Rice fermentation liquor for enhancing energy of skin cells and mitochondria as well as preparation method and application of rice fermentation liquor

Abstract

The invention discloses rice fermentation liquor for enhancing energy of skin cells and mitochondria as well as a preparation method and application of the rice fermentation liquor. The preparation method comprises the following steps: S1, enzymolysis: preparing rice milk, and adding a set amount of beta-glucanase for hydrolysis so as to release saccharides and oligosaccharides; step S2, a fermentation step: adding saccharomycetes Saccharomyces veronae and Lactiplantibacillus plantarum for co-culture, so that micro-aerobic or intermittent aeration is kept in a fermentation space, and nucleosides can be generated in a metabolic pathway; s3, a separation and enrichment step: performing filtration and screening to retain small molecule nucleoside / nucleotide; and then through selective enrichment, nucleoside enriched components are collected. According to the rice fermentation liquor for enhancing the energy of the skin cells and mitochondria and the preparation method and application thereof, the energy of the cells and the mitochondria can be improved through the rice fermentation liquor, the bioavailability of nutrient substances of the fermentation liquor is improved, and it is guaranteed that functional components penetrate into the submuscular layer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cosmetic ingredients, and relates to a rice fermentation liquor, in particular to a rice fermentation liquor for enhancing skin cell and mitochondrial energy, a preparation method thereof and application. BACKGROUND

[0002] Skin aging is divided into endogenous aging caused by age growth and exogenous aging caused by factors such as sun exposure, environmental pollution, smoking and lifestyle. These aging phenotypes include wrinkles, hair whitening and shedding, uneven pigmentation and slow wound healing. Aging is closely related to mitochondrial DNA damage. With the deepening of anti-aging research, it has been found that mitochondria in cells are the key factors affecting human aging. Skin aging is closely related to mitochondria. Mitochondria are organelles that exist in most cells, are wrapped by two membranes, and have a diameter of about 0.5-10 microns. In addition to participating in processes such as cell differentiation, cell information transmission and cell apoptosis, mitochondria are also important places for oxidative phosphorylation and ATP in cells. Mitochondria can improve the body's antioxidant capacity, inhibit the oxidative stress response of active oxygen, maintain the normal differentiation and growth of human cells, and further protect the cells in the human body and prevent aging.

[0003] As the energy center of cells, mitochondria are responsible for producing most of the energy molecules required by cells, adenosine triphosphate (Adenosine triphosphate, ATP). Adenosine triphosphate is a nucleotide that acts as a "molecular currency" for energy transfer within cells, responsible for storing and transferring chemical energy, and also plays an important role in nucleic acid synthesis. ATP transports chemical energy within cells to promote metabolism, and is one of the final products of photophosphorylation, aerobic respiration and fermentation, and participates in many cellular processes, including biosynthetic reactions, cell activity and cell division. As the direct source of energy for various life activities in the body, an increase in the content of ATP in cells can promote cell repair and regeneration, and the level of cell ATP content directly reflects the vitality of cells.

[0004] NAD+(Nicotinamide adenine dinucleotide) is the full name of "nicotinamide adenine dinucleotide" in Chinese, and is also called coenzyme I. It is a hydrogen ion coenzyme that appears in multiple metabolic reactions in cells and can convert ADP to ATP through chemical osmotic coupling in the electron transport chain.

[0005] The existing invention related to mitochondrial energy in the field of cosmetics mainly adds the precursor of NAD+, such as reduced nicotinamide adenine dinucleotide (NADH), so as to achieve the effects of anti-oxidation and anti-aging. NADH has a large molecular weight and is difficult to be directly absorbed by the human body, so the bioavailability is low.

[0006] The existing anti-aging functional components such as retinol have the disadvantages of strong irritation, poor stability, low bioavailability, limited application population, poor product stability, easy influence by environmental factors and use conditions, and inability to guarantee the product efficacy, so the liposome wrapping technology is often used for modification, and the production process is complex and the cost is high.

[0007] Therefore, there is an urgent need to design a new cosmetic component capable of enhancing mitochondrial energy in order to overcome at least part of the above-mentioned defects of the existing cosmetic components. SUMMARY

[0008] The present application provides a rice fermentation liquid for enhancing skin cell and mitochondrial energy, a preparation method and application thereof. The rice fermentation liquid can enhance cell and mitochondrial energy, improve the bioavailability of nutrients in the fermentation liquid, and ensure that the functional components reach the dermal layer.

[0009] To solve the above technical problems, according to one aspect of the present application, the following technical solution is adopted:

[0010] A preparation method of a rice fermentation liquid for enhancing skin cell and mitochondrial energy, the preparation method comprising:

[0011] Step S1, enzymolysis step: preparing rice slurry, adding a certain amount of beta-glucanase for hydrolysis to release saccharides and oligosaccharides;

[0012] Step S2, fermentation step: adding Saccharomyces veronae and Lactiplantibacillus plantarum for co-culture, keeping the fermentation space in micro-aerobic or intermittent aeration to facilitate the production of nucleosides in the metabolic pathway;

[0013] Step S3, separation and enrichment step: retaining small molecule nucleosides / nucleotides by filtration and screening, and then collecting nucleoside-enriched components by selective enrichment.

[0014] As an embodiment of the present application, in step S1, the mass concentration of the rice slurry is 10-60%, the enzymolysis temperature is 55-70°C, and the enzymolysis time is 4-10h.

[0015] As an embodiment of the present application, in step S1, the dosage of beta-glucanase accounts for 0.001-1% of the mass of the rice slurry.

[0016] In one embodiment of the present invention, in step S2, the volume ratio of yeast Saccharomyces veronae to Lactiplantibacillus plantarum is 3:1 to 8:1, and the mixture is inoculated.

[0017] In one embodiment of the present invention, in step S2, the fermentation conditions are: temperature of 30-34°C, pH value controlled at 4.5-6.5, and fermentation time of 24-72 hours; the aeration rate of the fermentation space is controlled to maintain microaerophilicity or intermittent aeration in the fermentation space, so as to facilitate the production of nucleosides by metabolic pathways.

[0018] As one embodiment of the present invention, step S3 includes:

[0019] Step S3A, Primary Decellularization and Coarse Filtration: Remove solids by mechanical filtration of 0.5–5 μm.

[0020] Step S3B, Small molecule sieving step: Use an ultrafiltration membrane with a cutoff of 0.5-1.5 kDa to remove large peptides / proteins and retain small molecule nucleosides / nucleotides; retain the permeate;

[0021] Step S3C, Selective Enrichment Step: The permeate is loaded into a weak cation exchange resin (Amberlite IRC-50 type) at pH 4.5-5.0, and then eluted sequentially with 2-4 BV of deionized water and 10-40 mM NaCl solution to collect the fraction rich in nucleosides.

[0022] As one embodiment of the present invention, the preparation method further includes a raw material pretreatment step before step S1: washing, removing impurities, drying, crushing and sieving rice.

[0023] According to another aspect of the present invention, the following technical solution is adopted: a rice fermentation broth that enhances the energy of skin cells and mitochondria, prepared by the above preparation method.

[0024] According to another aspect of the present invention, the following technical solution is adopted: the application of the above-mentioned rice fermentation liquid that enhances the energy of skin cells and mitochondria in the preparation of skin care products.

[0025] The beneficial effects of this invention are as follows: The rice fermentation liquid, its preparation method, and its application, which enhance the energy of skin cells and mitochondria, can fill the gap in the application of rice fermentation liquid in the field of skin care by improving cell and mitochondrial energy. The rice fermentation liquid in this invention has a synergistic penetration-enhancing effect, improving the bioavailability of nutrients in the fermentation liquid and ensuring that the active ingredients penetrate deep into the skin's basal layer. The rice fermentation liquid in this invention can indirectly increase NAD+ levels by replenishing cellular energy. It has a mild composition and good stability, making it suitable for various cosmetic formulations. It is also less affected by environmental factors and usage conditions, and the preparation process is relatively simple.

[0026] This invention employs a dual-strain co-culture system with a complementary relationship between nucleoside synthesis and lactic acid metabolism. By controlling fermentation conditions to create a specific microaerophilic environment, it is the first to combine ion exchange, ultrafiltration, and low-temperature concentration for the screening and enrichment of small molecules in rice fermentation broth, achieving highly efficient retention and concentration of thermosensitive small molecules such as NR / NMN. Compared with traditional reverse osmosis concentration processes, this enrichment method effectively removes polysaccharides and large peptide impurities, avoids activity loss, and significantly increases NAD5 content in the resulting product. + Precursor molecule content.

[0027] The fermentation target of this invention is no longer limited to "total active substances" or "moisturizing factors", but rather to NAD. + The precursor is product-oriented, and a closed-loop control route has been established from substrate design and strain selection to metabolic enrichment.

[0028] The process of this invention does not require special rice varieties or proprietary strains. All strains are safe strains that can be obtained industrially. The process conditions are mild, stable, and scalable. The enriched products can be directly applied to various cosmetic formulations such as serums, freeze-dried powders, and soluble microneedle patches, and have excellent process compatibility and commercial application potential. Attached Figure Description

[0029] Figure 1 This is a flowchart of a method for preparing rice fermentation broth in one embodiment of the present invention.

[0030] Figure 2 This is a schematic diagram of mitochondrial morphology test results in one embodiment of the present invention. Detailed Implementation

[0031] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0032] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention.

[0033] The description in this section pertains to only a few typical embodiments, and the present invention is not limited to the scope of the embodiments described. Substitution of identical or similar prior art methods with some technical features in the embodiments is also within the scope of the description and protection of this invention.

[0034] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions disclosed in these documents concerning synthetic techniques, product and processing design, polymers, comonomers, initiators, or catalysts in the art. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0035] The numerical ranges in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. A numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided there is an interval of at least two units between any lower and any higher value. For example, if a component, physical, or other property (such as molecular weight, melt index, etc.) is described as 100 to 1000, this means that all individual values, such as 100, 101, 102, etc., are explicitly listed, as well as all subranges, such as 100 to 166, 155 to 170, 198 to 200, etc. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly stated in this application. It should also be noted that the terms "first," "second," etc., used herein are not intended to specify a particular order, but are merely used to distinguish substances with different structures.

[0036] When referring to chemical compounds, unless explicitly stated otherwise, the singular includes all isomers and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). Additionally, unless explicitly stated otherwise, nouns described with "an," "a," or "the" also include their plural forms.

[0037] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0038] The steps described in the various embodiments in the specification are for illustrative purposes only, and the implementation of this application is not limited by the order of the steps.

[0039] This invention discloses a method for preparing rice fermentation broth that enhances the energy of skin cells and mitochondria. Figure 1 This is a flowchart of a method for preparing rice fermentation broth according to an embodiment of the present invention; please refer to [link / reference]. Figure 1 The preparation method includes:

[0040]

Step S1

[0041] In one embodiment of the present invention, in step S1, the mass concentration of rice slurry is 10-60%, the enzymatic hydrolysis temperature is 55-70°C, and the enzymatic hydrolysis time is 4-10 h; the addition of β-glucanase accounts for 0.001-1% of the mass of rice slurry.

[0042]

Step S2

[0043] In one embodiment of the present invention, in step S2, the volume ratio of yeast *Saccharomyces veronae* to *Lactiplantibacillus plantarum* is 3:1 to 8:1, and they are mixed and inoculated. In one embodiment, the volume ratio of yeast *Saccharomyces veronae* to *Lactiplantibacillus plantarum* is 5:1, and they are mixed and inoculated. Fermentation conditions: temperature 30–34°C, pH value controlled at 4.5–6.5, fermentation time 24–72 h; the aeration rate of the fermentation space is controlled to maintain microaerophilicity or intermittent aeration in the fermentation space to facilitate the production of nucleosides through metabolic pathways.

[0044]

Step S3

[0045] In one embodiment of the present invention, step S3 is a post-fermentation treatment step for enriching NR / NMN small molecules; step S3 includes:

[0046] Step S3A, Primary Decellularization and Coarse Filtration: Remove solids by mechanical filtration of 0.5–5 μm.

[0047] Step S3B, Small molecule sieving step: Use an ultrafiltration membrane with a cutoff of 0.5-1.5 kDa (e.g., 0.5 kDa, 1 kDa, 1.5 kDa) to remove large peptides / proteins and retain small molecule nucleosides / nucleotides; retain the permeate;

[0048] Step S3C, Selective Enrichment Step: The permeate is loaded into a weak cation exchange resin (Amberlite IRC-50 type) at pH 4.5-5.0, and then eluted sequentially with 2-4 BV of deionized water and 10-40 mM NaCl solution to collect the fraction rich in nucleosides.

[0049] Step S3C, Selective Enrichment Step: Ion Exchange / Weak Ion Exchange Resin Adsorption. The permeate is loaded onto a weak cation exchange resin (Amberlite IRC-50 type) at a pH of 4.5–5.0 (e.g., 4.5, 4.8, 5.0). Gradient elution is performed with deionized water at 2–4 BV (e.g., 2 BV, 3 BV, 4 BV) and 10–40 mM (e.g., 10 mM, 20 mM, 40 mM) NaCl solutions, and the fraction rich in nucleosides is collected.

[0050] In one embodiment of the present invention, the preparation method further includes a raw material pretreatment step before step S1: washing, removing impurities, drying, crushing and sieving rice.

[0051] This invention also discloses a rice fermentation broth that enhances the energy of skin cells and mitochondria, prepared using the above-described method.

[0052] This invention further discloses the application of the above-mentioned rice fermentation broth that enhances the energy of skin cells and mitochondria in the preparation of skin care products.

[0053] This rice fermented liquid is made from carefully selected rice through yeast fermentation, enriching it with active substances. It contains various effective ingredients such as α-KG, adenosine, guanosine, nucleotides, amino acids, polysaccharides, and minerals. Specifically, α-KG helps promote keratinocyte proliferation, strengthens the skin barrier function, enhances skin's water retention capacity, and increases the expression of barrier-related genes (FLG, SPT, and IVL), helping to soothe and strengthen the skin barrier, keeping skin hydrated for longer and reducing fine lines and wrinkles caused by dryness. Guanosine, commonly used in skincare and health supplements, can directly and quickly replenish cellular energy when applied topically, promoting the biosynthesis of collagen, elastin, and fibrin. It can inhibit MMP enzyme activity, thereby strengthening the skin barrier and resisting external environmental pollution. It has excellent diffusion and permeability; due to its small contact angle, it can quickly penetrate the deeper layers of the skin and achieve a continuous moisturizing effect. In particular, experiments have shown that rice fermented liquid has a strong effect on skin cell energy and NAD+ levels, enabling anti-aging at the cellular level.

[0054] Existing inventions related to rice fermentation liquid, such as patent CN202311510483.5, mainly focus on enriching active ingredients through fermentation to repair photodamage. However, there is no research on using rice fermentation liquid to enhance cell and mitochondrial energy to achieve anti-aging effects.

[0055] In one embodiment of the present invention, the method for preparing the rice fermentation broth of the present invention includes the following steps:

[0056] Example 1: Co-culture fermentation to prepare NAD-rich products + Rice fermentation liquid of the precursor

[0057] This embodiment provides a method for preparing NAD-rich preparations using a co-culture approach. + A method for obtaining rice fermentation broth precursors. This method utilizes a combination of microbial communities with both sugar metabolism and nucleoside synthesis functions, combined with membrane separation and ion exchange enrichment processes, to obtain fermentation products containing high concentrations of nicotinamide ribose (NR) and nicotinamide mononucleotide (NMN), which can be used to enhance mitochondrial energy in skin cells.

[0058] Raw materials and pretreatment

[0059] Take 1000g of commercially available high-quality rice flour, add 2000g of purified water (solid-liquid ratio 1:2, w / w), and stir well to obtain rice slurry. Add 0.5% β-glucanase (w / w, enzyme activity 150U / g) to the rice slurry, and react at 65℃ for 8 hours while maintaining stirring. After the reaction is complete, quickly cool to 32℃ and adjust the pH to 5.5.

[0060] Fermentation steps

[0061] The yeasts *Saccharomyces veronae* and *Lactiplantibacil lus plantarum* were co-cultured at a volume ratio of 5:1, with the total inoculum accounting for 1.0% of the fermentation broth volume. Fermentation was carried out at 30–32℃, pH controlled at 5.0–5.5, and cultured under microaerophilic conditions for 48 h, with aeration to maintain dissolved oxygen concentration below 5 mg / L.

[0062] Separation and enrichment

[0063] After fermentation, the bacterial cells and solid impurities were removed by filtration through a 0.45 μm membrane. The filtrate was then treated with a 1 kDa ultrafiltration membrane, and the permeate was retained. The permeate was loaded onto a weak cation exchange resin (Amberlite IRC-50 type), the pH was adjusted to 4.8, and after loading, it was eluted with deionized water for 3 BV, followed by elution with a 10–40 mM NaCl gradient. The nucleoside-enriched fraction was collected.

[0064] Comparative Example 1: Single-strain fermentation group

[0065] To verify the effect of co-culture system on promoting NAD + The role of the precursor in the synthesis was the same as in Example 1, using rice milk enzymatic hydrolysis conditions, with Kluyveromyces marxianus inoculated alone (inoculation amount 1.0%, v / v), and other parameters (temperature 32℃, pH 5.5, fermentation 48h) kept consistent.

[0066] Comparative Example 2: Original fermentation broth not enriched

[0067] The original liquid from the end of fermentation in Example 1 was directly filtered for sterilization without ultrafiltration or resin enrichment.

[0068] Comparative Example 3: Fermentation group without enzymatic hydrolysis of substrate

[0069] Untreated rice flour was directly mixed with water (1:2, w / w) and fermented. The bacterial strains and fermentation conditions were the same as in Example 1.

[0070] Comparative Example 4: Heat Inactivation Treatment Group

[0071] The fermentation broth obtained in Example 1 was treated at 90°C for 30 minutes to degrade some of the small molecule active ingredients.

[0072] Efficacy evaluation test

[0073] Based on the detection of mitochondrial morphology, mitochondrial membrane potential and adenosine triphosphate (ATP) content in fibroblasts

[0074] (1) Main reagents

[0075] Mito-Tracker Green mitochondrial green light probe (C1048, Beyotime), Hoechst33342 (B8040, Solarbio), DMEM culture medium (Gibco), PBS (Solarbio), ATP assay kit (Beyotime), JC-1 mitochondrial membrane potential assay kit (Beyotime).

[0076] (2) Main instruments

[0077] CO2 incubator (Thermo), clean bench, UVA irradiator (Philips), inverted white light / fluorescence imaging microscope (Olympus IX51), upright fluorescence imaging microscope (Eclipse Ci-LNikon), and imaging system MicroPublisher Q-IMAGING.

[0078] (3) Sample grouping

[0079] A - Control group; B - UVA model group (10 J / cm2); C - Example 1 (0.025%); D - Comparative example 1 (0.025%); E - Comparative example 2 (0.025%); F - Comparative example 3 (0.025%); G - Comparative example 4 (0.025%)

[0080] (4) Test methods

[0081] ① Cell seeding: After reviving the cells, when the plating rate reaches about 60%, seed the cells into 6-well plates and incubate overnight in a CO2 incubator (37℃, 5% CO2).

[0082] ② Solution preparation: Prepare the working solution of the test substance according to the test group in 5.3.

[0083] ③ Drug administration: According to the above test groupings, when the cell deposition rate in the 6-well plate reaches 30%-50%, drug administration is performed in groups, with 3 replicates per group. 2 mL of culture medium is added to each well of the blank control group and negative control group, and 2 mL of culture medium containing the corresponding concentration of the test sample is added to each well of the sample group. After drug administration, the 6-well plate is placed in an incubator and incubated for 24 hours.

[0084] ④ UVA irradiation: According to the test groups, except for the blank control group, all other groups were subjected to UVA irradiation at a dose of 10 J / cm2. After irradiation, they were placed in a CO2 incubator for 24 hours.

[0085] ⑤ JC-1 staining test: After culture, wash cells three times with PBS. Perform the test according to the JC-1 detection kit instructions, taking pictures under a microscope and using Image-... Image processing software is used for analysis.

[0086] ⑥ Mitochondrial morphology staining: After culture, cells were washed three times with PBS. Detection was performed according to the instructions of the Mito-Tracker Green assay kit and Hoechst 33342. Images were taken under a microscope and analyzed using Image-J image processing software.

[0087] ⑦ ATP content detection: After culture, wash twice with 2 mL / well PBS, add 200 μL of lysis buffer to each well, lyse the cells by pipetting, and collect the samples. Detect the ATP content according to the kit instructions.

[0088] ⑧ Calculation of enhancement rate and inhibition rate:

[0089] Improvement rate (%) = (sample group - model group) / model group × 100%;

[0090] Inhibition rate (%) = (Model group - Sample group) / Model group × 100%.

[0091] Figure 2 This is a schematic diagram of mitochondrial morphology test results in one embodiment of the present invention; please refer to [link / reference]. Figure 2 In normal fibroblasts, mitochondria typically exhibit a spindle-shaped or elongated morphology, indicating structural integrity and normal function. Under UVA stress, mitochondria show significant damage, transforming into spherical or short rod-shaped structures. After treatment with C-Example 1 (0.025%), the mitochondrial morphology significantly recovered, gradually returning to its normal spindle-shaped or elongated form. This indicates that the sample, to some extent, repaired UVA-induced mitochondrial damage, restoring mitochondrial structural integrity and function. This result suggests that the sample has the potential to protect mitochondrial morphology and function, helping cells maintain normal physiological states under stress.

[0092] Table 2 Summary of Adenosine Triphosphate (ATP) Concentration (μM·mL / mg) Test Results for Each Group

[0093] Group Average Improvement rate (%) A 31.92 - B 10.26 - C 24.82 141.91 D 16.82 63.94 E 15.22 48.34 F 16.14 57.31 G 14.94 45.61

[0094] Experimental results showed that UVA irradiation significantly reduced the ATP concentration in fibroblasts, indicating a significant inhibitory effect on cellular energy metabolism. After sample treatment, the ATP concentration of cells recovered to varying degrees, showing that some samples could effectively alleviate UVA-induced cell damage and restore cellular energy metabolism. In particular, after treatment with C-Example 1 (0.025%), the sample showed the most significant recovery effect on ATP levels, reaching 141.91%, indicating that this sample can effectively resist UVA damage to cells.

[0095] Table 3. Improvement rate of mitochondrial membrane potential

[0096] Group Average Improvement rate (%) A 125.59 - B 117.98 - C 137.85 16.84 D 121.90 2.47 E 127.11 7.74 F 121.61 3.08 G 125.48 6.36

[0097] The experimental results showed that the effects of the test samples on mitochondrial membrane potential were assessed using the JC-1 staining method. Some test samples effectively improved the decrease in mitochondrial membrane potential, suggesting that they have a protective effect on mitochondrial function. Among them, C-Example 1 (0.025%) showed the most significant improvement effect, with an improvement rate of 16.87%.

[0098] Skin elasticity and firmness test

[0099] Skin elasticity was measured using a Cutometer dual MPA580 skin elasticity tester in each embodiment, and skin firmness was measured after 0, 14, and 28 days of use (comparison example). Skin elasticity and firmness were also measured after 0, 14, and 28 days of use. The rate of change in skin elasticity and firmness relative to before use was calculated. The rate of change is calculated as follows: (Measurement after product use - Measurement before product use) ÷ Measurement before product use × 100%. The rate of change in skin elasticity is shown in Table 3, and the rate of change in skin firmness is shown in Table 4. A higher skin elasticity measurement indicates increased skin elasticity, and a higher skin firmness measurement indicates increased skin firmness.

[0100] Table 4. Change rate of skin elasticity

[0101]

[0102] Table 5. Change rate of skin firmness

[0103]

[0104] As shown in Tables 4 and 5, compared with before use, after 14 and 28 days of using Example 1, the skin elasticity and firmness increased significantly. However, the increase in skin elasticity and firmness after using Examples 2 to 6 and Control Examples 1 to 3 was lower than that after using Example 1. It can be inferred that Example 1 helps to improve skin firmness and elasticity and exert anti-aging effects.

[0105] In summary, the rice fermentation broth, its preparation method, and its applications proposed in this invention, which enhances the energy of skin cells and mitochondria, can fill the gap in the application of rice fermentation broth in the field of skin care. The rice fermentation broth in this invention has a synergistic penetration-enhancing effect, improving the bioavailability of nutrients in the fermentation broth and ensuring that the active ingredients penetrate deep into the skin's basal layer. The rice fermentation broth in this invention can indirectly increase NAD+ levels by replenishing cellular energy. Its components are mild and have good stability, making it suitable for various cosmetic formulations. It is also less affected by environmental factors and usage conditions, and the preparation process is relatively simple.

[0106] This invention employs a dual-strain co-culture system with a complementary relationship between nucleoside synthesis and lactic acid metabolism. By controlling fermentation conditions to create a specific microaerophilic environment, it is the first to combine ion exchange, ultrafiltration, and low-temperature concentration for the screening and enrichment of small molecules in rice fermentation broth, achieving highly efficient retention and concentration of thermosensitive small molecules such as NR / NMN. Compared with traditional reverse osmosis concentration processes, this enrichment method effectively removes polysaccharides and large peptide impurities, avoids activity loss, and significantly increases NAD5 content in the resulting product. + Precursor molecule content.

[0107] The fermentation target of this invention is no longer limited to "total active substances" or "moisturizing factors", but rather to NAD. + The precursor is product-oriented, and a closed-loop control route has been established from substrate design and strain selection to metabolic enrichment.

[0108] The process of this invention does not require special rice varieties or proprietary strains. All strains are safe strains that can be obtained industrially. The process conditions are mild, stable, and scalable. The enriched products can be directly applied to various cosmetic formulations such as serums, freeze-dried powders, and soluble microneedle patches, and have excellent process compatibility and commercial application potential.

[0109] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The description and application of the present invention herein are illustrative and not intended to limit the scope of the invention to the embodiments described above. Effects or advantages involved in the embodiments may not be apparent due to various factors, and the description of effects or advantages is not intended to limit the embodiments. Variations and modifications of the embodiments disclosed herein are possible, and various substitutions and equivalents of the components in the embodiments are well known to those skilled in the art. It should be apparent to those skilled in the art that the invention can be implemented in other forms, structures, arrangements, proportions, and with other components, materials, and parts without departing from the spirit or essential characteristics of the invention. Other variations and modifications can be made to the embodiments disclosed herein without departing from the scope and spirit of the invention.

Claims

1. A method of preparing a rice ferment liquor for enhancing skin cell and mitochondrial energy, characterized by, The preparation method comprises: Step S1, an enzymatic hydrolysis step: preparing rice slurry, adding a certain amount of β-glucanase for hydrolysis to release sugars and oligosaccharides; Step S2, a fermentation step: co-culturing Saccharomyces veronae and Lactiplantibacillus plantarum, and keeping the fermentation space in a micro-aerobic or intermittent aeration state to facilitate the production of nucleosides in the metabolic pathway; Step S3, a separation and enrichment step: through filtration and screening, small molecule nucleosides / nucleotides are retained; and then through selective enrichment, nucleoside-enriched components are collected.

2. The preparation method according to claim 1, characterized in that: In the step S1, the mass concentration of the rice slurry is 10-60%, the enzymatic hydrolysis temperature is 55-70℃, and the enzymatic hydrolysis time is 4-10h.

3. The preparation method according to claim 1, characterized in that: In the step S1, the dosage of β-glucanase accounts for 0.001-1% of the mass of the rice slurry.

4. The preparation method according to claim 1, characterized in that: In the step S2, the volume ratio of Saccharomyces veronae to Lactiplantibacillus plantarum is 3:1 to 8:1, and mixed inoculation is performed.

5. The preparation method according to claim 1, characterized in that: In the step S2, the fermentation conditions are as follows: the temperature is 30-34℃, the pH value is controlled at 4.5-6.5, and the fermentation time is 24-72h; the ventilation amount of the fermentation space is controlled to keep the fermentation space in a micro-aerobic or intermittent aeration state to facilitate the production of nucleosides in the metabolic pathway.

6. The preparation method according to claim 1, characterized in that: The step S3 comprises: Step S3A, a primary cell removal and coarse filtration step: removing solids through 0.5-5μm mechanical filtration; Step S3B, a small molecule screening step: a 0.5-1.5kDa cutoff ultrafiltration membrane is selected to remove large peptides / proteins and retain small molecule nucleosides / nucleotides; the permeate is retained; Step S3C, a selective enrichment step: the permeate is loaded on a weak cation exchange resin at a pH of 4.5-5.0, sequentially eluted with 2-4BV of deionized water and 10-40mM NaCl solution for gradient elution, and the fraction rich in nucleosides is collected.

7. The preparation method according to claim 6, characterized in that: The weak cation exchange resin is Amberlite IRC-50 type weak cation exchange resin.

8. The preparation method according to claim 1, characterized in that: The preparation method further comprises a raw material pretreatment step before step S1: washing, removing impurities, drying, crushing and screening the rice.

9. A rice fermentation liquor for enhancing skin cells and mitochondrial energy, which is prepared by the preparation method according to any one of claims 1-8.

10. Use of the rice fermentation liquor for enhancing skin cells and mitochondrial energy according to claim 9 in the preparation of skin care products.

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