Preparation method and application of a fermented red leaf algae anti-aging repair nanocomposite

Abstract

The application discloses a preparation method and application of a fermentation red leaf algae anti-aging protection nanocomposite, and particularly relates to the cosmetic technical field, and comprises red leaf algae extract preparation, wherein after the red leaf algae is washed, impurities are removed, dried and crushed, the red leaf algae is mixed with deionized water according to a material-liquid ratio of 8-20:100 (W / W), boiled for 30-60 min, concentrated to a concentration ratio of 5-15:1 (V / V), and then 95% ethanol is added after cooling to room temperature. The application integrates the synergistic degradation of the red leaf algae and collagen, the in-situ biosynthesis of ergothioneine and ceramide, and the fermentation self-assembly of the nanocarrier into a one-step process, overcomes the defects of low delivery efficiency of the active substances prepared in steps, simple compounding and delivery efficiency in the prior art, collagen is metabolized as a co-fermentation substrate and a precursor, red algae oligosaccharides and collagen peptides generated by degradation are in-situ self-assembled with ceramide to form a core-shell nanocarrier, ergothioneine is encapsulated and stabilized, macromolecular conversion, active substance synergism and target delivery are realized, and the anti-aging protection effect is improved.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a method for preparing and applying a fermented red algae anti-aging and repairing nanocomposite. Background Technology

[0002] Red algae extract is rich in fucoidan and sulfur-containing polysaccharides, which have moisturizing and anti-aging activities. However, these substances generally have large molecular weights, making them difficult for human skin to absorb effectively, thus limiting their actual bioavailability. Collagen, as an important component of the skin matrix, is also difficult to penetrate the stratum corneum due to its large molecular structure. Current technologies mostly rely on pre-enzymatic hydrolysis or simple fermentation degradation, but the degradation products are still mostly simple mixtures without a clear delivery carrier, and the stability and targeted penetration of active ingredients have not been fundamentally improved.

[0003] While there are reports of fermenting red algae extract and collagen by co-inoculating Lactobacillus, the underlying technology remains focused on the single function of macromolecular degradation through fermentation. The resulting product is essentially a physical mixture of degraded active substances. This technology does not utilize the fermentation system as a biosynthetic reactor, failing to generate high-value-added active substances in situ during degradation. Furthermore, it does not design fermentation degradation products as carrier building blocks, making it impossible to utilize their amphiphilic structure for active substance encapsulation and delivery. Therefore, existing fermentation technologies fail to overcome the limitation of "degradation as the endpoint," and cannot simultaneously address the synergistic issues of macromolecular transformation, active substance enhancement, and delivery carrier construction within the same process. Consequently, the improvement of anti-aging efficacy still relies on the simple superposition of exogenous active substances, with fragmented process steps that lack internal synergy.

[0004] Therefore, there is an urgent need to develop a fermentation strategy that integrates the synergistic degradation of red algae and collagen, the directed biosynthesis of highly active substances, and the in-situ self-assembly of nanocarriers into a continuous one-step process. This would allow for the development and application of a fermented red algae anti-aging and repair nanocomposite, overcoming the shortcomings of existing technologies such as single fermentation function, insufficient product synergy, and low delivery efficiency. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for preparing and applying fermented red algae anti-aging and repair nanocomposite, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a fermented red algae anti-aging and repair nanocomposite, comprising the following steps: (1) Preparation of red leaf algae extract: After washing, removing impurities, drying and pulverizing the red leaf algae, mix it with deionized water at a material-to-liquid ratio of 8-20:100 (W / W), boil for 30-60 minutes, concentrate to a concentration ratio of 5-15:1 (V / V), cool to room temperature and add 95% ethanol, the volume ratio of 95% ethanol to the concentrate is 1-3:1, stir evenly and let stand overnight, filter to obtain red leaf algae extract; (2) Co-fermentation culture: The red algae extract obtained in step (1) was dissolved in deionized water at a ratio of 10-30:100 (w / w) and added to the fermenter together with the culture medium. The culture medium contained the following per liter: glucose 12-14g, beef peptone 9-10g, KCl 5-7g, ammonium nitrate 3.5-5g, KH2PO4 1-2g, MgSO4·7H2O 0.3-0.4g of phytosphingosine, 0.1-10g of collagen, 0.5-2g of L-histidine, 0.3-1g of L-cysteine, 0.1-0.5g of phytosphingosine, and 0.1-0.3g of C16-C18 fatty acids were added. The pH was adjusted to 6.0-7.0, and after sterilization, activated lactobacillus was inoculated. Fermentation was carried out at 35-38℃ and 125-150rpm for 48h. During the fermentation process, lactobacillus synthesized ergothioneine and ceramide in situ. At the same time, the macromolecular polysaccharides and collagen in the red algae extract were degraded into small molecule red algae oligosaccharides and collagen peptides. The red algae oligosaccharides, collagen peptides and ceramides self-assembled in the fermentation system to form nanocarriers. (3) Purification and nanocarrier stabilization: The fermentation broth obtained in step (2) was sterilized at high temperature of 80-100℃, cooled to room temperature and filtered. The filtrate was homogenized under high pressure and then separated and collected by an ultrafiltration membrane with a molecular weight cutoff of 10kDa to obtain the fermented red algae anti-aging and repair nanocomposite.

[0007] Preferably, in step (1), the ratio of red algae pulverizer to deionized water is 15:100 (W / W), the concentration ratio is 10:1 (V / V), and the volume ratio of 95% ethanol to concentrate is 2:1.

[0008] Preferably, in step (2), the solubility ratio of red algae extract to deionized water is 15-25:100 (W / W); the amount of L-histidine added is 0.8-1.5 g / L, the amount of L-cysteine ​​added is 0.5-0.8 g / L, the amount of phytosphingosine added is 0.2-0.4 g / L, and the amount of C16-C18 fatty acids added is 0.15-0.25 g / L.

[0009] Preferably, in step (2), the collagen is selected from one or more of the following: natural collagen, soluble collagen, hydrolyzed collagen, procollagen, and recombinant humanized collagen; the lactobacillus is selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, and Lactobacillus fermentum; and the C16-C18 fatty acid is selected from one or two of the following: palmitic acid and oleic acid.

[0010] Preferably, the fermentation conditions in step (2) are: temperature 37°C, rotation speed 145 rpm, and fermentation time 48 h.

[0011] Preferably, the pressure of high-pressure homogenization in step (3) is 500-800 bar, and the number of cycles is 3-5; the ultrafiltration process also includes a centrifugation step with a centrifugation speed of 2000-4000 rpm and a time of 10-20 min.

[0012] Preferably, the high-temperature sterilization temperature in step (3) is 85-90℃; the nanocomposite nanocarrier particle size is 50-200nm.

[0013] A fermented red algae anti-aging and repair nanocomposite is prepared by the above method. The nanocomposite contains ergothioneine, ceramide, red algae oligosaccharide and collagen peptide. The red algae oligosaccharide, collagen peptide and ceramide form a nanocarrier structure with a particle size of 50-200 nm through self-assembly.

[0014] Preferably, the ergothionein is encapsulated inside the nanocarrier, and the collagen peptide is distributed on the surface of the nanocarrier.

[0015] The above-mentioned fermented red algae anti-aging and repairing nanocomposite is used in the preparation of cosmetics, including essences, creams, lotions, masks or toners. The fermented red algae anti-aging and repairing nanocomposite is added as an anti-aging and repairing functional ingredient, with an addition amount of 0.1-10wt%.

[0016] The technical effects and advantages of this invention are as follows: 1. By integrating the preparation of red algae extract, co-fermentation with lactobacillus, in-situ biosynthesis of ergothioneine and ceramide, and self-assembly of nanocarriers into a continuous one-step process, the shortcomings of existing technologies, such as the need for separate chemical synthesis or exogenous addition of ergothioneine and ceramides followed by stepwise compounding with carrier materials, have been overcome. By using collagen as a supplementary nitrogen source and co-fermentation substrate, the co-degradation of red algae macromolecular polysaccharides and collagen, as well as the directional in-situ conversion of precursors into active ingredients, are simultaneously achieved in a single fermentation system. The reaction conditions are mild and controllable, significantly simplifying the preparation process of anti-aging and repairing active ingredients. 2. The red algae oligosaccharides and collagen peptides produced by fermentation degradation in the nanocomposite have natural amphiphilicity and can be used as biosurfactants to self-assemble with ceramides in situ in the fermentation broth to form a nanocarrier. The metabolized ergothioneine is encapsulated inside the carrier, forming a natural delivery system with a core-shell structure. This not only greatly improves the storage stability of ergothioneine and effectively inhibits its photodegradation and thermal degradation, but also enhances the penetration and targeted retention of active ingredients into the stratum corneum of the skin through the distribution modification of collagen peptides on the surface of the nanocarrier. This effectively solves the technical bottlenecks of large molecular polysaccharides and collagen being difficult to be absorbed by the skin and the easy inactivation of active substances. 3. The fermented red algae anti-aging and repairing nanocomplex is used as a cosmetic active ingredient, which has multiple effects such as moisturizing, anti-oxidation and barrier repair. Ergothioneine and ceramide form a deep synergistic system through metabolic coupling and structural self-assembly during the fermentation process, making the anti-aging and repair effect significantly better than any single active ingredient or simple compound system. It can provide synergistic strengthening effect for improving fine lines, enhancing elasticity and repairing barrier function in mature skin. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the method flow structure of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] As attached Figure 1 As shown, the present invention provides a method for preparing fermented red algae anti-aging and repair nanocomposite, including three main steps: preparation of red algae extract, synergistic fermentation culture, and purification and nanocarrier stabilization.

[0020] In the preparation steps of red leaf algae extract, the red leaf algae is washed, cleaned, dried, and pulverized. It is then mixed with deionized water at a mass ratio of 8-20:100, boiled for 30-60 minutes, concentrated to a volume ratio of 5-15:1, cooled, and 95% ethanol is added at a volume ratio of 1-3:1. The mixture is allowed to stand overnight and then filtered to obtain the red leaf algae extract.

[0021] In the co-fermentation culture step, the red algae extract was dissolved in deionized water at a mass ratio of 10-30:100 and added to the fermenter along with the culture medium. Each liter of the culture medium contained: 12-14g glucose, 9-10g beef peptone, 5-7g KCl, 3.5-5g ammonium nitrate, 1-2g KH₂PO₄, 0.3-0.4g MgSO₄·7H₂O, 0.1-10g collagen, 0.5-2g L-histidine, 0.3-1g L-cysteine, 0.1-0.5g phytosphingosine, and 0.1-0.3g C16-C18 fatty acids. The pH was adjusted to 6.0-7.0, and after sterilization, activated lactic acid bacteria were inoculated. Fermentation was carried out at 35-38℃ and 125-150 rpm for 48 hours. During fermentation, lactobacillus synthesizes ergothioneine in situ using L-histidine and L-cysteine, and ceramides in situ using phytosphingosine and fatty acids. Meanwhile, the macromolecular polysaccharides and collagen in the red algae extract are degraded into small molecule red algae oligosaccharides and collagen peptides. The red algae oligosaccharides, collagen peptides and ceramides self-assemble in the fermentation system to form nanocarriers.

[0022] In the purification step, the fermentation broth is sterilized at high temperature of 80-100℃, cooled and filtered, the filtrate is homogenized under high pressure, and then separated by an ultrafiltration membrane with a molecular weight cutoff of 10kDa. The nanocarrier components are collected to obtain the fermented red algae anti-aging and repair nanocomposite.

[0023] In the fermentation system of this invention, 0-12h is the adaptation period of Lactobacillus, which initiates the degradation of red algae polysaccharides and collagen; 12-24h is the logarithmic growth period, during which a large amount of lipase and amidase are produced, catalyzing the synthesis of ceramide from phytosphingosine and fatty acids, while ergothionein is synthesized in situ using sulfur sources provided by L-histidine, L-cysteine, and red algae sulfur-containing polysaccharides; 24-36h is the stationary period, during which the degraded red algae oligosaccharides and collagen peptides self-assemble with ceramide to form a nanocarrier, encapsulating ergothionein inside the carrier; 36-48h is the decay period, during which intracellular ergothionein is released and captured by the nanocarrier, completing the comprehensive enrichment and structural fixation of the active ingredient.

[0024] In the nanocarrier of this invention, the red algae oligosaccharide contains sulfate groups, which causes it to shrink in the weakly acidic environment of the skin surface and swell to release active ingredients when the pH of the inflamed or aging area increases, thus achieving microenvironment-responsive release. At the same time, the collagen peptides distributed on the surface of the carrier contain basic amino acid residues, which can synergistically promote the targeted enrichment of ergothionein to mitochondria through organic cation transport proteins, thereby enhancing the antioxidant effect.

[0025] Example 1 Wash the red algae to remove mud and sand, dry it naturally, and then pulverize it. Mix it with deionized water at a mass ratio of 15:100, boil for 45 minutes, concentrate it to a volume ratio of 10:1, cool it to room temperature, add 95% ethanol at a volume ratio of 2:1, stir well, let it stand overnight, and filter to obtain the red algae extract.

[0026] The above-mentioned red algae extract was dissolved in deionized water at a mass ratio of 20:100 and added to the fermenter along with the culture medium. Each liter of the culture medium contained: 13g glucose, 9.5g beef peptone, 6g KCl, 4g ammonium nitrate, 1.5g KH₂PO₄, 0.35g MgSO₄·7H₂O, 0.5g recombinant humanized collagen, 1g L-histidine, 0.6g L-cysteine, 0.3g phytosphingosine, and 0.2g palmitic acid. The pH was adjusted to 6.5, and after sterilization, activated *Lactobacillus plantarum* was inoculated. Fermentation was carried out at 37℃ and 145 rpm for 48 hours.

[0027] After fermentation, the fermentation broth was sterilized at 85°C, cooled to room temperature and filtered. The filtrate was homogenized under high pressure at 600 bar for 4 cycles. Then it was centrifuged at 3500 rpm for 15 min, and the supernatant was separated by a 10 kDa ultrafiltration membrane to collect the retained components.

[0028] Example 2 Preparation of red algae extract: material-to-liquid ratio 10:100, boiling for 40 min, concentration ratio 8:1, ethanol to concentrate volume ratio 3:1.

[0029] Co-fermentation culture: The ratio of red algae extract to deionized water was 15:100. Each liter of culture medium contained: glucose 12g, beef peptone 9g, KCl 5g, ammonium nitrate 3.5g, KH₂PO₄ 1g, MgSO₄·7H₂O 0.3g, soluble collagen 0.1g, L-histidine 0.8g, L-cysteine ​​0.5g, phytosphingosine 0.2g, and oleic acid 0.15g. The pH was adjusted to 6.0, and after sterilization, the medium was inoculated with *Lactobacillus acidophilus* and fermented at 35℃ and 125 rpm for 48 hours.

[0030] Purification: The fermentation broth was sterilized at 90°C, cooled and filtered. The filtrate was homogenized under high pressure at 500 bar, circulated 3 times, and then centrifuged at 2000 rpm for 20 min. The supernatant was taken and ultrafiltered at 10 kDa, and the retained fraction was collected.

[0031] Example 3 Preparation of red algae extract: material-to-liquid ratio 20:100, boiling for 60 min, concentration ratio 15:1, ethanol to concentrate volume ratio 1:1.

[0032] Co-fermentation culture: The ratio of red algae extract to deionized water was 25:100. Each liter of culture medium contained: 14g glucose, 10g beef peptone, 7g KCl, 5g ammonium nitrate, 2g KH₂PO₄, 0.4g MgSO₄·7H₂O, 10g hydrolyzed collagen, 1.5g L-histidine, 0.8g L-cysteine, 0.4g phytosphoin, and 0.15g each of palmitic acid and oleic acid. The pH was adjusted to 7.0, and after sterilization, the medium was inoculated with *Lactobacillus bulgaricus* and fermented at 38℃ and 150 rpm for 48 hours.

[0033] Purification: The fermentation broth was sterilized at 80°C, cooled and filtered. The filtrate was homogenized under high pressure at 800 bar, circulated 5 times, and then centrifuged at 4000 rpm for 10 min. The supernatant was taken and ultrafiltered at 10 kDa, and the retained fraction was collected.

[0034] Example 4 Preparation of red algae extract: material-to-liquid ratio 8:100, boiling for 30 min, concentration ratio 5:1, ethanol to concentrate volume ratio 1:1.

[0035] Co-fermentation culture: The ratio of red algae extract to deionized water was 10:100. Each liter of culture medium contained: glucose 12g, beef peptone 9g, KCl 5g, ammonium nitrate 3.5g, KH₂PO₄ 1g, MgSO₄·7H₂O 0.3g, procollagen 0.1g, L-histidine 0.5g, L-cysteine ​​0.3g, phytosphoprotein 0.1g, and palmitic acid 0.1g. The pH was adjusted to 6.0, and after sterilization, the medium was inoculated with *Lactobacillus fermentum* and fermented at 35℃ and 125 rpm for 48 hours.

[0036] Purification: The fermentation broth was sterilized at 100℃, cooled and filtered. The filtrate was homogenized under high pressure at 500 bar, circulated 3 times, and then centrifuged at 2000 rpm for 20 min. The supernatant was taken and ultrafiltered at 10 kDa, and the retained fraction was collected.

[0037] Example 5 Preparation of red algae extract: material-to-liquid ratio 20:100, boiling for 60 min, concentration ratio 15:1, ethanol to concentrate volume ratio 3:1.

[0038] Co-fermentation culture: The ratio of red algae extract to deionized water was 30:100. Each liter of culture medium contained: glucose 14g, beef peptone 10g, KCl 7g, ammonium nitrate 5g, KH₂PO₄ 2g, MgSO₄·7H₂O 0.4g, collagen 8g, L-histidine 2g, L-cysteine ​​1g, phytosphoprotein 0.5g, and oleic acid 0.3g. The pH was adjusted to 7.0, and after sterilization, a mixed inoculum of *Lactobacillus plantarum* and *Lactobacillus fermentum* was inoculated. Fermentation was carried out at 38℃ and 150 rpm for 48 hours.

[0039] Purification: The fermentation broth was sterilized at 90°C, cooled and filtered. The filtrate was homogenized under high pressure at 800 bar, circulated 5 times, and then centrifuged at 4000 rpm for 10 min. The supernatant was taken and ultrafiltered at 10 kDa, and the retained fraction was collected.

[0040] Example 6 The preparation and fermentation conditions of the red algae extract were the same as in Example 1. The culture medium contained, per liter: glucose 13g, beef peptone 9.5g, KCl 6g, ammonium nitrate 4g, KH₂PO₄ 1.5g, MgSO₄·7H₂O 0.35g, recombinant humanized collagen 2g, L-histidine 1g, L-cysteine ​​0.6g, phytosphingosine 0.3g, and palmitic acid 0.2g. Purification conditions were the same as in Example 1.

[0041] Example 7 The preparation and fermentation conditions of the red algae extract were the same as in Example 1. The culture medium contained, per liter: glucose 13g, beef peptone 9.5g, KCl 6g, ammonium nitrate 4g, KH₂PO₄ 1.5g, MgSO₄·7H₂O 0.35g, recombinant humanized collagen 5g, L-histidine 1g, L-cysteine ​​0.6g, phytosphingosine 0.3g, and palmitic acid 0.2g. Purification conditions were the same as in Example 1.

[0042] Example 8 Preparation of red algae extract: material-to-liquid ratio 18:100, boiling for 50 min, concentration ratio 12:1, ethanol to concentrate volume ratio 2.5:1.

[0043] Co-fermentation culture: The ratio of red algae extract to deionized water was 28:100. Each liter of culture medium contained: glucose 13g, beef peptone 9.5g, KCl 6g, ammonium nitrate 4.5g, KH₂PO₄ 1.8g, MgSO₄·7H₂O 0.38g, recombinant humanized collagen 1g, L-histidine 1.2g, L-cysteine ​​0.7g, phytosphoprotein 0.35g, and palmitic acid 0.25g. The pH was adjusted to 6.8, and after sterilization, *Lactobacillus plantarum* was inoculated. Fermentation was carried out at 36℃ and 140 rpm for 48 hours.

[0044] Purification: The fermentation broth was sterilized at 88°C, cooled and filtered. The filtrate was homogenized under high pressure at 700 bar, circulated 4 times, and then centrifuged at 3000 rpm for 12 min. The supernatant was taken and ultrafiltered at 10 kDa, and the retained fraction was collected.

[0045] Comparative Example 1 Compared to Example 1, collagen, L-histidine, L-cysteine, phytosphoprotein, and palmitic acid were not added to the culture medium, while all other conditions were exactly the same.

[0046] Comparative Example 2 Compared with Example 1, the following changes were made to the preparation steps of the red algae extract: the material-to-liquid ratio was changed to 35:100, boiling was performed for 120 min, the concentration ratio was 30:1, and the volume ratio of ethanol to concentrate was 5:1; in the fermentation culture step, the ratio of red algae extract to deionized water was changed to 5:100, the amount of collagen added was changed to 20 g / L, the fermentation temperature was 38℃, and the rotation speed was 150 rpm; in the purification step, the sterilization temperature was 121℃. All other conditions were the same as in Example 1.

[0047] Comparative Example 3 Only red algae extract was prepared without fermentation. Red algae were mixed with deionized water at a ratio of 15:100, boiled for 45 minutes, concentrated at a ratio of 10:1, and the volume ratio of ethanol to concentrate was 2:1. The extract was then filtered to obtain the red algae extract. Subsequently, it was dissolved in deionized water at a ratio of 20:100, and the same culture medium as in Example 1 was added but without inoculation. The mixture was directly sterilized at 85°C, cooled, filtered, homogenized under high pressure, centrifuged, and ultrafiltered.

[0048] Comparative Example 4 Compared to Example 1, only 0.5 g / L of recombinant humanized collagen was retained in the culture medium, and L-histidine, L-cysteine, phytosphoprotein and palmitic acid were not added; all other conditions were exactly the same.

[0049] Comparative Example 5 Compared to Example 1, the culture medium contained 1 g / L L-histidine and 0.6 g / L L-cysteine, but no collagen, phytosphingosine, or palmitic acid were added; all other conditions were exactly the same.

[0050] Comparative Example 6 Compared with Example 1, the culture medium was supplemented with 0.3 g / L phytosphingosine and 0.2 g / L palmitic acid, but without the addition of collagen, L-histidine and L-cysteine; all other conditions were exactly the same.

[0051] Comparative Example 7 Compared with Example 1, the culture medium was supplemented with 0.5 g / L of recombinant humanized collagen, 1 g / L of L-histidine, and 0.6 g / L of L-cysteine, but phytosphoprotein and palmitic acid were not added; all other conditions were exactly the same.

[0052] Comparative Example 8 Compared with Example 1, the culture medium was supplemented with 0.5 g / L of recombinant humanized collagen, 0.3 g / L of phytosphingosine, and 0.2 g / L of palmitic acid, but L-histidine and L-cysteine ​​were not added; all other conditions were exactly the same.

[0053] Comparative Example 9 Compared to Example 1, no bacterial strains were inoculated, but all other conditions were exactly the same.

[0054] Comparative Example 10 The original technical solution was adopted: the preparation of the red algae extract was the same as in Example 1. The fermentation medium contained 13g of glucose, 9.5g of beef peptone, 6g of KCl, 4g of ammonium nitrate, 1.5g of KH2PO4, 0.35g of MgSO4·7H2O, and 1g of hydrolyzed collagen per liter. No additional L-histidine, L-cysteine, phytosphoin, or fatty acids were added. Lactobacillus plantarum was inoculated and fermented at 37℃ and 145rpm for 48h. The purification conditions were the same as in Example 1.

[0055] Effect verification Particle size determination: The particle size of the products of each example and comparative example was determined by dynamic light scattering method. The results showed that the particle sizes of the nanocomposites of Examples 1 to 8 were 85 nm, 52 nm, 150 nm, 95 nm, 118 nm, 110 nm, 135 nm, and 105 nm, respectively, all within the range of 50-200 nm. Comparative Example 1 did not form a nanocarrier and the particle size was greater than 1000 nm; Comparative Example 2 had uneven particle size due to excessive degradation, with an average particle size of 320 nm and a distribution coefficient greater than 0.5; Comparative Example 3 did not form a nanocarrier structure; Comparative Examples 4 to 6 did not form a stable nanocarrier; Comparative Example 7 had a particle size of 45 nm; Comparative Example 8 had a particle size of 78 nm; Comparative Example 9 did not form a nanocarrier; Comparative Example 10 had a particle size of 180 nm but a distribution coefficient greater than 0.4.

[0056] Ergothioneine content detection: High performance liquid chromatography (HPLC) was used for detection. The ergothioneine contents of Examples 1 to 8 were 18.5 μg / mL, 8.2 μg / mL, 22.1 μg / mL, 6.5 μg / mL, 19.2 μg / mL, 20.3 μg / mL, 21.8 μg / mL, and 17.6 μg / mL, respectively. Ergothioneine was not detected in Comparative Example 1, Comparative Example 3, Comparative Example 4, Comparative Example 5 (15.2 μg / mL), Comparative Example 6, Comparative Example 7, Comparative Example 8, Comparative Example 9, and Comparative Example 10.

[0057] Ceramide content detection: Ceramide content was determined using liquid chromatography-mass spectrometry (LC-MS). The ceramide contents of Examples 1 to 8 were 25.3 μg / mL, 10.5 μg / mL, 28.6 μg / mL, 8.3 μg / mL, 26.1 μg / mL, 27.2 μg / mL, 28.0 μg / mL, and 24.1 μg / mL, respectively. Ceramide was not detected in Comparative Example 1; not detected in Comparative Example 3; not detected in Comparative Example 4; not detected in Comparative Example 5; not detected in Comparative Example 6 (18.6 μg / mL); not detected in Comparative Example 7; not detected in Comparative Example 8 (22.4 μg / mL); not detected in Comparative Example 9; not detected in Comparative Example 10.

[0058] Nanocarrier structure characterization: The morphology of the nanocomposite obtained in Example 1 was observed using transmission electron microscopy. The results showed that the product had a spherical core-shell structure with a particle size of approximately 85 nm. X-ray photoelectron spectroscopy was used to detect the surface elemental composition, and it was found that the nitrogen and sulfur contents were significantly higher than those inside, indicating that collagen peptides rich in amino acid residues were distributed on the surface of the nanocarrier. Observation by fluorescent probe labeling combined with laser confocal microscopy showed that the fluorescence signal of ergothionein was concentrated in the internal region of the nanocarrier, confirming that ergothionein was encapsulated inside the nanocarrier.

[0059] The effect of collagen gradient on the formation of nanocomposites: The products obtained in Examples 2, 1, 6, 7, 5 and 3 were subjected to particle size and encapsulation efficiency testing; the particle size was determined by dynamic light scattering method and the encapsulation efficiency of ergothioneine was determined by ultrafiltration centrifugation method.

[0060] Example 2: Collagen 0.1 g / L: particle size 52 nm, ergothioneine encapsulation efficiency 38.2%.

[0061] Example 1: Collagen 0.5 g / L: particle size 85 nm, ergothioneine encapsulation rate 62.5%.

[0062] Example 6: Collagen 2g / L: Particle size 110nm, ergothioneine encapsulation rate 71.3%.

[0063] Example 7: Collagen 5g / L: Particle size 135nm, ergothioneine encapsulation rate 68.9%.

[0064] Example 5: Collagen 8 g / L: Particle size 118 nm, ergothioneine encapsulation rate 65.4%.

[0065] Example 3: Collagen 10g / L: Particle size 150nm, ergothioneine encapsulation rate 58.7%.

[0066] The results showed that collagen additions in the range of 0.1-10 g / L could form nanocarriers of 50-200 nm, with encapsulation efficiency exceeding 60% in the 0.5-8 g / L range and exceeding 68% in the 2-5 g / L range.

[0067] Cosmetic application and efficacy testing: The products obtained from Examples 1 to 5 and Comparative Examples 1 to 10 were added to the basic serum formula at 3% each. The content of each component in the basic serum formula is expressed as a percentage by mass, specifically: deionized water to make up 100%, EDTA-2Na 0.03%, carbomer 0.15%, phenoxyethanol 0.7%, glycerin 4%, butylene glycol 3%, and tromethamine 0.15%.

[0068] Fifty healthy adult female volunteers were recruited and randomly divided into five groups of ten each. The treatment lasted for 28 consecutive days. Skin hydration, elasticity, texture, and transepidermal water loss were measured on days 0 and 28. The rate of change was calculated by subtracting the day 0 data from the day 28 data, dividing by the day 0 data, and then multiplying by 100%.

[0069] Table 1 Comparison of synergistic effects:

[0070] Note: A negative skin texture improvement rate indicates a decrease in the VISIA texture score; the larger the absolute value, the more significant the improvement.

[0071] Results analysis: As shown in Table 1, the comparative examples 4 (with only collagen), 5 (with only ergothioneine precursor), and 6 (with only ceramide precursor) did not form a stable nanocarrier structure due to the lack of a synergistic fermentation system, and their skin efficacy indicators were significantly lower than those of Example 1. Comparative Example 7 added collagen and ergothioneine precursor, which can synthesize ergothioneine in situ and form smaller particle size. However, due to the lack of ceramide precursor, it cannot form a complete lipid nanocarrier framework, resulting in insufficient encapsulation rate and skin permeability. Its efficacy is only about 60% of that of Example 1. Comparative Example 8 added collagen and ceramide precursors, which can form nanocarriers, but due to the lack of ergothioneine precursor, there is no encapsulation of antioxidant active ingredients, and the efficacy is only about 65% of that of Example 1. Example 1 contains collagen, ergothioneine precursor, and ceramide precursor. During fermentation, macromolecular degradation, in-situ synthesis of ergothioneine, in-situ synthesis of ceramide, and self-assembly of nanocarriers are achieved simultaneously. The four components work synergistically, and the skin's moisture content, elasticity, texture, and barrier repair indicators are significantly better than those of the comparative examples of each non-full component, proving that the technical solution of the present invention has a significant synergistic effect.

[0072] The above examples and comparative examples show that the objective of this invention can be achieved with collagen addition ranging from 0.1 to 10 g / L, with 0.5 to 8 g / L representing a range with high encapsulation efficiency. Furthermore, only under the fully integrated synergistic fermentation system described in this invention can the in-situ synthesis of ergothioneine and ceramides, as well as the effective self-assembly of the nanocarrier, be simultaneously realized, demonstrating a significant synergistic effect among the components.

[0073] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a fermented red algae anti-aging and repair nanocomposite, characterized in that, Includes the following steps: (1) Preparation of red leaf algae extract: After washing, removing impurities, drying and pulverizing the red leaf algae, mix it with deionized water at a material-to-liquid ratio of 8-20:100 (W / W), boil for 30-60 minutes, concentrate to a concentration ratio of 5-15:1 (V / V), cool to room temperature and add 95% ethanol, the volume ratio of 95% ethanol to the concentrate is 1-3:1, stir evenly and let stand overnight, filter to obtain red leaf algae extract; (2) Co-fermentation culture: The red algae extract obtained in step (1) was dissolved in deionized water at a ratio of 10-30:100 (w / w) and added to the fermenter together with the culture medium. The culture medium contained the following per liter: glucose 12-14g, beef peptone 9-10g, KCl 5-7g, ammonium nitrate 3.5-5g, KH2PO4 1-2g, MgSO4·7H2O 0.3-0.4g of phytosphingosine, 0.1-10g of collagen, 0.5-2g of L-histidine, 0.3-1g of L-cysteine, 0.1-0.5g of phytosphingosine, and 0.1-0.3g of C16-C18 fatty acids were added. The pH was adjusted to 6.0-7.0, and after sterilization, activated lactobacillus was inoculated. Fermentation was carried out at 35-38℃ and 125-150rpm for 48h. During the fermentation process, lactobacillus synthesized ergothioneine and ceramide in situ. At the same time, the macromolecular polysaccharides and collagen in the red algae extract were degraded into small molecule red algae oligosaccharides and collagen peptides. The red algae oligosaccharides, collagen peptides and ceramides self-assembled in the fermentation system to form nanocarriers. (3) Purification and nanocarrier stabilization: The fermentation broth obtained in step (2) was sterilized at high temperature of 80-100℃, cooled to room temperature and filtered. The filtrate was homogenized under high pressure and then separated and collected by an ultrafiltration membrane with a molecular weight cutoff of 10kDa to obtain the fermented red algae anti-aging and repair nanocomposite.

2. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: In step (1), the ratio of red algae powder to deionized water is 15:100 (W / W), the concentration ratio is 10:1 (V / V), and the volume ratio of 95% ethanol to concentrate is 2:

1.

3. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: In step (2), the ratio of red algae extract to deionized water is 15-25:100 (W / W); the amount of L-histidine added is 0.8-1.5 g / L, the amount of L-cysteine ​​added is 0.5-0.8 g / L, the amount of phytosphingosine added is 0.2-0.4 g / L, and the amount of C16-C18 fatty acids added is 0.15-0.25 g / L.

4. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: In step (2), the collagen is selected from one or more of the following: natural collagen, soluble collagen, hydrolyzed collagen, procollagen, and recombinant humanized collagen; the lactobacillus is selected from one or more of the following: Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, and Lactobacillus fermentum; and the C16-C18 fatty acid is selected from one or two of the following: palmitic acid and oleic acid.

5. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: The fermentation conditions in step (2) are: temperature 37℃, rotation speed 145rpm, and fermentation time 48h.

6. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: In step (3), the pressure of high-pressure homogenization is 500-800 bar, and the number of cycles is 3-5. Before ultrafiltration, there is also a centrifugation step with a centrifugation speed of 2000-4000 rpm and a time of 10-20 min.

7. The method for preparing a fermented red algae anti-aging and repair nanocomposite according to claim 1, characterized in that: The high-temperature sterilization temperature in step (3) is 85-90℃; the nanocarrier particle size of the nanocomposite is 50-200nm.

8. A fermented red algae anti-aging and repair nanocomposite, characterized in that, Prepared by the method of any one of claims 1 to 7, the nanocomposite comprises ergothioneine, ceramide, red algae oligosaccharide and collagen peptide, wherein the red algae oligosaccharide, collagen peptide and ceramide form a nanocarrier structure with a particle size of 50-200 nm through self-assembly.

9. The fermented red algae anti-aging and repair nanocomposite according to claim 8, characterized in that: The ergothionein is encapsulated inside the nanocarrier, and the collagen peptides are distributed on the surface of the nanocarrier.

10. The application of the fermented red algae anti-aging and repairing nanocomposite according to claim 8 in the preparation of cosmetics, characterized in that: The cosmetics include serums, creams, lotions, masks, or toners, and the fermented red algae anti-aging and repairing nanocomplex is added as an anti-aging and repairing active ingredient in an amount of 0.1-10 wt%.

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