Rose fermentation liquor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fermentation, and particularly relates to rose fermentation liquor as well as a preparation method and application thereof. Comprising the following steps: performing enzymolysis on rose pulp by utilizing pectinase and cellulase to obtain rose enzymatic hydrolysate; mixing the rose enzymatic hydrolysate, saccharides and yeast peptone to obtain a mixed solution, sterilizing the mixed solution, and fermenting the mixed solution by using plant lactobacillus LP-Only to obtain a rose fermentation solution; the rotating speed of the fermentation is 30-50r / min, the fermentation temperature is 36-38 DEG C, and the fermentation time is 20-28h. The rose fermentation liquor prepared by the invention has whitening and anti-oxidation effects, and when the rose fermentation liquor is sealed and placed at room temperature for 9 months, the viable bacteria of plant lactobacillus are kept at 106 CFU / mL or above.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a rose fermentation broth, its preparation method, and its application. Background Technology

[0002] Skin whitening and anti-oxidation are popular concepts in both the health food and cosmetics industries. As people's living standards improve, they are no longer limited to simply having enough to eat and wear; they are paying more attention to their appearance, making truly effective skin whitening and anti-oxidation products a key focus. However, the fast pace of modern life and the high prices and difficulty in frequently using skincare products negatively impact the effectiveness of whitening products. Therefore, developing affordable, more effective, and easier-to-consistently-used whitening products meets market demand.

[0003] Roses, belonging to the Rosaceae family, possess not only high ornamental value but also significant nutritional and medicinal value. Fermented roses, with their combined floral aroma and potential health benefits, have garnered considerable attention. However, fermented products contain a large number of live microorganisms. Direct packaging can lead to spoilage due to the proliferation of unwanted microorganisms, and the number of live bacteria cannot be maintained stably during storage, severely impacting product quality and efficacy. Current technologies typically sterilize the fermentation broth to kill all live bacteria and halt metabolic activity, thus achieving long-term product preservation. Therefore, how to maximize the retention of functional live bacteria during long-term storage has become a pressing technical challenge in the industrial production of rose-fermented beverages. Summary of the Invention

[0004] The purpose of this invention is to provide a rose fermentation broth, its preparation method, and its application. The resulting rose fermentation broth has whitening and antioxidant effects, and when sealed and stored at room temperature for 9 months, the viable bacteria count of *Lactobacillus plantarum* remains at 10%. 6 CFU / mL or higher.

[0005] This invention provides a method for preparing rose fermentation broth, comprising the following steps: enzymatically hydrolyzing rose petal slurry using pectinase and cellulase to obtain rose enzymatic hydrolysate; mixing and sterilizing the rose enzymatic hydrolysate, sugars, and yeast peptone, and then using *Lactobacillus plantarum* (… Lactobacillus plantarum LP-Onlly was used for fermentation to obtain rose fermentation broth; the fermentation speed was 30~50 r / min, the fermentation temperature was 36~38℃, and the fermentation time was 20~28 h; The pectinase accounts for 0.03% to 0.05% of the rose petal sap by mass; the cellulase accounts for 0.03% to 0.05% of the rose petal sap by mass. The preservation number of *Lactobacillus plantarum* LP-Onlly is CGMCC No. 1258; the inoculation volume of *Lactobacillus plantarum* LP-Onlly is 1%~2% of the volume of the rose enzymatic hydrolysate; the viable count of *Lactobacillus plantarum* LP-Onlly is ≥1.0×10⁻⁶. 9 CFU / mL.

[0006] As a preferred embodiment, the fermentation pressure is 0.03~0.07 MPa.

[0007] As a preferred embodiment, the mass-to-volume ratio of the sugars to the rose hydrolysate is 8-12 g / L; the mass-to-volume ratio of the yeast peptone to the rose hydrolysate is 8-12 g / L.

[0008] As a preferred embodiment, the enzymatic hydrolysis temperature is 40~45℃ and the enzymatic hydrolysis time is 3~4h.

[0009] As a preferred embodiment, the sterilization process further includes adjusting the pH of the mixture to 6.5-7.0.

[0010] As a preferred embodiment, the method for preparing the rose petal slurry includes: grinding rose petals with water to obtain rose petal slurry; wherein the mass ratio of rose petals to water is 1:25~35.

[0011] As a preferred embodiment, the grinding tool includes a colloid mill.

[0012] As a preferred embodiment, the pretreatment of the roses includes: soaking the roses in water for 1-2 hours at a water temperature of 30-40°C.

[0013] This invention provides a rose fermentation broth prepared using the above-described method, wherein the pH of the rose fermentation broth is 3-4, and the viable count of the rose fermentation broth is ≥6×10⁻⁶. 6 CFU / mL.

[0014] This invention provides the application of the above preparation method or the above rose fermentation broth in the preparation of whitening and antioxidant products.

[0015] Beneficial effects: This invention provides a method for preparing rose fermentation broth, comprising the following steps: enzymatically hydrolyzing rose slurry using pectinase and cellulase to obtain rose enzymatic hydrolysate; mixing and sterilizing the rose enzymatic hydrolysate, sugars, and yeast peptone, and then using *Lactobacillus plantarum* (… Lactobacillus plantarumRose fermentation broth was obtained by fermentation with LP-Onlly; the fermentation speed was 30-50 r / min, the fermentation temperature was 36-38℃, and the fermentation time was 20-28 h; the pectinase content was 0.03%-0.05% of the rose broth mass; the cellulase content was 0.03%-0.05% of the rose broth mass; the preservation number of *Lactobacillus plantarum* LP-Onlly was CGMCC No. 1258; the inoculation volume of *Lactobacillus plantarum* LP-Onlly was 1%-2% of the rose enzymatic hydrolysate volume; the viable count of *Lactobacillus plantarum* LP-Onlly was ≥1.0 × 10⁻⁶. 9 CFU / mL. The preparation method of this invention is simple, utilizing rose petals and *Lactobacillus plantarum* LP-Onlly to prepare rose fermentation broth. The fermentation broth is not sterilized, thus preserving the viable bacteria properties relatively well. The resulting rose fermentation broth can inhibit tyrosinase activity, thereby reducing melanin production and achieving a whitening effect, while also possessing strong antioxidant capabilities. The pH of the rose fermentation broth of this invention is 3-4, which can inhibit the growth of other bacteria, ensuring the safety of the live bacteria product within its shelf life. The rose fermentation broth prepared by this invention, when stored at room temperature under sealed conditions for 9 months, still maintains a viable count of *Lactobacillus plantarum* of 10. 6 With a CFU / mL or higher, it can retain the maximum number of functional live bacteria during long-term storage and remains safe and effective within its shelf life.

[0016] This invention provides a rose fermentation broth prepared using the above-described method, wherein the pH of the rose fermentation broth is 3-4, and the viable count of the rose fermentation broth is ≥6×10⁻⁶. 6 CFU / mL. The pH of the rose fermentation broth described in this invention is 3-4, which can inhibit the growth of other bacteria, ensuring the safety of the live bacteria product within its shelf life. Furthermore, even after being stored at room temperature under sealed conditions for 9 months, the viable count of *Lactobacillus plantarum* remains at 10. 6 The CFU / mL level is above 1. The rose fermentation broth described in this invention has both whitening and antioxidant effects, and its bacterial count remains high even after long-term storage.

[0017] This invention provides the application of the above-described preparation method or the above-described rose fermentation broth in the preparation of whitening and antioxidant products. The rose fermentation broth prepared by the method of this invention can inhibit tyrosinase activity, thereby reducing melanin production and achieving a whitening effect, while also possessing strong antioxidant capacity. The results of the examples show that after treatment with the rose fermentation broth prepared by the method of this invention, the melanin synthesis rate of cells is only 71.80%±0.55%, significantly lower than that of rose fermentation broth prepared by other methods; the DPPH free radical scavenging rate reaches 65.16%±0.96%, and the hydroxyl free radical scavenging rate reaches 81.43%±0.61%, significantly higher than that of rose fermentation broth prepared by other methods.

[0018] Biological Preservation Information Lactobacillus plantarum ( Lactobacillus plantarum LP-Onlly was deposited on December 6, 2004, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 1258. Detailed Implementation

[0019] This invention provides a method for preparing rose fermentation broth, comprising the following steps: enzymatically hydrolyzing rose petal slurry using pectinase and cellulase to obtain rose enzymatic hydrolysate; mixing and sterilizing the rose enzymatic hydrolysate, sugars, and yeast peptone, and then using *Lactobacillus plantarum* (… Lactobacillus plantarum LP-Onlly was used for fermentation to obtain rose fermentation broth; the fermentation speed was 30~50 r / min, the fermentation temperature was 36~38℃, and the fermentation time was 20~28 h; The pectinase accounts for 0.03% to 0.05% of the rose petal sap by mass; the cellulase accounts for 0.03% to 0.05% of the rose petal sap by mass. The preservation number of *Lactobacillus plantarum* LP-Onlly is CGMCC No. 1258; the inoculation volume of *Lactobacillus plantarum* LP-Onlly is 1%~2% of the volume of the rose enzymatic hydrolysate; the viable count of *Lactobacillus plantarum* LP-Onlly is ≥1.0×10⁻⁶. 9 CFU / mL.

[0020] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0021] This invention preferably selects roses free from mold and impurities, removes the stems, and pre-treats the rose petals by soaking them in water. The roses used in this invention can be fresh or dried. The soaking time can be any value within the range of 1 to 2 hours, for example, 1, 1.2, 1.5, 1.7, or 2 hours; the water temperature can be any value within the range of 30 to 40°C, for example, 30, 35, or 40°C. Soaking softens the fibrous tissue, relaxes the cell walls, and improves pulping efficiency and pulp fineness.

[0022] In this invention, the soaked rose petals are preferably ground with water to obtain a rose petal slurry. The mass ratio of rose petals to water in this invention can be any value within the range of 1:25 to 35, for example, 1:25, 1:27, 1:30, 1:32, or 1:35. As a preferred embodiment, the grinding tool used in this invention includes a colloid mill; the ultrafine grinding by the colloid mill allows for a more complete release of the anthocyanins, vitamin C, and other effective components in the rose petals.

[0023] This invention utilizes pectinase and cellulase to enzymatically hydrolyze the aforementioned rose petal sap, obtaining a rose petal hydrolysate. The mass of the pectinase can be any value within the range of 0.03% to 0.05% of the rose petal sap mass, for example, 0.03%, 0.04%, or 0.05%; the mass of the cellulase can also be any value within the range of 0.03% to 0.05% of the rose petal sap mass, for example, 0.03%, 0.04%, or 0.05%. In a specific embodiment of this invention, both the pectinase and cellulase were purchased from Henan Ganchun Food Co., Ltd., and both had an enzyme activity of 50,000 U. The enzymatic hydrolysis temperature can be any value within the range of 40 to 45°C, for example, 40, 42, 43, 44, or 45°C, and the hydrolysis time can be any value within the range of 3 to 4 hours, for example, 3, 3.5, or 4 hours. The pectinase first decomposes the pectin to reduce viscosity, while the cellulase destroys the cellulose skeleton; together, they achieve complete degradation of the cell wall.

[0024] This invention mixes the above-mentioned rose enzymatic hydrolysate, sugars, and yeast peptone to obtain a mixed solution. The mass-to-volume ratio of sugars to rose enzymatic hydrolysate can be any value within the range of 8-12 g / L, for example, 8, 9, 10, 11, or 12 g / L; the mass-to-volume ratio of yeast peptone to rose enzymatic hydrolysate can also be any value within the range of 8-12 g / L, for example, 8, 9, 10, 11, or 12 g / L. As a preferred embodiment, the sugars include sucrose and / or glucose. Because rose juice contains a large amount of tannins and other phenolic substances, and only a small amount of sugars, it lacks adequate carbon and nitrogen sources. Furthermore, when *Lactobacillus plantarum* LP-Onlly is inoculated with sugars or yeast peptone alone, its growth in the fermentation broth is slow or nonexistent. However, by simultaneously adding sugars and yeast peptone within a suitable range, the rapid growth of *Lactobacillus plantarum* LP-Onlly can be facilitated. As a preferred option, the pH of the above mixture is adjusted to any value within the range of 6.5 to 7.0, such as 6.5, 6.8, or 7.0. Adjusting the pH provides suitable fermentation conditions for *Lactobacillus plantarum* LP-Onlly.

[0025] This invention sterilizes the above-mentioned mixture. As a preferred embodiment, the sterilization method of this invention includes pasteurization; pre-fermentation sterilization can eliminate contaminating microorganisms. The pasteurization temperature of this invention can be any value within the range of 75~85℃, for example, 75, 80, or 85℃; the pasteurization time can be any value within the range of 3~7 minutes, for example, 3, 5, or 7 minutes.

[0026] This invention utilizes *Lactobacillus plantarum* LP-Onlly, preferably involving activation of *Lactobacillus plantarum* LP-Onlly before fermentation to obtain an activated solution. The *Lactobacillus plantarum* LP-Onlly described in this invention has the preservation number CGMCC No. 1258. As a preferred embodiment, *Lactobacillus plantarum* LP-Onlly is inoculated onto MRS liquid medium for activation, with two consecutive generations of activation, adjusting the viable cell count in the activated solution to 1.0 × 10⁻⁶. 9CFU / mL. This invention preferably utilizes the activated *Lactobacillus plantarum* LP-Onlly for fermentation to obtain rose fermentation broth. The inoculation volume of the activated *Lactobacillus plantarum* LP-Onlly solution can be any value within the range of 1% to 2% of the rose enzymatic hydrolysate volume, for example, 1%, 1.5%, or 2%. In a specific embodiment of this invention, the *Lactobacillus plantarum* LP-Onlly was purchased from Shanghai Nord Biotechnology Co., Ltd.; the *Lactobacillus plantarum* LP-Onlly has a good auxiliary effect in enhancing the whitening effect. The fermentation pressure of this invention can be any value within the range of 0.03 to 0.07 MPa, for example, 0.03, 0.04, 0.05, 0.06, or 0.07 MPa. As a preferred embodiment, the fermentation equipment includes: a fermentation tank; the fermentation tank can provide sterile air pressure for static fermentation. The fermentation speed of this invention can be any value within the range of 30 to 50 r / min, for example, 30, 40, or 50 r / min; a suitable speed is beneficial for the homogenization of substances and more thorough fermentation. The fermentation temperature described in this invention can be any value within the range of 36~38℃, for example, 36, 37, or 38℃; the fermentation time can be any value within the range of 20~28h, for example, 20, 22, 24, 26, or 28h. Suitable fermentation conditions are beneficial to improving the whitening and antioxidant effects of the fermentation liquid. The results of the examples show that extending the fermentation time worsens both the whitening and antioxidant effects of the rose fermentation liquid. As a preferred embodiment, the fermentation process further includes filtration; the mesh size of the filter cloth used for filtration is any value within the range of 750~850 mesh, for example, 750, 800, or 850 mesh; the collected filtrate is the rose fermentation liquid.

[0027] This invention provides a rose fermentation broth prepared using the above-described method, wherein the pH of the rose fermentation broth is any value within the range of 3 to 4, for example, 3, 3.5, or 4; and the viable count of the rose fermentation broth is ≥6 × 10⁻⁶. 6 CFU / mL, for example 6×10 6 9.5×10 6 7.3×10 7 Or 8.9×10 8 CFU / mL.

[0028] This invention provides the application of the above-described preparation method or the above-described rose fermentation broth in the preparation of whitening and antioxidant products. As a preferred embodiment, the products of this invention include at least one of the following: food, health products, and cosmetics. The rose fermentation broth prepared by the method of this invention can inhibit tyrosinase activity, thereby reducing melanin production and achieving a whitening effect, while also possessing strong antioxidant capacity. The results of the examples show that after treatment with the rose fermentation broth prepared by the method of this invention, the cellular melanin synthesis rate is only 71.80%±0.55%, significantly lower than that of rose fermentation broth prepared by other methods; the DPPH free radical scavenging rate reaches 65.16%±0.96%, and the hydroxyl free radical scavenging rate reaches 81.43%±0.61%, significantly higher than that of rose fermentation broth prepared by other methods.

[0029] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a rose fermentation broth, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Reagents used in the experiment: Lactobacillus plantarum ( Lactobacillus plantarum LP-Onlly, accession number CGMCCNO.1258; Lactobacillus plantarum 299V Lactobacillus plantarum subsp. plantarum The strain numbers are: SHIMCC (SHBCC) D24745, DSM9843, purchased from Shanghai Jiachu Bioengineering Co., Ltd. Pectinase and cellulase were both purchased from Henan Ganchun Food Co., Ltd., and their enzyme activity was 50,000 U. Fetal bovine serum (FBS), catalog number: 04-001-1ACS, purchased from Bioind, Israel. Penicillin-Streptomycin Solution, catalog number: 15140122, purchased from Thermo Fisher Scientific, USA; RPMI-1640 culture medium, catalog number: 11875093, purchased from Thermo Fisher Scientific; DMEM culture medium, batch number: 11965092, purchased from Thermo Fisher Scientific, USA; DPPH free radical scavenging reagent kit, catalog number: A153-1-1, purchased from Nanjing Jiancheng Biotechnology Institute; Hydroxyl radical assay kit, catalog number: A018-1-1, purchased from Nanjing Jiancheng Bioengineering Institute.

[0031] Example 1 Whitening and antioxidant rose fermented liquid is prepared by the following steps: (1) Preparation of seed culture: Take out Lactobacillus plantarum LP-Onlly from the -80℃ freezer, inoculate it into MRS liquid medium at 2% (V / V), and culture at 37℃ for 12h. Activate for two generations and adjust the viable count in the activation solution to 1.0×10⁻⁶. 9 CFU / mL was used to obtain the Lactobacillus plantarum LP-Onlly activation solution; The MRS liquid culture medium consisted of: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium citrate, 2.6 g / L K₂PO₄·3H₂O, 0.1 g / L MgSO₄·7H₂O, 0.05 g / L LmnSO₄, 1 mL / L Tween 80, and 0.5 g / L cysteine ​​phosphate, with a pH of 6.8.

[0032] (2) Screening: Select 1 kg of dried rose petals that are free from mold and impurities, remove the stems, crush the petals and soak them in warm water (40℃) for 2 hours.

[0033] (3) Pulping: After soaking, the crushed rose petals are added to 35kg of purified water according to the weight ratio, and then ground in a colloid mill to obtain rose petal pulp.

[0034] (4) Enzymatic hydrolysis: Add 18g of pectinase and 18g of cellulase to the rose petal slurry obtained in step (3), that is, the added mass of pectinase and cellulase accounts for 0.05% of the mass of rose petal slurry, and hydrolyze at 45℃ for 4h to obtain rose petal hydrolysate.

[0035] (5) Compound preparation: Add food-grade sucrose and yeast peptone to the rose enzymatic hydrolysate. Take 1L of rose enzymatic hydrolysate, add 10g of sucrose and 10g of yeast peptone, and add food-grade sodium citrate to adjust the pH to 6.5.

[0036] (6) Pasteurization: The compounded rose enzymatic hydrolysate is pasteurized, that is, sterilized in an 80°C water bath for 5 minutes.

[0037] (7) Fermentation culture: Inoculate the sterilized rose enzymatic hydrolysate with Bacillus plantarum LP-Onlly activation solution. The inoculation volume of Bacillus plantarum LP-Onlly activation solution is 2% (v / v) of the rose enzymatic hydrolysate. Fermentation is carried out in a fermenter, i.e., aseptic air pressure static fermentation, tank pressure 0.05MPa, rotation speed 50r / min, fermentation at 37℃ for 24h. After fermentation, the fermentation product is filtered through an 800 mesh filter cloth and the filtrate is collected as fermentation liquid.

[0038] (8) Filling: Fill and seal the fermentation liquid obtained in step (7).

[0039] Example 2 The process was carried out in accordance with Example 1, except that in step (7), the fermentation speed was 50 r / min.

[0040] Example 3 The process is carried out in accordance with Example 1, except that in step (7), the tank pressure is 0.03 MPa during fermentation.

[0041] Comparative Example 1 The process is carried out in accordance with Example 1, except that in step (7), the fermentation speed of 50 r / min is replaced with 0 r / min.

[0042] Comparative Example 2 The procedure was carried out in accordance with Example 1, except that in step (7), the inoculation volume of Lactobacillus plantarum LP-Onlly activation solution was replaced with 0.2% of the volume of rose enzymatic hydrolysate instead of 2%.

[0043] Comparative Example 3 The procedure was carried out in accordance with Example 1, except that in step (7), the inoculated Lactobacillus plantarum LP-Onlly was replaced with Lactobacillus plantarum 299V.

[0044] Comparative Example 4 The process is carried out in accordance with Example 1, except that no sucrose is added during the compounding in step (5).

[0045] Comparative Example 5 The process is carried out in accordance with Example 1, except that yeast peptone is not added during the compounding in step (5).

[0046] Comparative Example 6 The process is carried out in accordance with Example 1, except that in step (7), the fermentation time of 24h is replaced with 30h.

[0047] During the research, it was found that after adding sucrose or yeast peptone alone and then inoculating with *Lactobacillus plantarum* LP-Onlly for 24 hours of fermentation, the OD of Comparative Example 4 was significantly lower. 600 The OD of Comparative Example 5 is 0.5. 600 The value was 0.7. Because rose juice contains a large amount of tannins and other phenolic substances, only a small amount of sugars, and lacks adequate carbon and nitrogen sources, and because the fermentation broth obtained from comparative ratios 4 and 5 was not further tested after adding sucrose or yeast peptone alone and then inoculating with *Lactobacillus plantarum*, *Lactobacillus plantarum* LP-Onlly grew slowly or not at all in the fermentation broth.

[0048] Experimental Example 1: Whitening Effect Study 1.1 Culture and passage of B16 cells (mouse melanoma cells) B16 cells were cultured in RPMI-1640 medium (hereinafter referred to as antibiotic-containing RPMI-1640 medium) containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin antibiotic solution in cell culture flasks. The medium was 10% FBS and 1% penicillin-streptomycin antibiotic solution. The flasks were placed in a 37°C incubator containing 5% CO2. When the cells covered more than 80% of the bottom area of ​​the flask, the original culture medium was discarded. 1 mL of 0.25% trypsin was added to digest the adherent B16 cells for 15 seconds. The cells were then observed under an inverted microscope. When the cells became rounded, 4 mL of antibiotic-containing RPMI-1640 medium was added to stop the digestion. The cells were then resuspended to obtain B16 cells in the logarithmic growth phase. After adjusting the cell concentration, subsequent experiments could be performed.

[0049] 1.2 Effects of Rose Fermented Beverage on B16 Cell Viability Take the B16 cells in the logarithmic growth phase obtained in 1.1 and adjust the cell concentration to 5.0 × 10⁻⁶. 4100 μL of the culture medium was inoculated into each well of a 96-well plate at a concentration of 1 / mL and incubated at 37°C for 24 h. The original culture medium was removed, and 100 μL of RPMI-1640 complete medium containing rose fermentation broth from different treatment groups (2% of the total culture medium volume) was added to each well of the experimental groups. Specifically, the rose fermentation broths obtained in Example 1, Comparative Examples 1-3, and Comparative Example 6 were added, and the blank group was inoculated with an equal volume of medium. Each concentration was used in triplicate. After incubation at 37°C for another 24 h, 10 μL of MTT (thiazolyl blue, 5 mg / mL) was added in the dark, and the plate was then incubated at 37°C for another 4 h. The supernatant was removed, and 100 μL of DMSO (dimethyl sulfoxide) was added, followed by incubation in the dark for 10 min. The absorbance (A) of each well was measured at 570 nm using a microplate reader. The cell viability R was calculated using the following formula: R = (A1 / A3) × 100%; where A1 and A3 are the absorbance values ​​of the experimental group and the control group, respectively. The effect of different rose fermentation broths on the viability of B16 cells was determined using the MTT assay described above, and the results are shown in Table 1.

[0050] Table 1. Effects of different rose fermentation broths on the survival rate of B16 cells.

[0051] Note: Data are expressed as Mean ± SD (n=3), and different letters indicate significant differences between different groups. P <0.05.

[0052] As shown in Table 1, neither the examples nor the comparative examples had any effect on cell viability.

[0053] 1.3 Determination of Tyrosinase (TYR) Inhibition Rate Take the B16 cells in the logarithmic growth phase obtained in 1.1 and adjust the cell concentration to 5.0 × 10⁻⁶. 4100 μL of the culture medium was inoculated into each well of a 96-well plate at a concentration of 1 / mL and incubated at 37°C for 24 h. The original culture medium was removed, and 100 μL of RPMI-1640 complete medium containing different treatment group rose fermentation broths (2% of the total culture medium volume) was added to each well of the experimental groups. Specifically, the rose fermentation broths obtained in Example 1, Comparative Examples 1-3, and Comparative Example 6 were added, and the blank group was treated with an equal volume of medium. Each concentration was used in triplicate. The plates were incubated at 37°C for another 24 h. The 96-well plates were then removed, the supernatant was removed, and the plates were washed twice with PBS buffer. 100 μL of 1% Triton X-100 solution was added to each well, and the plates were sonicated and preheated at 37°C for 5 min. Then, 100 μL of PBS solution containing 1 mg / mL L-DOPA was added, and the plates were incubated at 37°C for 1 h. The absorbance (A) of each well was measured at 490 nm using a microplate reader, and the TYR inhibition rate (R) was calculated using the following formula. R = [1 - (A1 / A3)] × 100%; where A1 and A3 are the absorbance values ​​of the experimental group and the blank group, respectively. The inhibitory effect of different rose fermentation broths on TYR was determined, and the results are shown in Table 2.

[0054] Table 2. Inhibitory effects of different rose fermentation broths on TYR.

[0055] Note: Data are expressed as Mean ± SD (n=3), and different letters indicate significant differences between different groups. P <0.05.

[0056] As shown in Table 2, all different rose fermentation broths exhibited significant TYR inhibition. The inhibition effect of Example 1 was the strongest, while the inhibition effect of the comparative example was weaker. This indicates that the fermentation culture method, the amount of inoculum, the inoculated strain, and the fermentation time have a significant impact on the final TYR inhibition rate.

[0057] 1.4 Melanin Synthesis Inhibition Experiment B16 cells in the logarithmic growth phase obtained in step 1.1 were seeded and cultured in DMEM medium containing 200 nmol / L α-MSH and 10% fetal bovine serum. Here, 200 nmol / L is the concentration of α-MSH in the DMEM medium, and 10% is the volume percentage of fetal bovine serum in the DMEM medium. The culture was carried out at a density of 5.0 × 10⁶ cells per well. 5Cells were seeded at a density of 1 / mL into 6-well plates and cultured at 37°C for 24 hours. After cell attachment, 100 μL / well of DMEM complete medium (containing 10% fetal bovine serum, where 10% is the volume percentage of fetal bovine serum in the complete medium) containing 2% (v / v) rose fermentation broth obtained from different treatment groups was added to each well. Specifically, rose fermentation broths obtained in Examples 1, 1-3, and 6 were added, while the blank group received an equal volume of complete medium (containing 10% fetal bovine serum). After culturing at 37°C for another 24 hours, cell clumps were collected, and 100 μL of 2 mol / L NaOH solution (containing 10% DMSO) was added. The cells were then incubated at 80°C for 1 hour. The supernatant was collected, and the absorbance (A) of each well was measured at 405 nm using a microplate reader. The melanin synthesis rate was calculated using the following formula: R = (A1 / A3) × 100%, where A1 and A3 are the absorbance values ​​of the experimental and blank groups, respectively. The effects of different rose fermentation broths on melanin synthesis were determined, and the results are shown in Table 3.

[0058] Table 3. Effects of different rose fermentation broths on melanin synthesis

[0059] Note: Data are expressed as Mean ± SD (n=3), and different letters indicate significant differences between different groups. P <0.05.

[0060] α-MSH can promote melanin synthesis in B16 cells. The anti-melanin production efficacy of rose fermentation broths can be evaluated by measuring the effects of different rose fermentation broths on α-MSH-induced melanin synthesis. Table 3 shows that Example 1 showed the best inhibitory effect on melanin synthesis, while Comparative Example 3 showed the worst effect, indicating that *Lactobacillus plantarum* LP-Onlly is significantly more effective than *Lactobacillus plantarum* 299V.

[0061] Experimental Example 2: Study on Antioxidant Effect 2.1 Determination of DPPH free radical scavenging rate In the experiment, measurements were performed according to the instructions. The blank group consisted of a mixture of 80% (V / V) methanol and the working solution, the control group consisted of the test sample and a mixture of 80% (V / V) methanol, and the experimental group consisted of a mixture of the test sample and the working solution. The test samples were rose fermentation broths obtained in Examples 1, Comparative Examples 1-3, and Comparative Example 6. The DPPH free radical scavenging ability kit was tested according to the instructions, using a microplate reader to measure the absorbance (A) of each well at 517 nm. The DPPH free radical scavenging rate was calculated using the following formula: R = {1 - [(A1 - A2) / A3]} × 100%, where A1, A2, and A3 are the absorbance values ​​of the experimental group, control group, and blank group, respectively. The antioxidant capacity of different rose fermentation broths was evaluated by measuring the DPPH free radical scavenging rate, and the results are shown in Table 4.

[0062] Table 4. Effects of different rose fermentation broths on DPPH free radical scavenging rate

[0063] Note: Data are expressed as Mean ± SD (n=3), and different letters indicate significant differences between different groups. P <0.05.

[0064] As shown in Table 4, Example 1 showed the best DPPH scavenging effect, while Comparative Example 3 showed the worst effect. This indicates that the fermentation strain has a significant impact on the DPPH free radical scavenging rate, and the effect of *Lactobacillus plantarum* LP-Onlly is significantly better than that of *Lactobacillus plantarum* 299V.

[0065] 2.2 Determination of hydroxyl radical scavenging rate ddH2O was used as a negative control. Different samples were mixed with the substrate solution according to the instructions of the hydroxyl radical assay kit. The samples were rose fermentation broths obtained in Examples 1, Comparative Examples 1-3, and Comparative Example 6. After mixing, the mixture was added to a 96-well plate and reacted at 37°C for 1 min. The reaction was then immediately terminated by adding the chromogenic reagent, and the mixture was allowed to stand at room temperature (25°C) for 20 min. The absorbance (A) of each well was measured at 550 nm using a microplate reader. The hydroxyl radical scavenging rate was calculated according to the following formula: R = [1 - (A1 / A3)] × 100%, where A1 and A3 are the absorbance values ​​of the experimental group and the blank group, respectively. The results of the determination of the hydroxyl radical scavenging rate of different rose fermentation broths are shown in Table 5.

[0066] Table 5. Effects of different rose fermentation broths on hydroxyl radical scavenging rate

[0067] Note: Data are expressed as Mean ± SD (n=3), and different letters indicate significant differences between different groups. P <0.05.

[0068] As shown in Table 5, similar to the results of the DPPH free radical scavenging rate determination, the rose fermentation broth of Example 1 showed the best scavenging effect on hydroxyl free radicals, which was 43.6%, 4.22%, 369.06%, and 60.93% higher than that of Comparative Examples 1-3 and Comparative Example 6, respectively. This indicates that the inoculated strain had the greatest impact on the antioxidant effect of the rose fermentation broth.

[0069] Test Example 3: Stability Tracking Test The rose fermentation broths obtained in Example 1, Comparative Examples 1-3 and Comparative Example 6 were placed at 25°C for 9 months, and the viable bacteria count of the rose fermentation broth was tested every three months. The results are shown in Table 6.

[0070] Table 6. Changes in viable bacterial count during preservation of different rose fermentation broths.

[0071] As shown in Table 6, Example 1 had the highest number of viable bacteria, and the number of viable bacteria remained stable at 10 during long-term preservation. 6 The CFU / mL result indicates that the fermentation culture method, inoculum size, inoculum strain, and fermentation time have a significant impact on the number of viable bacteria during the storage period.

[0072] Experiment Example 4: Sensory Quality Analysis Referring to the sensory requirements in section 3.2 of GB 7101-2022 "National Food Safety Standard for Beverages", eight sensory evaluators (2 males and 6 females, aged 18-47) were selected to conduct sensory evaluations. The sensory scoring criteria are shown in Table 7, and the sensory evaluation results are shown in Table 8.

[0073] Table 7 Sensory Rating Criteria

[0074] Table 8 Sensory evaluation results of different rose fermentation broths

[0075] As shown in Table 8, Example 1 received the highest overall evaluation. It exhibited the characteristic red color of roses, was clear and transparent, had a uniform and stable consistency, with no obvious sediment or suspended matter, a balanced sweet and sour taste, a harmonious flavor, and no irritation. It had a slight astringent taste, but this was within an acceptable range. In summary, this invention provides a simple, low-cost rose fermentation liquid with whitening and antioxidant effects, and its viable bacterial count remains stable at 10 during long-term preservation. 6 It has a concentration of CFU / mL and enhances the color, aroma, and flavor of beverages when used.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing rose flower fermentation broth, characterized in that, The method comprises the following steps: Rose petal sap was enzymatically hydrolyzed using pectinase and cellulase to obtain rose petal hydrolysate; the rose petal hydrolysate, sugars, and yeast peptone were mixed, sterilized, and then processed using *Lactobacillus plantarum* (…). Lactobacillus plantarum LP-Onlly was used for fermentation to obtain rose fermentation broth; the fermentation speed was 30~50 r / min, the fermentation temperature was 36~38℃, and the fermentation time was 20~28 h; The mass of the pectinase accounts for 0.03%-0.05% of the mass of the rose flower slurry; the mass of the cellulase accounts for 0.03%-0.05% of the mass of the rose flower slurry. The preservation number of the plant Lactobacillus plantarum LP-Onlly is CGMCC No. 1258; the inoculation volume of the plant Lactobacillus plantarum LP-Onlly is 1% to 2% of the volume of the rose enzyme hydrolysate; the viable bacterial count of the plant Lactobacillus plantarum LP-Onlly is ≥1.0×10 9 CFU / mL.

2. The preparation method according to claim 1, characterized in that, The pressure of the fermentation is 0.03-0.07 MPa.

3. The preparation method according to claim 1, characterized in that, The mass-volume ratio of the saccharide to the rose flower enzymolysis liquid is 8-12 g / L; the mass-volume ratio of the yeast protein peptone to the rose flower enzymolysis liquid is 8-12 g / L.

4. The method of claim 1, wherein, The enzymolysis temperature is 40-45 DEG C, and the enzymolysis time is 3-4 h.

5. The method of claim 1, wherein the step of forming the first and second layers is performed by a process selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. Before the sterilization, the method further comprises adjusting the pH of the mixed liquid to 6.5-7.

0.

6. The preparation method according to claim 1, characterized in that, The preparation method of the rose flower slurry comprises: grinding rose flowers with water to obtain a rose flower slurry; the mass ratio of the rose flowers to water is 1:25-35.

7. The preparation method according to claim 6, characterized in that, The grinding tool comprises a colloid mill.

8. The preparation method according to claim 6, characterized in that, The pretreatment of the rose flowers comprises: soaking the rose flowers in water for 1-2 h, and the temperature of the water is 30-40 DEG C.

9. The rose flower fermentation broth prepared by the preparation method according to any one of claims 1-8, characterized in that, The pH of the rose flower fermentation liquor is 3-4; the viable bacterial count of the rose flower fermentation liquor is ≥6×10 6 CFU / mL.

10. The use of the rose flower fermentation liquid in claim 9 in the preparation of whitening and antioxidant products.

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