A method for preparing active raw materials by two-stage fermentation and its application

Abstract

This invention provides a method for preparing active ingredients through a two-stage fermentation process and its application, relating to the field of microbial fermentation technology. The invention provides a low-acid-producing *Bifidobacterium longum* strain, with accession number CGMCC No. 37872. The method includes: mixing *Polygonatum odoratum* and *Saussurea involucrata*, adding water and pulping, filtering and sterilizing to obtain a substrate; adding thermophilic bacteria lysate to the substrate for enzymatic hydrolysis and saccharification to obtain a mixed saccharified liquid; inoculating the aforementioned low-acid-producing *Bifidobacterium longum* strain into the mixed saccharified liquid and fermenting under anaerobic conditions to obtain a fermentation broth; subjecting the fermentation broth to low-temperature, high-pressure homogenization to cell disruption, centrifugation, and low-temperature pasteurization to obtain the active ingredient. This invention achieves synergistic effects between plant-derived active ingredients and microbial active ingredients through a two-stage fermentation process. The prepared active ingredient has excellent moisturizing, repairing, antioxidant, and soothing effects and can be widely used in the cosmetics field.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically a method for preparing active raw materials through two-stage fermentation and its application. Background Technology

[0002] Polygonatum odoratum ( Polygonum odoratum ) and Tibetan snow lotus ( Saussurea involucrata Polygonatum odoratum is a traditional plant used in both medicine and food. Modern research shows that it is rich in polysaccharides, steroidal saponins, and flavonoids, possessing excellent moisturizing, antioxidant, and soothing effects. Saussurea involucrata, on the other hand, is rich in flavonoids, polysaccharides, and terpenoids, exhibiting significant antioxidant, anti-inflammatory, and melanin-inhibiting activities. However, traditional plant extraction methods, such as hot water extraction or organic solvent extraction, often suffer from low extraction rates of active ingredients, easy inactivation of heat-sensitive substances, organic solvent residues, and difficulty in transdermal absorption of large molecular active ingredients, thus limiting their efficient application in cosmetics.

[0003] Plant fermentation technology utilizes the growth and metabolism of microorganisms to biotransform plant raw materials, effectively degrading plant cell walls, releasing and modifying endogenous active ingredients, and enriching microbial secondary metabolites to achieve synergistic effects of active substances. However, existing fermentation technologies mostly employ single-strain fermentation, which suffers from low substrate utilization, limited metabolites, and limited efficacy targets. While mixed-strain simultaneous fermentation attempts to overcome these shortcomings, significant differences in the optimal growth conditions of different strains can easily lead to strain competition and metabolic inhibition, resulting in uncontrollable fermentation processes, poor batch stability, and difficulty in achieving industrial-scale production. Bifidobacterium ( Bifidobacterium longum As a commonly used probiotic, its fermented lysate has good effects on skin repair and soothing. However, conventional lactic acid bacteria have a strong ability to produce acid during fermentation. If the pH at the end of fermentation is too low, it will not only lead to the hydrolysis and inactivation of plant-derived active ingredients, but also irritate the skin, making it difficult to meet the strict requirements of cosmetics for gentleness and formula stability.

[0004] Therefore, how to develop a preparation method that can efficiently release plant active ingredients while avoiding strain competition and achieving mild fermentation has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a low-acid-producing strain of Bifidobacterium longum, wherein Bifidobacterium longum ( Bifidobacterium longum The strain exhibits reduced lactic acid production and LDH enzyme activity, while increasing the retention rate of plant polysaccharide active substances, making it suitable for cosmetic fermentation needs.

[0006] Another objective of this invention is to provide a method for preparing active ingredients through two-stage fermentation. The two-stage fermentation process significantly improves substrate utilization, shortens the main fermentation cycle, and has high production efficiency. The prepared active ingredients can be directly used as core functional ingredients in cosmetics.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a low-acid-producing strain of Bifidobacterium longum, wherein Bifidobacterium longum ( Bifidobacterium long The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37872.

[0008] This invention provides a method for preparing active raw materials through a two-stage fermentation process, comprising the following steps: Polygonatum odoratum and Saussurea involucrata were mixed and then slurried with water to obtain a slurry. The slurry was then filtered and sterilized to obtain a substrate. Thermophilic thermophilic bacteria lysate was added to the substrate to carry out an enzymatic hydrolysis and saccharification reaction to obtain a mixed saccharified solution. The *Bifidobacterium longum* strain was inoculated into the mixed saccharification liquid and fermented under anaerobic conditions to obtain a fermentation broth. The fermentation broth was then subjected to low-temperature high-pressure homogenization to break up cells, centrifuged, and sterilized to obtain the active raw material.

[0009] Preferably, the amount of thermophilic thermophilic bacteria lysate added is 5%-30% of the substrate volume, the saccharification temperature is 30-80℃, and the saccharification time is 1-6h.

[0010] Preferably, the thermophilic bacterium has the preservation number CICC 10647.

[0011] Preferably, the inoculation amount of Bifidobacterium longum is 2%-10% of the volume of the mixed saccharification liquid, the fermentation temperature is 30-40℃, and the fermentation time is 1-14h.

[0012] More preferably, the pH of the system is maintained at 5.5-7.0 during the fermentation process.

[0013] Preferably, the mass ratio of the Solomon's seal and the Tibetan snow lotus is (1-20):1.

[0014] Preferably, the pressure of the low-temperature high-pressure homogenization cell disruption is 85-95 MPa, and the temperature is 30-37°C.

[0015] This invention provides an active raw material prepared by the method described above.

[0016] This invention provides the application of the aforementioned active ingredient in the preparation of cosmetics with moisturizing, repairing, antioxidant and / or soothing effects.

[0017] Compared with the prior art, the present invention has the following advantages: (1) This invention provides a novel low-acid-producing Bifidobacterium longum strain, CGMCC No. 37872, obtained through targeted domestication. Experiments show that this strain can stably grow and proliferate under weakly alkaline conditions at pH 7.4. After 6 hours of fermentation, the pH of the system remains at 6.15, and the lactic acid yield is only 5.47 mg / dL, a decrease of 70.8% compared to the original strain; the lactate dehydrogenase (LDH) activity decreases to 18.6 U / mL, a reduction of 85.3%. Simultaneously, the viable count of this strain can reach 1.8 × 10⁻⁶. 9 The CFU / mL concentration of this strain resulted in a plant polysaccharide retention rate of 94.7%, a 52 percentage point increase compared to the original strain's 62.3%. After 10 generations of subculturing, the performance indicators of each generation showed a deviation of ≤5%, with no reversion mutations and excellent genetic stability, fully meeting the requirements of industrial continuous fermentation. The successful domestication of this strain fundamentally solves the industry pain points of excessive acid production during traditional lactic acid bacteria fermentation, such as the inactivation of plant active ingredients and high skin irritation, providing a core strain resource for the preparation of mild cosmetic raw materials.

[0018] (2) The two-stage fermentation process constructed in this invention achieves a significant synergistic effect. In the pretreatment stage of thermophilic thermophilic bacteria lysate, the substrates of Solomon's seal and Tibetan snow lotus are enzymatically hydrolyzed and saccharified by the complex enzyme system such as cellulase and amylase abundant in its cells, so that the soluble polysaccharide content reaches 46.78 mg / mL and the solid content reaches 6.8%, which are 109.2% and 142.8% higher than the traditional hot water extraction, respectively, and 31.7% and 54.5% higher than the commercially available complex enzyme treatment, respectively, which efficiently releases the endogenous active ingredients of plants. After the second stage of low-acid fermentation using this saccharified broth, the DPPH free radical scavenging rate of the obtained product reached 58.26%, 79.54%, and 92.37% at concentrations of 2, 5, and 10 mg / mL, respectively. This was significantly higher than that of the commercially available enzyme-treated group (45.18%, 66.32%, and 81.65%) and the traditional water extraction group (32.57%, 50.29%, and 68.43%), fully demonstrating that there is a clear synergistic effect mechanism between the pretreatment of thermophilic thermophilic bacteria lysate and the fermentation of low-acid-producing Bifidobacterium longum.

[0019] (3) The active ingredient prepared by this invention exhibits excellent skincare efficacy. Efficacy evaluation using a 3D human epidermal skin model shows that a 3% concentration of the active ingredient of this invention can significantly reduce the number of UVB-induced sunburned cells, with an inhibition rate of 65.19%. P <0.01); significantly increased the content of lobelin (LOR), filaggrin (FLG), and hyaluronic acid (HA), with increases of 102.94%, 131.25%, and 116.22%, respectively. PThe p-value <0.01 indicates that it has a clear skin-soothing and repairing effect. Regarding moisturizing effects, the active ingredient of this invention can significantly increase the content of aquaporin AQP3, with an increase rate of 54.55% (p<0.01). In terms of antioxidant effects, the active ingredient of this invention can significantly upregulate the expression levels of 11 target genes, including Claudin1, Claudin4, Occludin, and SOD2, with the core antioxidant gene SOD2 upregulated by 87.23% (p<0.01). P <0.01). The above-mentioned multi-dimensional and multi-target superior efficacy fully demonstrates the technical advantages of this invention in achieving synergistic enrichment of plant-derived active ingredients and microbial active ingredients through two-stage fermentation.

[0020] (4) This invention employs a biological enzymatic hydrolysis and low-temperature anaerobic fermentation process throughout, without high-temperature extraction or the addition of organic solvents. This avoids the risks of inactivation of heat-sensitive active ingredients and solvent residue. The pH value of the prepared active raw materials is stable within a mild range of 5.5-7.0, making it suitable for all skin types, especially sensitive skin. The process is controllable in stages, completely avoiding strain competition and metabolic inhibition. It has good batch stability and is easy to scale up for industrial production. The obtained active raw materials can be directly used as core active ingredients in various cosmetics such as serums, creams, toners, and masks, and have good market application prospects.

[0021] Biological Preservation Information The *Bifidobacterium longum* strain described in this invention is classified and named as follows: Bifidobacterium longum Depository: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Accession Number: CGMCC No. 37872; Date of Deposit: March 9, 2026. Attached Figure Description

[0022] Figure 1 This is a bar chart showing the average values ​​of sunburn cells in different treatment groups. In the chart, ## represents the difference between the treatment group and the control group. P <0.01; compared with the negative control group, represent P <0.01; Figure 2 This is a bar chart showing the average relative integrated optical density (IOD) of the LOR under different treatment groups. In the chart, ## represents the difference from the blank control group. P <0.01; compared with the negative control group, represent P <0.01; Figure 3 This is a bar chart showing the average relative integrated optical density (IOD) of FLG in different treatment groups. In the chart, ## represents the difference between the control group and the control group. P<0.01; compared with the negative control group, represent P <0.01; Figure 4 This is a bar chart showing the average relative integrated optical density (IOD) of HA in different treatment groups. In the chart, ## represents the difference between the treatment group and the control group. P <0.01; compared with the negative control group, represent P <0.01; Figure 5 This is a bar chart showing the average relative integrated optical density (IOD) of AQP3 in different treatment groups. In the chart, ## represents the difference between the treatment group and the control group. P <0.01; compared with the negative control group, represent P <0.01; Figure 6 This is a bar chart showing the SOD2 gene expression levels in different treatment groups. Compared to the blank control group, ## represents... P <0.01; compared with the negative control group, represent P <0.01. Detailed Implementation

[0023] This invention provides a low-acid-producing strain of Bifidobacterium longum, wherein Bifidobacterium longum ( Bifidobacterium long The strain described in this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37872. The strain was obtained through directed gradient domestication of a primitive *Bifidobacterium longum* strain. The domestication steps are as follows: After activating the primitive *Bifidobacterium longum* strain, it was placed in a pH-controlled fermenter with pH 6.8 as the initial condition. After the strain stabilized, the pH was increased by 0.1-0.3 units each time. After each increase, the strain was passaged for 3-5 generations until it stabilized at that pH. This process was repeated until the strain could stably proliferate at pH 7.3-7.5. Strains with reduced lactic acid production and reduced lactate dehydrogenase (LDH) activity were screened and cultured stably to obtain the low-acid-producing *Bifidobacterium longum* CGMCC No. 37872. When this strain ferments under anaerobic conditions using enzymatic hydrolysates of Polygonatum odoratum and Saussurea involucrata as substrates, it can maintain a high number of viable bacteria while keeping the fermentation system within a mild pH range, effectively preventing the hydrolysis and inactivation of plant-derived active ingredients, and significantly enhancing the antioxidant activity of the fermentation products.

[0024] This invention provides a method for preparing active raw materials through a two-stage fermentation process, comprising the following steps: Polygonatum odoratum and Saussurea involucrata were mixed and then slurried with water to obtain a slurry. The slurry was then filtered and sterilized to obtain a substrate. Thermophilic thermophilic bacteria lysate was added to the substrate to carry out an enzymatic hydrolysis and saccharification reaction to obtain a mixed saccharified solution. The *Bifidobacterium longum* strain was inoculated into the mixed saccharification liquid and fermented under anaerobic conditions to obtain a fermentation broth. The fermentation broth was then subjected to low-temperature high-pressure homogenization to break up cells, centrifuged, and sterilized to obtain the active raw material.

[0025] In this invention, the authentic medicinal herb Polygonatum odoratum (Yu Zhu) is selected. Polygonum odoratum ) and Tibetan snow lotus ( Saussurea wrapped The dried rhizomes of Polygonatum odoratum and Saussurea involucrata are pulverized and sieved separately for later use. Polygonatum odoratum and Saussurea involucrata are mixed in a mass ratio of (1-20):1, preferably 4:1, i.e., 400g of Polygonatum odoratum and 100g of Saussurea involucrata are weighed and added to 1-20 times the total volume of the solid medicinal materials with deionized water, preferably 10 times the volume (5L). The mixture is placed in a high-speed pulverizer and pulped at 20,000 rpm for 3-8 minutes, preferably 5 minutes, to fully break down the medicinal materials and evenly mix them with water to form a slurry. The resulting slurry is filtered under pressure through a filter cloth to remove unbroken coarse particles and insoluble fiber residue, and the filtrate is collected. The filtrate is placed in an autoclave and sterilized at 80-125℃ for 0.1-5 hours, preferably at 121℃ for 20 minutes, to kill any possible contaminants in the raw materials and avoid the risk of contamination during subsequent fermentation. After sterilization, the filtrate is cooled to room temperature to obtain the substrate that can be used for subsequent enzymatic hydrolysis and saccharification. Through the above steps, macromolecules such as starch, cellulose, and hemicellulose in Polygonatum odoratum and Saussurea involucrata are fully exposed, providing accessible sites of action for the complex enzyme system in the subsequent thermophilic bacteria lysate. At the same time, the sterilization process ensures the aseptic state of the substrate, laying the foundation for the stability and reproducibility of the two-stage fermentation process.

[0026] In this invention, after the substrate preparation is completed and cooled to room temperature, thermophilic thermocline lysate is added to the substrate. The thermophilic thermocline lysate is prepared by adding thermophilic thermocline (accession number CICC 10647) to the substrate. Thermae thermophilic The bacteria were inoculated into the fermentation medium and fermented at high density for 24 hours at 65℃ and 200 rpm. After centrifugation to collect the bacterial sludge, it was resuspended in physiological saline to a concentration of 10% (w / v). Then, it was homogenized 3-5 times at 80-100 MPa pressure and 4℃ using a high-pressure homogenizer, with a cell disruption rate ≥95%. The resulting lysate had a protein content ≥8 mg / mL and a total enzyme activity ≥1500 U / mL, including cellulase activity ≥300 U / mL and amylase activity ≥500 U / mL. The thermophilic bacteria (… Thermae thermophilicThis can be obtained through conventional purchasing channels. The above-mentioned thermophilic thermophilic bacteria lysate is added to the substrate at a dosage of 5%-30% of the substrate volume, preferably 20% (v / v). The mixture is placed in an enzymatic reaction vessel and subjected to a saccharification reaction at 30-80℃ for 1-6 hours, preferably at 65℃ for 3 hours. During the reaction, the mixture is stirred at 100 rpm to ensure sufficient contact between the enzyme and the substrate. Under these conditions, the abundant intracellular enzyme system in the thermophilic thermophilic bacteria lysate, including cellulase, amylase, pectinase, and glycosidase, can efficiently degrade structural components such as cellulose, hemicellulose, and pectin in the cell walls of Polygonatum odoratum and Saussurea involucrata, disrupting the dense structure of the cell walls and allowing the full release of intracellular polysaccharides, flavonoids, saponins, and other active ingredients into the reaction system. Meanwhile, glycosidases and methyltransferases in the lysate can chemically modify the released active ingredients, improving their bioavailability; superoxide dismutase can scavenge reactive oxygen free radicals in the reaction system, creating a stable microenvironment for subsequent fermentation. After the saccharification reaction is completed, a mixed saccharified liquid containing a large amount of soluble polysaccharides, flavonoids and other active ingredients is obtained, which can be directly used for the second stage of fermentation.

[0027] In this invention, the mixed saccharified liquid obtained by the above-mentioned enzymatic hydrolysis and saccharification reaction is cooled to the fermentation temperature, and the initial pH value is adjusted to a suitable range for the growth of Bifidobacterium longum. The low-acid-producing Bifidobacterium longum strain CGMCC No. 37872 preserved in this invention is activated and cultured to a high-density culture to achieve a viable count of 1 × 10⁻⁶ cells / mL. 10 CFU / mL or higher. Inoculate the above high-density bacterial culture into the mixed saccharification liquid at an inoculum volume of 2%-10%, preferably 5% (v / v). After inoculation, immediately create a strictly anaerobic environment, which can be achieved using an anaerobic incubator. Control the fermentation temperature at 30-40℃, preferably 32℃; during fermentation, stir at a low speed of 50-100 rpm to avoid increased dissolved oxygen affecting bacterial growth. Control the fermentation time within 1-14 hours, preferably 6-8 hours, with 7 hours being optimal. Throughout the fermentation process, because the low-acid-producing Bifidobacterium longum CGMCC No. 37872 used has undergone targeted acclimatization, its lactate dehydrogenase activity is significantly reduced, resulting in a substantial decrease in lactic acid production. Therefore, the pH of the fermentation system can be stably maintained within a mild range of 5.5-7.0, without the need for additional acid-base regulators. During fermentation, low-acid-producing Bifidobacterium longum utilizes the soluble polysaccharides, oligosaccharides, and small-molecule carbon sources released in the mixed saccharification liquid for growth and metabolism. On the one hand, it further degrades the plant-derived macromolecular active ingredients into smaller molecules that are more easily absorbed through the skin. On the other hand, it enriches the probiotic active lysate components unique to Bifidobacterium, such as extracellular polysaccharides, short-chain fatty acids, small-molecule polypeptides, and nucleic acid fragments.

[0028] In this invention, after fermentation, the obtained fermentation broth undergoes low-temperature, high-pressure homogenization to break down cells. The preferred steps are as follows: The fermentation broth is placed in a low-temperature, high-pressure homogenizer and homogenized under a pressure of 80-100 MPa. During homogenization, the temperature is controlled at ≤4℃ to prevent the inactivation of heat-sensitive active ingredients. Homogenization is repeated 2-3 times to achieve a cell disruption rate of over 90%. Through this treatment, the cell walls of *Bifidobacterium longum* (low-acid-producing bacteria) are effectively broken, and intracellular active substances such as peptidoglycan, DNA fragments, small polypeptides, intracellular enzymes, and metabolic intermediates are fully released into the fermentation broth, forming a complex system with plant-derived active ingredients. After cell disruption, the disrupted broth is transferred to a refrigerated centrifuge for low-temperature centrifugation to remove impurities. The centrifugation temperature is controlled at 4-10℃, the centrifugation speed is 8000-12000 rpm, and the centrifugation time is 10-20 minutes to remove broken bacterial residues, incompletely broken cell fragments, and insoluble plant fibers, and the supernatant is collected. This centrifugation condition effectively removes solids while maximizing the retention of soluble active ingredients in the supernatant. The supernatant collected by centrifugation is then sterilized, preferably by low-temperature pasteurization at a temperature of 60-85°C for 20-30 minutes, preferably at 80°C for 25 minutes. This sterilization condition effectively kills residual live bacteria in the system, ensuring the microbial safety of the raw materials, while maximizing the retention of the bioactivity of heat-sensitive active ingredients such as polysaccharides, flavonoids, and peptides, avoiding activity loss caused by high-temperature sterilization. After sterilization, once the material has cooled, appropriate amounts of commonly used cosmetic preservatives, such as 1,2-hexanediol and 1,2-pentanediol, are added at 2% (w / v) and 1% (w / v), respectively. After thorough mixing, the mixture is aseptically packaged to obtain the final cosmetic active ingredient.

[0029] This invention provides an active raw material prepared by the method described above.

[0030] This invention provides the application of the aforementioned active ingredient in the preparation of cosmetics with moisturizing, repairing, antioxidant and / or soothing effects.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0033] Example 1 A method for preparing active raw materials through a two-stage fermentation process includes the following steps: (1) Pretreatment of Solomon's Seal and Tibetan Snow Lotus substrate: Weigh 400g of Solomon's Seal and 100g of Tibetan Snow Lotus (feed ratio 4:1), add 5L of deionized water, grind in a high-speed pulverizer at 20000rpm for 5min, filter under pressure through a 30-mesh filter cloth, place the filtrate in an autoclave, sterilize at 121℃ for 20min, cool to room temperature, and obtain the nutrient substrate; (2) Saccharification preconversion: Add 20% (v / v) thermophilic thermophilic bacteria lysate to the nutrient substrate, stir at 65℃ and 100rpm for 3h until the saccharification endpoint is reached to obtain a mixed saccharified liquid; (3) Inoculation and fermentation: The mixed saccharification solution was cooled to 32°C, the initial pH was adjusted to 7.4, and low-acid-producing Bifidobacterium longum was inoculated at an inoculation rate of 5% (v / v). Bifidobacterium longum High-density bacterial culture (viable count ≥10) of CGMCC No. 37872 10 (CFU / mL), sterile nitrogen gas was introduced to replace the air, and fermentation was carried out at 32℃, 80 rpm and under strict anaerobic conditions for 7 hours until the fermentation endpoint was reached to obtain the fermentation broth; (4) Post-processing: The fermentation broth was homogenized twice at 90 MPa and 35℃, centrifuged at 4℃ and 10000 rpm for 15 min, and the supernatant was collected; pasteurized at 80℃ for 25 min, 2% 1,2-hexanediol and 1% 1,2-pentanediol were added, stirred evenly and then aseptically packaged to obtain the active raw material.

[0034] The preparation method of *Thermophilus thermophilus* lysate is as follows: *Thermophilus thermophilus* with preservation number CICC 10647 was inoculated into fermentation medium (5 g / L peptone, 3 g / L yeast extract, 2 g / L sodium chloride, pH 7.0) and fermented at high density for 24 h at 65℃ and 200 rpm. The fermentation broth was centrifuged at 4℃ and 8000 rpm for 10 min to collect the bacterial sludge, which was resuspended in sterile physiological saline to a bacterial sludge concentration of 10% (w / v). The lysate was homogenized four times at 4℃ under high pressure using a high-pressure homogenizer, with a cell disruption rate ≥95%, thus obtaining *Thermophilus thermophilus* lysate. The lysate was found to have a protein content of 9.2 mg / mL and a total enzyme activity of 1680 U / mL, including 350 U / mL cellulase and 580 U / mL amylase.

[0035] Comparative Example 1 Unlike Example 1, in step (2), a complex enzyme of amylase and saccharifying enzyme was added to the nutrient substrate. The amylase and saccharifying enzyme were purchased from Novozymes (China) Biotechnology Co., Ltd., and saccharification was carried out at 65°C and 100 rpm for 4 hours.

[0036] Comparative Example 2 Unlike Example 1, in step (2), the obtained nutrient substrate was heated at 100°C for 3 hours for extraction.

[0037] Example 2 This embodiment evaluates the liquefaction and saccharification capabilities of different methods on the mixed slurry of medicinal materials. The effects of thermophilic bacteria fermentation lysate, amylase + saccharifying enzyme (commercially available), and heat extraction methods on the soluble polysaccharide content in the nutrient substrate of *Polygonatum odoratum*-*Saussurea involucrata* under optimal conditions were compared. The polysaccharide content was determined using the phenol-sulfuric acid method, referring to the method described in *SN / T 4260-2015 Determination of Crude Polysaccharides in Exported Plant-Derived Foods - Phenol-Sulfuric Acid Method*. The results are shown in Table 1.

[0038] Table 1 Comparison of saccharification effects of different methods on the nutrient substrate of Polygonatum odoratum-Saussurea involucrata

[0039] According to the results in Table 1, the saccharification treatment of thermophilic bacterium lysate used in this invention increases the solid content by 142.8% and the soluble polysaccharide content by 109.2% compared with traditional hot water extraction; compared with commercially available compound enzymes, the solid content increases by 54.5% and the polysaccharide content increases by 31.7%, which can release plant endogenous active substances more efficiently.

[0040] Example 3: Low-acid-producing Bifidobacterium longum ( Bifidobacterium longum Screening and domestication of CGMCC No. 37872 1. Filtering Five commercially available freeze-dried bacterial powders of lactic acid bacteria—*Lactobacillus thermophilus*, *Lactobacillus bulgaricus*, *Lactobacillus plantarum*, *Bifidobacterium longum*, and *Bifidobacterium breve*—were activated and inoculated into the mixed saccharification broth prepared in Example 1. Anaerobic fermentation was carried out at 37°C for 6 hours, and the pH value and total acid content of the fermentation broth were measured. The pH value of the fermentation supernatant was measured using a pH meter, and the total acid (TAC) was determined according to GB12456-2021, "National Food Safety Standard - Determination of Total Acid in Food". The results are shown in Table 2.

[0041] Table 2 Comparison of acid-producing capacity of different lactic acid bacteria in mixed saccharification solution

[0042] As shown in Table 2, thermophilic lactic acid bacteria, Lactobacillus bulgaricus, and Lactobacillus plantarum all exhibited extremely strong acid-producing capabilities, with the pH value dropping sharply to 2.47-2.89 after 6 hours of fermentation, corresponding to a total acid (TAC) content as high as 10.62-12.35 g / L. This highly acidic environment can lead to the inactivation of plant active ingredients and cause strong skin irritation, failing to meet the mildness requirements of cosmetic raw materials. Among them, Bifidobacterium longum showed the mildest acid-producing capability, with a pH value of 3.82 after 6 hours of fermentation, making it an ideal starting strain for subsequent low-acid-producing acclimatization.

[0043] 2. Domestication After activating the original strain of Bifidobacterium longum, it was inoculated into MRS medium at pH 6.8 and cultured for 24 hours before measuring the growth OD. 600 The final pH, lactic acid yield, and LDH enzyme activity were used as initial baselines. Gradual acclimatization was then performed by controlling the pH in the fermenter: the initial pH was set at 6.8, and after the strain stabilized, the pH was gradually increased in increments of 0.1-0.3 units each time, with 3-5 generations after each increase until the strain stabilized at that pH. This process was repeated until the strain could stably proliferate at pH 7.4, yielding low-acid-producing *Bifidobacterium longum* CGMCC No. 37872. The lactic acid content in the fermentation broth was determined according to GB 1886.173-2016 National Food Safety Standard for Food Additives - Lactic Acid; LDH enzyme activity was determined using ultraviolet spectrophotometry, specifically examining the catalytic conversion of NADH to NAD within 1 minute by lactic acid bacteria lysate under optimal conditions. + The results are shown in Table 3.

[0044] Table 3 Comparison of fermentation performance of different lactic acid bacteria at pH 7.4

[0045] As shown in Table 3, the domesticated low-acid-producing Bifidobacterium longum strain exhibited a 70.8% reduction in lactic acid production and an 85.3% reduction in LDH enzyme activity. The fermentation system maintained a stable pH within a skin-friendly range, while allowing for normal growth and proliferation. The retention rate of plant polysaccharide active ingredients increased by 52%, perfectly meeting the fermentation requirements for cosmetics. Thermophilic lactic acid bacteria, Lactobacillus bulgaricus, and Lactobacillus plantarum, among others, have a natural optimal growth pH of 5.0–6.5, belonging to the acid-tolerant type of lactic acid bacteria. A weakly alkaline environment of pH 7.4 directly inhibits their cell membrane permeability and the activity of key metabolic enzymes, preventing the strains from initiating core metabolic pathways such as glycolysis, ultimately hindering their growth and proliferation.

[0046] This invention involves subculturing a domesticated, low-acid-producing *Bifidobacterium longum* in MRS medium for 10 consecutive generations. Lactic acid production and pH were measured for 6 hours of fermentation in each generation. Results showed that the performance indicators of each generation exhibited a deviation of ≤5%, with no reversion mutations, demonstrating good genetic stability and meeting the requirements for industrial continuous fermentation. This strain was deposited and obtained the accession number CGMCC No. 37872.

[0047] Example 4 Take the saccharification solutions prepared in Example 1, Comparative Example 1, and Comparative Example 2 respectively, adjust the initial pH to 7.4, and inoculate them with a high-density bacterial culture of low-acid-producing Bifidobacterium longum CGMCC No. 37872 (viable count ≥ 1 × 10⁻⁶) obtained in Example 3 at a 5% (v / v) inoculation rate. 10The concentration of CFU / mL was increased, and sterile nitrogen was introduced to replace the air. Fermentation was carried out at 32℃ and 80 rpm under strictly anaerobic conditions for 7 hours. After fermentation, the fermentation broth was homogenized twice under low temperature and high pressure at 90 MPa to break down cells. The supernatant was collected by centrifugation at 4℃ and 10000 rpm for 15 min and then pasteurized at 80℃ for 25 min to obtain the active raw material sample. The sample was tested according to GB / T 31740.2-2015 Determination of Antioxidant Activity of Plant Extracts Part 2: DPPH Free Radical Scavenging Method. Three replicates were set up for each sample, and the average value was taken. The formula for calculating the DPPH free radical scavenging rate is: In the formula: A control is the absorbance of the solution without sample; A sample is the absorbance of the solution with sample; A blank is the absorbance of the sample solvent; the detection wavelength is 517 nm.

[0048] The free radical scavenging rate of each sample at concentrations of 2 mg / mL, 5 mg / mL, and 10 mg / mL was determined by the DPPH method, and the results are shown in Table 4.

[0049] Table 4 Comparison of DPPH free radical scavenging rates of fermentation products prepared from different saccharification solutions (%)

[0050] As shown in Table 4, the DPPH free radical scavenging rate of the substrate treated with thermophilic thermophilic bacteria lysate after fermentation was significantly higher than that of the fermentation products of commercially available enzymatic hydrolysis and traditional water extraction saccharification substrates at various concentration gradients. This indicates that the saccharification method can efficiently release and retain the antioxidant active ingredients in Solomon's seal-snow lotus, and synergistically enhance the antioxidant capacity of the fermentation products with the metabolites of domesticated Bifidobacterium longum fermentation.

[0051] Example 5: Evaluation of the skincare efficacy of the active ingredients of the present invention Using the 3D human epidermal skin model EpiKutis®, a skin photodamage model was established by UVB irradiation to evaluate the skin care efficacy of the active ingredient prepared in Example 1 (hereinafter named Lyfida® 3.0).

[0052] 1. Experimental Methods (1) The experiment was conducted using a 3D human epidermal skin model (EpiKutis®). The model was randomly placed in a 6-well plate, with 3 replicates per group. The experiment included a blank control group, a negative control group, a positive control group, and a 3% (m / m) Lyfida® 3.0 sample group, all incubated at 37℃ in a 5% CO2 incubator. Except for the blank control group, all other groups were given 600 mJ / cm². 2 A skin photodamage model was established using UVB irradiation, and the experiment was repeated three times throughout.

[0053] (2) After irradiation, the blank control group and negative control group were added with model-specific EpiGrowth culture medium, the positive control group was added with culture medium containing the corresponding positive control (PC1 was 50 μM WY14643, PC2 was 7 μg / mL vitamin E), and the sample group was added with culture medium containing 3% Lyfida® 3.0; and incubated in a 37℃, 5% CO2 incubator in the dark for 24 h.

[0054] (3) After incubation, the model was cleaned and samples were collected and tested in batches: ① H&E staining was used to observe and count the number of sunburned cells under an upright microscope to evaluate the soothing and repairing effects of the samples; ② Immunofluorescence was used to detect the relative contents of lobelin (LOR), filaggrin (FLG), and aquaporin (AQP3) under a fluorescence microscope to evaluate the repair and moisturizing effects of the samples; ③ Biotinylation was used to detect the relative contents of hyaluronic acid (HA) to evaluate the moisturizing and repairing effects of the samples; ④ qRT-PCR was used to extract total RNA from the model and reverse transcribe it into cDNA, and then the relative expression levels of 11 target genes, including Claudin1, Claudin4, and Occludin, were detected. △△CT The results were calculated to evaluate the antioxidant effect of the samples.

[0055] 2. Experimental Results (1) Evaluation of soothing and repairing effects: The number of sunburned cells was counted by H&E staining; the relative contents of lobe protein (LOR) and filaggrin (FLG) were detected by immunofluorescence; and the contents of hyaluronic acid (HA) were detected by biotin colorimetric method.

[0056] The results are as follows Figure 1-Figure 4 The results showed that a 3% concentration of Lyfida® 3.0 significantly reduced the number of UVB-induced sunburned cells, with an inhibition rate of 65.19%. P <0.01), while significantly increasing the content of LOR, FLG, and HA, with increases of 102.94%, 131.25%, and 116.22%, respectively. P <0.01), possessing clear skin soothing and repairing effects.

[0057] (2) The relative content of aquaporin AQP3 was detected by immunofluorescence.

[0058] The results are as follows Figure 5 The results showed that the relative content of AQP3 in the sample group was 154.55% of that in the negative control group, with an increase rate of 54.55%. P The result of <0.01 indicates that the active ingredient of the present invention has excellent moisturizing effect.

[0059] (3) The relative expression levels of 11 target genes, including Claudin1, Claudin4, Occludin, DSC1, DSG1, SOD2, BGT-1, TAUT, SMIT, HMIT and CerS3, were detected by qRT-PCR.

[0060] The results are as follows Figure 6 The results showed that 3% Lyfida® 3.0 significantly upregulated the expression of all the above-mentioned target genes, with the core antioxidant gene SOD2 upregulated by 87.23%. P <0.01), indicating that the active ingredient of the present invention has a clear antioxidant effect.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A low-acid-producing Bifidobacterium longum strain, characterized in that, The Bifidobacterium longum ( Bifidobacterium longum The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37872.

2. A method for preparing active raw materials through a two-stage fermentation process, characterized in that, Includes the following steps: Polygonatum odoratum and Saussurea involucrata were mixed and then slurried with water to obtain a slurry. The slurry was then filtered and sterilized to obtain a substrate. Thermophilic thermophilic bacteria lysate was added to the substrate to carry out an enzymatic hydrolysis and saccharification reaction to obtain a mixed saccharified solution. The *Bifidobacterium longum* strain described in claim 1 is inoculated into the mixed saccharification liquid and fermented under anaerobic conditions to obtain a fermentation broth; the fermentation broth is then subjected to low-temperature high-pressure homogenization to break up cells, centrifuged, and sterilized to obtain the active raw material.

3. The method according to claim 2, characterized in that, The amount of thermophilic thermophilic bacteria lysate added is 5%-30% of the substrate volume, the saccharification temperature is 30-80℃, and the saccharification time is 1-6h.

4. The method according to claim 2 or 3, characterized in that, The preservation number of the thermophilic bacterium is CICC10647.

5. The method according to claim 2, characterized in that, The inoculation amount of Bifidobacterium longum is 2%-10% of the volume of the mixed saccharification liquid, the fermentation temperature is 30-40℃, and the fermentation time is 1-14h.

6. The method according to claim 5, characterized in that, The pH of the system was maintained between 5.5 and 7.0 during the fermentation process.

7. The method according to claim 2, characterized in that, The mass ratio of Solomon's seal and Tibetan snow lotus is (1-20):

1.

8. The method according to claim 2, characterized in that, The pressure for the low-temperature high-pressure homogenization cell disruption is 85-95 MPa, and the temperature is 30-37℃.

9. The active raw material prepared by the method according to any one of claims 2-8.

10. The use of the active ingredient of claim 9 in the preparation of cosmetics having moisturizing, repairing, antioxidant and / or soothing effects.

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