Preparation method of Kushui rose fermentation liquor and application of Kushui rose fermentation liquor in anti-aging cosmetics

Abstract

The invention discloses a preparation method of Kushui rose fermentation liquor and application of the Kushui rose fermentation liquor in anti-aging cosmetics. According to the preparation method, Kushui rose is taken as a substrate, lactobacillus plantarum TS337821 is taken as zymophyte, and fermentation is carried out under optimal conditions (for example, the inoculation amount is 3%, and fermentation is carried out at 37 DEG C for 45 hours). According to the method, the content of active ingredients such as chlorogenic acid and cyanidin-3-O-glucoside in the fermentation liquor can be remarkably increased. Experiments prove that the obtained fermentation liquor has a definite anti-aging effect, and can effectively improve cell viability, promote synthesis of collagen I, inhibit matrix metalloproteinase-I and enhance activity of antioxidant enzymes in cells. The fermentation liquor can be directly used for preparing anti-aging cosmetics, and the recommended volume fraction in a formula is 0.75%. The invention provides a new technical scheme and a high-quality raw material for high-value development of the Kushui rose.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation engineering, and specifically relates to a method for directional fermentation of Rosa sertata x Rosa rugosa Yu.et.Ku. by using specific Lactiplantibacillus plantarum and application thereof in anti-aging cosmetics. BACKGROUND

[0002] Rosa sertata x Rosa rugosa Yu.et.Ku. is a characteristic plant resource in China, which is rich in flavonoids, polyphenols, chlorogenic acid, cyanidin glycoside and other active ingredients, and has excellent antioxidant and anti-aging potential. However, due to its dense plant cell wall structure, a large amount of active ingredients are wrapped in the cell in the form of combined state, and the traditional water extraction or organic solvent extraction method has the problems of low extraction efficiency, low bioavailability and the like, which seriously limits the high-value application of Rosa sertata x Rosa rugosa Yu.et.Ku. in the fields of functional food and high-end cosmetics.

[0003] Microbial fermentation technology is considered to be an effective means to break the plant cell wall and release and transform active ingredients. In the prior art, there have been attempts to ferment rose raw materials by using lactic acid bacteria, yeast and the like. For example: Patent document CN119614431A discloses a method for fermenting rose flowers by using Lactiplantibacillus plantarum, but it needs to be pretreated by complex papain, cellulase and other multi-step enzymolysis to break the wall before fermentation, which is complicated and increases the production cost and control difficulty.

[0004] Patent document CN119351288B discloses the fermentation of Grasse rose by using Lactiplantibacillus plantarum, which uses a single strain, but the rose variety used is Grasse rose, and does not involve the unique Rosa sertata x Rosa rugosa Yu.et.Ku. in terms of active ingredient composition. In addition, the anti-aging efficacy verification of the fermentation product only stays at the level of in vitro antioxidant, and fails to reveal its regulation effect on the core aging pathway such as skin cell collagen metabolism.

[0005] Another type of patent such as CN110200275A uses rose flower residue after extraction of essential oil as a substrate and uses a composite strain for fermentation, aiming to realize waste reuse. However, the active ingredient content of the substrate itself has been greatly lost due to the previous extraction, resulting in limited activity improvement of the final fermentation product and low economic value.

[0006] In summary, the existing rose fermentation technology generally has one or more of the following technical defects: Although there have been reports of using Lactiplantibacillus plantarum to ferment roses in the prior art, there are generally the following problems: (1) The strain did not have a specific match with the bitter rose; (2) It requires complex pretreatment steps such as enzymatic hydrolysis, making the process difficult to scale up; (3) The increase in key active ingredients is limited; (4) Lack of effective validation of skin aging pathways.

[0007] Therefore, there is an urgent need in this field to develop a fermentation technology and product that is simple in process, has strong specificity for strains and substrates, can significantly multiply the key active ingredients of bitter rose, and has a clear cellular-level anti-aging mechanism. Summary of the Invention

[0008] This invention aims to overcome the shortcomings of existing fermentation technologies for bitter rose, such as poor compatibility between strains and substrates, low release efficiency of active ingredients, complex fermentation processes, and unclear anti-aging mechanisms. It provides a method for targeted fermentation of bitter rose using a specific *Lactobacillus plantarum* strain TS337821. This method is simple, highly controllable, and can significantly increase the content of key active ingredients. Ultimately, it provides a bitter rose fermentation broth with clear anti-aging effects, its freeze-dried powder, and its applications in cosmetics and functional foods.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: First, this invention provides a method for preparing fermented broth from bitter rose, comprising the following steps: (1) Preparation of fermentation substrate: Take dried flower buds of bitter rose (Rosa sertata×Rosa rugosa Yu.et.Ku.), crush them through an 80-mesh sieve, add purified water at a material-to-liquid ratio of 1:25~35 (g / mL), mix well, sterilize at 115~125℃ for 15~30 minutes, cool to room temperature, and obtain fermentation substrate; (2) Activation of Lactobacillus plantarum: Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C for 12-16 hours to induce it to enter the logarithmic growth phase, thus preparing a highly active seed liquid; (3) Inoculation and fermentation: Under aseptic conditions, the activated Lactobacillus plantarum seed liquid in step (2) is inoculated into the fermentation substrate obtained in step (1) at an inoculation amount of 2%~4% (v / v), and fermented in a sealed environment at 35~40℃ for 40~50 hours. (4) Post-processing: After fermentation, the fermentation liquid was centrifuged at 8000 rpm for 10 minutes, the supernatant was taken, and then filtered through a 0.45μm microporous membrane to obtain the fermentation liquid of the bitter rose. As a further preferred option, the optimal fermentation conditions in step (3) are: fermentation temperature 37°C, fermentation time 45 hours, and inoculum amount 3% (v / v).

[0010] The key active ingredients in the fermented broth of bitter rose prepared by the above method were significantly enhanced, with the chlorogenic acid content increasing to 2.5 to 3.5 times that before fermentation and the cyanidin-3-O-glucoside content increasing to 2.0 to 3.0 times that before fermentation.

[0011] Secondly, the present invention provides a fermented liquid of bitter rose, which is prepared by the above method.

[0012] Finally, the present invention provides an anti-aging cosmetic composition comprising the aforementioned bitter rose ferment broth and a cosmetically acceptable carrier; wherein the bitter rose ferment broth has a mass fraction of 0.1% to 2.0% in the composition.

[0013] Compared with the prior art, the present invention has the following significant advantages: 1. Strong strain specificity and significant synergistic effect: This invention is the first to discover that Lactobacillus plantarum TS337821 has a unique substrate compatibility with Rose floridulus. Under conditions without enzymatic hydrolysis, the enzyme system secreted by Lactobacillus plantarum TS337821, such as β-glucosidase and pectinase, can efficiently break through the dense cell wall of Rose floridulus, achieving targeted release and transformation of active ingredients. This results in a multiple increase in the levels of key components such as chlorogenic acid and cyanidin glycoside (chlorogenic acid increased by 3.16 times and cyanidin-3-O-glucosinolate increased by 2.54 times), which is an unexpected technical effect. 2. The process is simple and controllable, can be stably scaled up, and is easy to industrialize: The method of this invention does not require complicated enzymatic pretreatment steps, and directly utilizes the strain's own enzyme system for biological cell disruption, greatly simplifying the process flow. At the same time, through precise optimization and control of key process parameters such as the material-liquid ratio, sterilization, activation, fermentation temperature, time, and inoculum size, the stability, reproducibility, and good industrial scale-up potential of the production process are ensured. 3. Clear Product Activity and High Application Value: The fermentation broth prepared by this invention not only significantly increases the content of active ingredients, but also demonstrates, through cell experiments, a clear anti-aging effect, effectively scavenging free radicals and promoting collagen synthesis. Cell experiments have verified that it can significantly increase COL-I, decrease MMP-1, enhance SOD / CAT / T-AOC, and reduce ROS, proving its multi-target anti-aging effect. The fermentation broth only requires a low addition amount (0.1%~2.0%) in cosmetics to achieve optimal results. The fermentation broth and freeze-dried powder developed in this invention can be used as high-efficiency raw materials, widely applied in anti-aging serums, creams, masks, and other cosmetics, as well as antioxidant functional foods, at low addition amounts (0.1%~2.0%), providing a new technical path and product form for the high-value development and utilization of bitter rose. Attached Figure Description

[0014] Figure 1 This is the standard curve of rutin drawn in Embodiment 1 of the present invention.

[0015] Figure 2 This is the standard curve of gallic acid plotted in Example 1 of the present invention.

[0016] Figure 3 This is a graph showing the effect of different bacterial strains (Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus oryzae) fermentation on the total flavonoid content in the fermentation broth of bitter rose over time in Example 1 of the present invention.

[0017] Figure 4 This is a graph showing the effect of different bacterial strains (Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus oryzae) fermentation on the total polyphenol content in the fermentation broth of bitter rose over time in Example 1 of the present invention.

[0018] Figure 5 The figure shows the results of a single-factor experiment in Example 2 of this invention to investigate the effect of fermentation time on the total flavonoid and total polyphenol content in the fermentation broth of bitter rose.

[0019] Figure 6 The figure shows the results of a single-factor experiment in Example 2 of this invention to investigate the effect of fermentation temperature on the total flavonoid and total polyphenol content in the fermentation broth of bitter rose.

[0020] Figure 7 The figure shows the results of a single-factor experiment in Example 2 of this invention to investigate the effect of inoculum size on the total flavonoid and total polyphenol content in the fermentation broth of *Rosa chinensis*.

[0021] Figure 8 This is a graph showing the results of detecting the effect of bitter rose fermentation broth on human fibroblast viability using the CCK-8 assay in Example 3 of the present invention.

[0022] Figure 9 The images shown are representative pictures (AD) of SA-β-Gal staining and a statistical chart (E) of the proportion of SA-β-Gal positive cells in Example 3 of this invention, used to show the effect of the fermentation broth of *Rosa chinensis* on cell senescence; (wherein, A: control group; B: fermentation broth group of *Rosa chinensis*; C: senescence model group; D: senescence model + fermentation broth group of *Rosa chinensis*).

[0023] Figure 10 This is a statistical graph showing the effect of the fermentation broth of bitter rose on the content of collagen I (coI-I) in cells of each group in Example 3 of the present invention.

[0024] Figure 11 This is a statistical chart showing the effect of the fermentation broth of *Rosa chinensis* on the content of matrix metalloproteinase-I (MMP-I) in each group of cells in Example 3 of the present invention.

[0025] Figure 12This is a statistical chart showing the effect of the fermentation broth of bitter rose on the superoxide dismutase (SOD) content of cells in each group in Example 3 of the present invention.

[0026] Figure 13 This is a statistical chart showing the effect of the fermentation broth of bitter rose on the catalase (CAT) content of cells in each group in Example 3 of the present invention.

[0027] Figure 14 This is a statistical chart showing the effect of the fermentation broth of bitter rose on the content of glutathione peroxidase (GSH-PX) in cells of each group in Example 3 of the present invention.

[0028] Figure 15 This is a statistical chart showing the effect of the fermentation broth of bitter rose on the total antioxidant capacity (T-AOC) of cells in each group in Example 3 of the present invention.

[0029] Figure 16 This is a statistical chart showing the effect of the fermentation broth of bitter rose on the content of reactive oxygen species (ROS) in each group of cells in Example 3 of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0031] Example 1: Screening of microbial strains and optimization of fermentation process 1. Experimental Materials The dried flower buds of the Kushui rose were harvested in Kushui Town, Yongdeng County, Gansu Province, and were identified by Professor Yang Fude of Gansu University of Traditional Chinese Medicine as dried flower buds of the Kushui rose (Rosa serata×Rosa rugosa Yu.et.Ku.), a plant of the Rosaceae family.

[0032] Lactobacillus plantarum TS337821, Saccharomyces cerevisiae TS345658, and Aspergillus oryzae TS3452630 were all purchased from Ningbo Taisto Biotechnology Co., Ltd.

[0033] MRS medium, malt extract medium, rutin standard, gallic acid standard, and other reagents are all commercially available.

[0034] 2. Experimental Methods 2.1 Preparation of fermentation substrate for bitter rose The dried flower buds of the bitter rose were pulverized using a pulverizer and passed through an 80-mesh sieve. 2.0 g of the powder was accurately weighed and placed in a 250 mL Erlenmeyer flask. 60 mL of ultrapure water was accurately added and thoroughly mixed. The mixture was then autoclaved at 121°C for 30 min. After cooling, the weight loss during sterilization was replenished with sterile ultrapure water to obtain the fermentation substrate.

[0035] 2.2 Preparation of Standard Curve Rutin Standard Curve: Accurately weigh 10 mg of rutin standard into a 10 mL volumetric flask, dissolve thoroughly, and dilute to the mark with ultrapure water. Then, pipette different volumes of the rutin standard solution into a set of 10 mL colorimetric tubes. Add 0.5 mL of 5% sodium nitrite solution to each tube, shake well, and let stand for 6 min; add 0.5 mL of 10% aluminum nitrate solution, shake well, and let stand for 6 min; add 2 mL of 4% sodium hydroxide solution, dilute to the mark with distilled water, shake well, and let stand for 15 min. Measure the absorbance at 510 nm. Plot the standard curve with concentration on the x-axis and absorbance on the y-axis as shown below. Figure 1 As shown, Y = 0.0835X + 0.0374, R 2 =0.9987.

[0036] Gallic acid standard curve: Accurately weigh 1.2 mg of gallic acid standard sample into a 10 mL volumetric flask, dissolve it completely, and dilute to the mark with ultrapure water. Then, pipette different volumes of gallic acid standard solution into a set of 10 mL colorimetric tubes, add 5 mL of 10% Folin-Ciocalteu reagent to each tube, shake well, and let stand for 6 min. Add 5 mL of 7.5% sodium carbonate solution, shake well, and let stand at room temperature for 60 min. Measure the absorbance at a wavelength of 765 nm. Plot the standard curve with concentration on the x-axis and absorbance on the y-axis as shown below. Figure 2 As shown, the linear equation and correlation coefficient are: Y = 0.0835X + 0.0374, R0 2 =0.9987.

[0037] 2.3 Preparation of microbial seed culture Lactobacillus plantarum seed culture: In a clean bench, Lactobacillus plantarum TS337821 was inoculated onto solid MRS medium and cultured at 37°C for 24 h. Single colonies with good growth were picked and transferred to MRS liquid medium and cultured at 37°C in a shaker for 16 h to obtain activated Lactobacillus plantarum seed culture.

[0038] Saccharomyces cerevisiae seed culture: Saccharomyces cerevisiae TS345658 was inoculated into solid malt extract medium and cultured in a constant temperature incubator at 35℃ for 3 days. Single colonies were picked and transferred into liquid malt extract medium and cultured in a constant temperature shaker at 35℃ for 36 hours to obtain the seed culture.

[0039] Aspergillus oryzae seed culture: The spore suspension of Aspergillus oryzae TS3452630 was inoculated into solid malt extract medium and cultured in a constant temperature incubator at 28℃ for 5 days. The spores were washed with sterile physiological saline, and the spore suspension was transferred to malt extract liquid medium and cultured in a constant temperature shaker at 28℃ for 48 h to obtain the seed culture.

[0040] 2.4 Fermentation and Sample Collection 3% (v / v) of the above three seed solutions were inoculated into the fermentation substrate of *Rosa kusnezoffii* prepared under section “2.1”.

[0041] Lactobacillus plantarum group: incubated statically at 37℃.

[0042] Saccharomyces cerevisiae group: incubated statically at 35℃.

[0043] Aspergillus oryzae group: incubated statically at 28℃.

[0044] Samples were taken at 0 h, 5 h, 10 h, 15 h, 20 h, 25 h, 35 h, 45 h, and 55 h of fermentation. The samples were sterilized again and the weight loss was made up with sterile ultrapure water. The samples were centrifuged at 8000 r / min for 10 min, and the supernatant was collected as the rose fermentation broth to be tested.

[0045] 2.5 Evaluation Index Determination The total flavonoid content was determined using the aluminum nitrate colorimetric method. The sample solution prepared in section 2.4 was diluted with water to a certain factor, and 1 mL was placed in a 10 mL volumetric flask. Subsequent experimental procedures were the same as those used in section 2.2 for plotting the rutin standard curve. The total flavonoid content in the sample was calculated based on the standard curve.

[0046] The total polyphenol content was determined using the Folin-Ciocalteu colorimetric method. The sample solution prepared in section 2.4 was diluted with water to a certain factor, and 1 mL was transferred to a 10 mL volumetric flask. Subsequent experimental procedures were the same as those used in section 2.2 for plotting the gallic acid standard curve. The total polyphenol content in the sample was calculated based on the standard curve.

[0047] 3. Experimental Results 3.1 Results of determination of total flavonoid content in fermentation broth The results are as follows Figure 3As shown in the figure, the total flavonoid content in the fermentation broth after fermentation with *Lactobacillus plantarum* increased compared to before fermentation within 0-55 hours. Rapid enrichment of total flavonoids was achieved between 0-45 hours, reaching a maximum at 45 hours, an increase of 39.47% compared to before fermentation. After 45 hours, the total flavonoid content began to decline. Similarly, the total flavonoid content in the fermentation broth after fermentation with *Saccharomyces cerevisiae* increased compared to before fermentation within 0-55 hours. The content gradually increased between 0-35 hours, reaching a maximum at 35 hours, an increase of 20.36% compared to before fermentation. After 35 hours, the total flavonoid content began to decline. After fermentation with *Aspergillus oryzae*, the total flavonoid content showed a trend of first slowly increasing and then slowly decreasing within 0-55 hours, reaching a maximum at 35 hours, an increase of 7.99% compared to before fermentation. After 35 hours, the total flavonoid content began to decline.

[0048] 3.2 Results of determination of total polyphenol content in fermentation broth The results are as follows Figure 4 As shown in the figure, the total polyphenol content in the fermentation broth after fermentation with *Lactobacillus plantarum* was higher than that before fermentation during the 0-55 h period. Rapid enrichment of total polyphenols was achieved between 0-20 h, with a slower increase between 20-35 h, reaching a maximum at 45 h, an increase of 35.81% compared to before fermentation, and then decreasing after 45 h. Similarly, the total polyphenol content in the fermentation broth after fermentation with *Saccharomyces cerevisiae* was also higher than that before fermentation during the 0-55 h period. The increase was faster between 0-5 h, with a slower increase between 5-35 h, reaching a maximum at 35 h, an increase of 16.90% compared to before fermentation, and then decreasing after 35 h. After fermentation with Aspergillus oryzae, the total polyphenol content showed a trend of first slowly increasing and then slowly decreasing between 0 and 55 hours, reaching a maximum at 35 hours, which was 12.52% higher than before fermentation. After 35 hours, the total polyphenol content began to decrease.

[0049] Conclusion: Using total flavonoids and total polyphenols as evaluation indicators, the most suitable strains for fermenting bitter rose were screened from Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae. The experimental results showed that the contents of total flavonoids and total polyphenols in the fermentation broth of bitter rose increased after fermentation by Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus oryzae, but the fermentation effect of Lactobacillus plantarum TS337821 was the best.

[0050] Example 2: Optimization of Lactobacillus plantarum fermentation process Based on the results of Example 1, using Lactobacillus plantarum TS337821 as the fermentation strain and the comprehensive score of total flavonoids and total polyphenols as the index, the fermentation process was optimized through single-factor experiments and response surface methodology.

[0051] 1. Single-factor experiment 1.1 Effect of fermentation time The effects of different fermentation times on the total flavonoid and total polyphenol content in the fermentation broth of bitter rose are shown in Figure 5. The figure shows that fermentation time has a certain impact on the content of total flavonoids and total polyphenols in the fermentation broth. Between 25 and 45 hours, the content of total flavonoids and total polyphenols gradually increases, reaching its maximum at 45 hours. Afterward, the content of total flavonoids and total polyphenols begins to decrease with increasing time. Therefore, 45 hours is selected as the optimal fermentation time.

[0052] 1.2 Effect of Fermentation Temperature The effects of different fermentation temperatures on the total flavonoid and total polyphenol content in the fermentation broth of bitter rose are shown in Figure 6. The figure shows that changes in fermentation temperature have a certain impact on the accumulation of total flavonoids and total polyphenols in the fermentation broth. Between 33℃ and 37℃, the contents of total flavonoids and total polyphenols gradually increase, reaching their maximum values ​​at 37℃. Subsequently, the contents of total flavonoids and total polyphenols gradually decrease with increasing temperature. Therefore, 37℃ was selected as the optimal fermentation temperature.

[0053] 2.3 Effect of inoculation amount The effects of different inoculum sizes of *Lactobacillus plantarum* on the total flavonoid and total polyphenol content in the fermentation broth of *Rosa kusnezoffii* are shown in Figure 7. The figure shows that the inoculum size between 2% and 3% had the greatest impact on the total flavonoid and total polyphenol content in the fermentation broth. At an inoculum size of 3%, the total polyphenol content in the fermentation broth reached its maximum. Afterward, with increasing inoculum size, the total flavonoid and total polyphenol content gradually decreased. Therefore, 3% was selected as the optimal inoculum size.

[0054] 2. Response Surface Optimization Experiment Based on the results of single-factor experiments, a three-factor, three-level response surface methodology was designed (fermentation time (A): 35 h, 45 h, 55 h; fermentation temperature (B): 35℃, 37℃, 39℃; inoculum size (C): 2%, 3%, 4%).

[0055] Regression analysis was performed using Design-Expert 13 software, and the quadratic regression model equation with the comprehensive score (Y) of total flavonoids and total polyphenols content as the response value was obtained as follows: Y=0.9315-0.0410A-0.0500B-0.0540C-0.0417AB-0.0585AC-0.0357BC-0.4280A 2 -0.3194B 2 - 0.2695C 2 The results of the analysis of variance are shown in Table 1. Table 1 Regression Analysis of Response Surface Experiment Table 1 shows that the model P < 0.0001, indicating that the regression model is highly significant; the lack-of-fit term P = 0.1243 > 0.05 is not significant, indicating that the model fits well, the data is accurate and reliable, and the model is valid; R 2 =0.9857, indicating that this model can explain 98.57% of the response value variation. In the model, R0... 2 pred=0.8267 and R 2 The adj=0.9672 value is close to the ideal level, indicating that the model has a small error and can be used to analyze and predict the fermentation process of bitter rose. The p-value for the linear term factor C is <0.05, while the p-values ​​for A and B are >0.05. The interaction terms AB, AC, and BC are all insignificant. 2 B 2 C 2 The p-value was <0.0001, indicating a highly significant difference.

[0056] In summary, this model and regression equation can fit the changing patterns of the response values ​​well, illustrating the interaction between the variables and the response values. The order of influence of each factor on the total flavonoid and total polyphenol content in the fermentation broth is: inoculum size > fermentation temperature > fermentation time.

[0057] 3. Optimal process verification Using Design Expert 13 software, a regression model was used for predictive analysis. The maximum predicted value of the comprehensive score was 0.94. The optimal process conditions were a fermentation time of 45 h, a fermentation temperature of 37℃, and an inoculum size of 3%. Under these conditions, three sets of validation experiments were conducted. The comprehensive scores of the three sets of validation experiments were 0.93, 0.89, and 0.91, respectively, with a mean of 0.91 and an RSD of 2.20%. This indicates that the established model has good predictive ability, and the optimized process is stable and feasible.

[0058] Under this optimal process, the content of basic components of bitter rose before and after fermentation was determined. The total flavonoid content was 57.98 mg / g, and the total polyphenol content was 117.86 mg / g. The total flavonoid content increased by 40.17% compared to before fermentation; the total polyphenol content increased by 36.51%; the total sugar content decreased by 9.7%; the reducing sugar content increased by 9.5%; and the crude protein content increased by 88.55%. The amino acid content analysis results before and after fermentation showed that a total of 24 amino acids were detected. Among them, the contents of histidine, 4-hydroxy-L-proline, arginine, glycine, aspartic acid, glutamic acid, alanine, γ-aminobutyric acid, L-ornithine, GABA, lysine, methionine, valine, isoleucine, leucine, and phenylalanine increased compared to before fermentation, by 189%, 15.6%, 309.7%, 493.3%, 7.41%, 154.6%, 36.6%, 12.8%, 460.3%, 9.31%, 459.8%, 21383.3%, 39.3%, 71.3%, and 243%, respectively. The percentages of aspartic acid, glutamic acid, serine, L-citrulline, threonine, proline, tyrosine, and tryptophan decreased after fermentation, by 21.8%, 25.8%, 20.7%, 54.4%, 20.6%, 13.7%, 21.8%, and 13.2%, respectively.

[0059] In addition, the contents of eight active ingredients of bitter rose before and after fermentation were determined, and the results are shown in Table 2 below. Table 2 Content of 8 active ingredients Table 2 shows that compared with the samples before fermentation, the contents of eight active ingredients—gallic acid, protocatechuic acid, cyanidin-3-O-glucoside, chlorogenic acid, rutin, ferulic acid, quercetin, and kaempferol—increased after fermentation. Among these, the increases in chlorogenic acid, cyanidin-3-O-glucoside, and protocatechuic acid were the most significant, increasing by 3.16 times, 2.54 times, and 1.61 times, respectively. The increases in rutin, gallic acid, ferulic acid, quercetin, and kaempferol were 1.39 times, 1.33 times, 1.09 times, 1.06 times, and 106 times, respectively. These results indicate that fermentation of bitter rose petals with *Lactobacillus plantarum* effectively increases the content of active ingredients.

[0060] Example 3: Verification of the anti-aging activity of fermented broth from bitter rose 1. Sample and model preparation Bitter rose fermentation broth was prepared according to the optimal process determined in Example 2 (Lactobacillus plantarum TS337821, inoculum amount 3%, fermentation at 37℃ for 45 h); a cell senescence model was prepared by inducing human fibroblasts with 10 g / L D-galactose for 72 h.

[0061] 2. Optimal volume fraction of fermented broth for bitter rose The CCK-8 test results show (e.g.) Figure 8 As shown in the figure, the cell activity of the control group was significantly higher than that of the senescent group, indicating that the cell senescence model was successfully prepared. Compared with the senescent group, the cell survival rate of the senescence model + bitter rose fermentation broth group was improved. The cell survival rate was the highest when the amount of bitter rose fermentation broth added was 0.75%. Therefore, the optimal volume fraction of bitter rose fermentation broth and the optimal treatment time for subsequent experiments were 0.75% and 24h.

[0062] 3. Effects of fermentation broth of *Rosa kusnezoffii* on senescence-specific SA-β-GaL activity in different groups SA-β-GaL staining results show (e.g.) Figure 9 As shown in the figure, there was no statistically significant difference in the proportion of SA-β-GaL positive cells between the bitter rose fermentation broth group and the control group (P=0.197); the proportion of SA-β-GaL positive cells in the aging model group was significantly higher than that in the control group (P=0.000); the proportion of SA-β-GaL positive cells in the aging model + bitter rose fermentation broth group was significantly lower than that in the aging model group (P=0.000).

[0063] 4. Effects of fermentation broth of *Rosa kusnezoffii* on the content of coI-I and MMP-I in cells of different groups. Human fibroblasts secrete collagen, and the amount of collagen is crucial for maintaining skin elasticity. As we age, the amount of collagen in the skin decreases, which is one of the main causes of skin aging. Results from coI-I content measurements show (e.g.) Figure 10 As shown in the figure, compared with the control group, the content of coi-I in the fermented broth group of bitter rose was significantly increased (P=0.000); compared with the control group, the content of coi-I in the aging model group was decreased (P=0.009); compared with the aging model group, the content of coi-I in the aging model + bitter rose fermented broth group was increased (P=0.013). This indicates that the fermented broth of bitter rose has a significant effect on promoting coi-I synthesis and can play a role in delaying aging.

[0064] MMP-I is a class of enzymes that degrade collagen. To achieve a better anti-aging effect, it's necessary to inhibit MMP-I activity while promoting collagenase synthesis. The results of MMP-I content determination show (e.g.) Figure 11 As shown in the figure, there was no statistically significant difference in MMP-I content between the Kushui rose fermentation broth group and the control group (P=0.674); compared with the control group, the aging model group had increased MMP-I content (P=0.001); compared with the aging model group, the aging model + Kushui rose fermentation broth group had decreased MMP-I content (P=0.001). This indicates that Kushui rose fermentation broth can significantly inhibit the synthesis of MMP-I and play a role in delaying aging.

[0065] 5. Effects of fermentation broth of *Rosa kusnezoffii* on the levels of SOD, CAT, GSH-PX, T-AOC, and ROS in cells of different groups. Superoxide dismutase (SOD) is an antioxidant enzyme present in the body, responsible for scavenging free radicals. As we age, the activity of this enzyme declines. Insufficient production of antioxidant enzymes leads to the inability to promptly eliminate free radicals, causing tissue damage and cell apoptosis—a major cause of aging. SOD content measurements show (e.g.) Figure 12 As shown in the figure, there was no statistically significant difference in SOD content between the fermented broth group and the control group (P=0.414); the SOD content in the aging model group was significantly lower than that in the control group (P=0.000); and the SOD content in the aging model + fermented broth group was significantly higher than that in the aging model group (P=0.019). This indicates that fermented broth can significantly enhance SOD activity and thus play a role in delaying aging.

[0066] CAT and GSH-PX are also antioxidant enzymes present in the body. The results of CAT content determination show (e.g.) Figure 13 As shown in the figure, there was no statistically significant difference in CAT content between the fermented broth group and the control group (P=0.727); the CAT content in the aging model group was significantly lower than that in the control group (P=0.000); and the CAT content in the aging model + fermented broth group was significantly higher than that in the aging model group (P=0.002). This indicates that fermented broth can significantly enhance CAT activity and thus play a role in delaying aging.

[0067] The results of GSH-PX content determination show (e.g.) Figure 14As shown in the figure, there was no statistically significant difference in GSH-PX content between the Bitter Rose Fermentation Broth group and the control group (P=0.113); the GSH-PX content in the aging model group was significantly lower than that in the control group (P=0.001); there was no statistically significant difference in GSH-PX content between the aging model + Bitter Rose Fermentation Broth group and the aging model group (P=0.071).

[0068] The strength of T-AOC is closely related to the body's ability to scavenge free radicals; the stronger the total antioxidant capacity, the stronger the ability to scavenge free radicals. Therefore, it is necessary to measure the total antioxidant capacity. The results of T-AOC measurement show (e.g.) Figure 15 As shown in the figure, there was no statistically significant difference in T-AOC between the Kushui rose fermentation broth group and the control group (P=0.371); compared with the control group, the TAOC in the aging model group was significantly lower (P=0.000); compared with the aging model group, the T-AOC in the aging model + Kushui rose fermentation broth group was significantly higher (P=0.002), indicating that Kushui rose fermentation broth can significantly enhance TAOC and play a role in delaying aging.

[0069] Because reactive oxygen species (ROS) have strong redox capabilities, they can cause cell damage and lead to aging. Therefore, the lower the ROS content in the body, the slower the aging process. Results of reactive oxygen species content measurements show (e.g.) Figure 16 As shown in the figure, compared with the control group, the ROS content of the fermented broth group of bitter rose was significantly increased (P=0.000); compared with the control group, there was no statistically significant difference in ROS content between the aging model group and the control group (P=0.006); compared with the aging model group, the ROS content of the aging model + bitter rose fermented broth group was significantly decreased (P=0.027), indicating that the fermented broth of bitter rose can significantly reduce the ROS content and play a role in delaying aging.

[0070] In summary, the fermented broth of bitter rose can reduce the activity of SA-β-GaL in human fibroblasts, enhance the activity of coi-I, inhibit the activity of MMP-I, restore the activity of SOD, CAT, and GSH-PX in cells, enhance the body's T-AOC, and reduce the content of ROS, thus achieving the effect of delaying aging through a multi-target mechanism.

[0071] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing fermented broth of bitter rose, characterized in that, Includes the following steps: (1) Preparation of fermentation substrate: Take dried flower buds of bitter rose, crush them and pass them through an 80-mesh sieve. Add purified water at a material-to-liquid ratio of 1:25–35 g / mL, sterilize at 115–125℃ for 15–30 min and then cool. (2) Activation of strains: Inoculate Lactobacillus plantarum into MRS medium and culture at 37°C for 12–16 h to bring it into the logarithmic growth phase; (3) Fermentation: The seed liquid obtained in step (2) is inoculated into the substrate obtained in step (1) at an inoculation rate of 2-4%, and fermented at 35-40℃ for 40-50 h; (4) Post-processing: The fermentation broth was centrifuged and filtered through a 0.45 μm microporous membrane to obtain the fermentation broth of bitter rose.

2. The method according to claim 1, characterized in that: The method also includes optimizing fermentation time, fermentation temperature, and inoculum size using response surface methodology to obtain the process conditions with the highest overall score for total flavonoid and total polyphenol content.

3. The method according to any one of claims 1 or 2, characterized in that, The fermentation conditions in step (3) are: fermentation temperature 37℃, fermentation time 45 hours, and inoculum size 3%.

4. The method according to claim 1, characterized in that: The fermentation broth prepared by the method described above contains 2.5 to 4.0 times more chlorogenic acid and 2.0 to 3.0 times more cyanidin-3-O-glucoside than before fermentation.

5. The method according to claim 1, characterized in that: In the fermentation broth, the content of protocatechuic acid increased by 1.3 to 2.0 times compared with that before fermentation, the total flavonoid content increased by ≥35%, and the total polyphenol content increased by ≥30%.

6. A fermented liquid of bitter rose, characterized in that, Prepared by the method according to any one of claims 1 to 5.

7. A fermented liquid of bitter rose as described in claim 6, characterized in that, It has at least two of the following features (a) to (c): (a) The chlorogenic acid content is not less than 2.5 times that before fermentation; (b) The content of cyanidin-3-O-glucoside is not less than 2.0 times that before fermentation; (c) It can reduce β-galactosidase activity, increase type I collagen expression and / or reduce matrix metalloproteinase-1 content in a human fibroblast aging model.

8. The fermentation broth according to claim 7, characterized in that: When the fermentation broth was used in a D-galactose-induced human fibroblast senescence model at a volume fraction of 0.75%, it significantly reduced the proportion of β-galactosidase-positive cells and significantly increased the intracellular activities of superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC).

9. An anti-aging cosmetic composition, characterized in that, The composition comprises the fermented broth of the bitter rose as described in claim 6 and a cosmetically acceptable carrier; wherein the volume fraction of the fermented broth of the bitter rose in the composition is 0.1% to 2.0%.

10. The anti-aging cosmetic composition as described in claim 9, characterized in that, The volume fraction of the fermented broth of the bitter rose in the composition is 0.75%.

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