian hormone AMH, luteinizing hormone LH, and follicle-stimulating hormone FSH. Rats were anesthetized and killed, and ovarian tissues were obtained for HE staining to observe the development of follicles.

[0103] The hormone levels of rats in each group were Figure 8 As shown, after feeding with the composition, the serum E2 and AMH levels of each administration group increased, and the levels of T, FSH and LH decreased compared with the model group. The hormone levels of each group were equivalent to the control, indicating that the composition has a certain ovarian protective effect and can prevent the occurrence of premature ovarian failure.

[0104] The experiment showed that the number of follicles at all levels in the model group was reduced, the number of granulosa layers was small, and the number of atretic follicles increased. The follicles at all levels in the combination group developed well, close to the blank group, and the follicle volume was larger. It can be seen that the primary follicles, growing follicles and mature follicles grew actively, and the number of granulosa layers was large and well-developed.

[0105] (3) Small-scale population trials:

[0106] 30 women aged 30-60 years old, who were willing to participate in the experiment and were able to complete the questionnaire and record, took 300 mg of the composition (the product prepared in Example 2) orally on an empty stomach every morning for 4 weeks. During the drinking period, they did not change their original eating habits and ate normally. After the end of the taking, a questionnaire survey was conducted.

[0107] After the cycle, 100% of the users said that the sensation was more obvious. According to the questionnaire results, the skin became smoother, the menstrual period became more regular, and the ovulation sensation was more obvious after taking the product.

[0108] Table 1 Statistics of the comprehensive anti-aging population test of the composition

[0109]

[0110]

[0111] The above effect verification experiments show that the composition preparation prepared by the present invention has a more comprehensive anti-aging and repairing function.

[0112] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to 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 by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. Application of postbiotics in the preparation of products that improve the anti-inflammatory, antioxidant capacity and bioavailability of astaxanthin; in, The postbiotics include inactivated lactobacillus rhamnosus ncxt-1; The lactobacillus rhamnosus ncxt-1 is deposited in the China Center for Type Culture Collection on November 6, 2024, and its biological preservation number is CCTCC NO: M 20242441.

2. A postbiotic astaxanthin composition, characterized in that: It contains postbiotics and astaxanthin; The mass ratio of the postbiotics to astaxanthin is 5-50:1, further 10-30:1; Wherein, the postbiotic comprises inactivated lactobacillus rhamnosus ncxt-1; The lactobacillus rhamnosus ncxt-1 is deposited in the China Center for Type Culture Collection on November 6, 2024, and its biological deposit number is CCTCC NO: M 20242441; Furthermore, the astaxanthin comprises astaxanthin derived from Haematococcus pluvialis, and a product rich in astaxanthin, further comprising Antarctic krill oil.

3. The postbiotic astaxanthin composition according to claim 2, characterized in that The postbiotics are prepared by the following method: S1. Activation of bacterial strains: streaking the lactobacillus rhamnosus ncxt-1 into an activation medium to obtain a pure strain; S2. Preparation of primary seed solution: Pick a single colony of Lactobacillus rhamnosus obtained in step S1 and place it in the first culture solution for static culture at 28-30° C. for 12-14 hours to obtain a primary seed solution; S3, preparation of secondary seed solution: inoculate the primary seed solution cultured above into the second culture solution, with an inoculation amount of 1-5% (preferably 0.5%, v / v), and culture at 28-35° C. (preferably 30° C.) for 12-14 hours to obtain the secondary seed solution; S4, Lactobacillus rhamnosus liquid fermentation: the above-mentioned secondary seed liquid is inoculated into the third culture liquid, the inoculation amount is 2-8% (preferably 5%, v / v), at 25-30° C. (preferably 28° C.), 30-50r / min (preferably 40r / min), and cultured for 3-4 hours; then the stirring speed is increased to 60-80r / min (preferably 70r / min), the fermentation temperature is increased to 28-35° C. (preferably 30° C.), the tank pressure is maintained at 0.05-0.09MPa (preferably 0.08Mpa), the pH is adjusted to weak acidity (pH 6.5), and fermented for 2-3 hours; then the stirring speed and temperature are kept unchanged, the tank pressure is increased to 0.1-0.12MPa (preferably 0.1Mpa), and the pH is maintained at weak acidity (pH 6.5), continue fermentation for 4-5 hours; then control 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 liquid to the fermentation liquid, and then sterilize at high temperature.

4. The postbiotic astaxanthin composition according to claim 3, characterized in that In step S1, the composition of the activation culture medium is as follows: 0.2%-1.0% yeast peptone, 0.3%-1.0% beef extract, 1.0%-2% glucose, 0.05%-0.2% yeast extract powder, 0.05-0.2% sodium acetate, 0.05-0.2% triammonium citrate, 0.05-0.2% potassium dihydrogen phosphate, 0.15-0.2% agar powder, and the pH is adjusted to weak acidity (pH 6.5).

5. The postbiotic astaxanthin composition according to claim 3, characterized in that In step S2, the first culture solution is composed of: 1.0%-2% glucose, 0.15-0.2% yeast peptone, 0.3-1.0% beef extract, 0.1-0.2% yeast extract powder, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, 0.05-0.2% triammonium citrate, and the pH is adjusted to weak acidity (pH 6.5).

6. The postbiotic astaxanthin composition according to claim 3, characterized in that In step S3, the second culture solution is composed of: 1.0%-2% glucose, 0.5-2% yeast peptone, 0.1-0.5% beef extract, 0.1-0.5% yeast extract powder, 0.05-0.2% triammonium citrate, 0.1-0.2% Tween-80, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, and the pH is adjusted to weak acidity (pH 6.5).

7. The postbiotic astaxanthin composition according to claim 3, characterized in that In the step S4, The third culture solution is composed of: 1.0%-2% oligofructose, 0.5-2% yeast peptone, 0.1-0.5% beef extract, 0.1-0.5% yeast extract powder, 0.05-0.2% triammonium citrate, 0.1-0.2% Tween-80, 0.1-0.2% sodium acetate, 0.1-0.2% potassium dihydrogen phosphate, and the pH is adjusted to weak acidity (pH 6.5); The high temperature sterilization specifically includes raising the temperature to 85° C. and maintaining it for 30 minutes for sterilization; further, the culture solution after high temperature sterilization is spray-dried to obtain the postbiotic solid preparation.

8. The method for preparing the postbiotic astaxanthin composition according to claims 2-7, characterized in that: The preparation method comprises the step of mixing the postbiotics with astaxanthin.

9. Use of the postbiotic astaxanthin composition according to any one of claims 2 to 7 in the preparation of anti-aging products.

10. The use according to claim 9, characterized in that: The anti-aging product is specifically a product that has anti-aging effects on the skin, ovaries and uterus.

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