Method for preparing fermented composition of honey, royal jelly, propolis, fermented composition and application

Abstract

This application discloses a method for preparing a fermented composition of honey, royal jelly, and propolis, the fermented composition itself, and its applications. The preparation method provided by this application includes: adding a first-stage substrate to a basic fermentation medium, inoculating with *Candida apis* and *Kluyveromyces martensii*, and carrying out a first-stage fermentation to obtain a first fermentation broth; adding a second-stage substrate to the first fermentation broth and carrying out a second-stage fermentation to obtain a second fermentation broth; and subjecting the second fermentation broth to cell disruption and post-treatment to obtain the fermented composition of honey, royal jelly, and propolis. This application uses *Candida apis* and *Kluyveromyces martensii* to carry out a two-stage synergistic fermentation of honey, royal jelly, and propolis as a triple substrate. After synergistic fermentation, cell disruption is performed to release yeast lysates. The resulting fermented composition is clear and transparent in appearance, has good stability, a rich rose aroma, and significantly inhibits inflammatory factors and promotes collagen synthesis.

Description

Technical Field

[0001] This application belongs to the field of cosmetic raw material technology, specifically relating to a method for preparing a fermented composition of honey, royal jelly, and propolis, the fermented composition of honey, royal jelly, and propolis prepared by this method, and the application of the fermented composition in cosmetics. Background Technology

[0002] Honey, royal jelly, and propolis are well-known and widely used as traditional natural nutritional products. Existing technologies have reported the use of microorganisms to ferment bee products to prepare beverages or health supplements; for example, yeast fermentation of honey or royal jelly can be used to obtain specific metabolites or improve product flavor.

[0003] Honey is rich in sugars, vitamins, and minerals; royal jelly contains abundant protein, royal jelly acid (10-HDA), and vitamins; and propolis contains flavonoids, terpenes, and other active ingredients. Therefore, bee products also have potential applications in cosmetics. However, there is currently a lack of research and practice on utilizing fermentation technology to enhance the application value of bee products in cosmetics. Summary of the Invention

[0004] This application provides a method for preparing a fermented composition of honey, royal jelly, and propolis, the fermented composition prepared by this method, and the application of the fermented composition in cosmetics. By subjecting honey, royal jelly, and propolis to a triple substrate of dual yeast synergistic fermentation and releasing yeast lysate after fermentation, a fermented composition with excellent appearance, odor, efficacy, and stability is obtained, thereby enhancing the application value of bee products in cosmetics.

[0005] In a first aspect, this application provides a method for preparing a fermented composition of honey, royal jelly, and propolis, comprising: adding a first-stage substrate to a basic fermentation medium, inoculating with *Candida apis* and *Kluyveromyces martensii*, and performing a first-stage fermentation for 36 to 40 hours to obtain a first fermentation broth; adding a second-stage substrate to the first fermentation broth and performing a second-stage fermentation for 18 to 20 hours to obtain a second fermentation broth; and subjecting the second fermentation broth to cell disruption and post-treatment to obtain the fermented composition of honey, royal jelly, and propolis; wherein, based on the mass of the basic fermentation medium, the first-stage substrate comprises 4% to 6% honey and 1.5% to 2.5% royal jelly, and the second-stage substrate comprises 4% to 6% honey, 1.5% to 2.5% royal jelly, and 0.02% to 0.05% propolis; and the viable count of *Candida apis* is 4.5 × 10⁻⁶ cells per volume of the basic fermentation medium. 5 cfu / mL ~5.5×10 6 The cfu / mL ratio of viable cells of Candida albicans and Kluyveromyces martensii was 1:(0.9~1.1).

[0006] In any embodiment of this application, the basic fermentation culture medium comprises, by mass percentage: 0.3-0.6% yeast extract, 0.1-0.3% dipotassium hydrogen phosphate, 0.1-0.2% sodium chloride, 0.1-0.3% glucose, 0.1-0.3% L-phenylalanine, with the remainder being water.

[0007] In any embodiment of this application, the first-stage substrate further includes water, ethanol, or a mixture thereof; the second-stage substrate further includes water, ethanol, or a mixture thereof.

[0008] In any embodiment of this application, the first-stage fermentation is carried out at 28-32°C, a stirring speed of 200-500 rpm, and a pH of 5.5-6.5.

[0009] In any embodiment of this application, the two-stage fermentation is carried out at 28-32°C, a stirring speed of 200-500 rpm, and a pH of 5.5-6.5.

[0010] In any embodiment of this application, cell disruption is performed using a high-pressure microjet, with the pressure of the high-pressure microjet being 1000~1500 bar.

[0011] In any embodiment of this application, the post-processing includes solid-liquid separation of the second fermentation broth after cell disruption, and purification, sterilization and preservation of the liquid portion.

[0012] Secondly, this application provides a fermented composition of honey, royal jelly, and propolis, prepared according to the method described in the first aspect.

[0013] Thirdly, this application provides an application of the fermentation composition prepared according to the method of the first aspect or the fermentation composition of the second aspect in cosmetics, wherein the amount of fermentation composition added is 0.1% to 10% of the total mass of the cosmetic.

[0014] This application employs *Candida albicans* and *Kluyveromyces martensii* to conduct staged synergistic fermentation of honey, royal jelly, and propolis as triple fermentation substrates. The amount and timing of substrate addition in the first and second stages of fermentation, as well as the fermentation time, are carefully controlled. After fermentation, cells are disrupted using high-pressure microfluidics to release yeast lysates, resulting in a fermented composition that is clear and transparent, possesses a rich rose aroma, and exhibits significant inhibitory effects on inflammatory factors and promotion of collagen synthesis. Stability studies show that this fermented composition exhibits good stability under various storage conditions and can be used as a raw material in cosmetics, demonstrating promising application prospects. Detailed Implementation

[0015] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the implementation details described in this specification are merely for illustrative purposes and are not intended to limit the scope of this application.

[0016] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0017] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0018] Unless otherwise stated, the values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this application). Unless otherwise stated, the test temperature for all parameters mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0019] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. The examples listed are merely representative and should not be construed as exhaustive.

[0020] In view of the aforementioned deficiencies of the prior art, this application provides a fermented composition of honey, royal jelly, and propolis, its preparation method, and its application.

[0021] The first aspect of this application provides a method for preparing a fermented composition of honey, royal jelly, and propolis, comprising: adding a first-stage substrate to a basic fermentation medium, inoculating with *Candida beemannii* and *Kluyveromyces martensii*, and performing a first-stage fermentation for 36 to 40 hours to obtain a first fermentation broth; adding a second-stage substrate to the first fermentation broth and performing a second-stage fermentation for 18 to 20 hours to obtain a second fermentation broth; and subjecting the second fermentation broth to cell disruption and post-treatment to obtain the fermented composition of honey, royal jelly, and propolis. The first-stage substrate comprises 4% to 6% honey and 1.5% to 2.5% royal jelly, and the second-stage substrate comprises 4% to 6% honey, 1.5% to 2.5% royal jelly, and 0.02% to 0.05% propolis, based on the mass of the basic fermentation medium; the viable count of *Candida beemannii* inoculated into the basic fermentation medium is 4.5 × 10⁻⁶ cells per volume of the basic fermentation medium. 5 cfu / mL ~5.5×10 6 The cfu / mL ratio of viable Candida albicans inoculated into the basal fermentation medium to viable Kluyveromyces inoculated into the basal fermentation medium was 1:(0.9~1.1).

[0022] In the embodiments of this application, *Candida apiacea* and *Kluyveromyces martensii* are used to conduct a two-stage synergistic fermentation of honey, royal jelly, and propolis as a triple fermentation substrate. On the one hand, honey serves as a high-quality carbon source, royal jelly provides a rich nitrogen source, and propolis provides lipids, complementing each other efficiently during fermentation and providing more comprehensive nutrients for the growth of *Candida apiacea* and *Kluyveromyces martensii*. On the other hand, *Kluyveromyces martensii* is characterized by rapid growth and vigorous metabolism, enabling it to quickly initiate fermentation and efficiently utilize small molecules in the fermentation system, such as sugars and amino acids. *Candida apiacea* grows relatively slowly but has a wider enzyme system, enabling it to utilize more complex substances in the fermentation system, such as macromolecular proteins and lipids. After synergistic fermentation, yeast lysate is further released through cell disruption. The final fermented composition of honey, royal jelly, and propolis is rich in small molecule amino acids, peptides, royal jelly acid, phenylethanol, fatty acids, and yeast-derived active substances, exhibiting significant abilities to inhibit inflammatory factors and promote collagen synthesis.

[0023] In addition, the embodiments of this application also produce a pleasant and soothing rose fragrance during fermentation. It is speculated that the main source of this natural fragrance is phenylalanine in the fermentation substrate (especially royal jelly), which is converted into phenylethanol, an aromatic alcohol compound with typical rose fragrance characteristics, under the fermentation action of Kluyveromyces martensii. The intensity of this fragrance is closely related to the content of phenylethanol in the fermentation composition and can be characterized by the content of phenylethanol in the fermentation composition. In recent years, the cosmetics industry has increasingly focused on the impact of emotions on the skin. The concept of emotional skincare has attracted the attention of cosmetic developers. Studies have shown that stimulating positive emotions in users through the visual, tactile, and olfactory aspects of cosmetics can influence users' psychological state and the effectiveness of skincare and makeup. Using the fermentation composition of this application as a raw material in cosmetics can give cosmetics a distinct and perceptible rose fragrance without adding fragrance ingredients, thereby enhancing the emotional value conveyed by cosmetics to users and improving the user experience and skincare effects.

[0024] Meanwhile, royal jelly's core functional component, royal jelly acid (10-hydroxy-2-decenoic acid), exhibits multi-target activity in the skin, but its stability and water solubility are poor, limiting its application in cosmetics. In the co-fermentation process of this application, beeswax contained in propolis, as an ester formed from higher fatty acids and higher monohydric alcohols, can be decomposed and utilized by *Candida albicans* to produce fatty acids. These fatty acids further combine with the sugars provided by honey, generating glycolipids through biosynthesis. Glycolipids are a type of amphoteric surfactant, which helps improve the solubility and stability of royal jelly acid in water.

[0025] Therefore, the fermentation composition prepared according to the embodiments of this application is not only clear and transparent in appearance, with good stability and a rich rose aroma, but also has a significant ability to inhibit inflammatory factors and promote collagen synthesis, and has a good application prospect in cosmetics.

[0026] As an example, the first-stage fermentation time can be 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, or any range of the above values.

[0027] As an example, the second-stage fermentation time can be 18 hours, 19 hours, 20 hours, or any range of the above values.

[0028] The implementation method of this application abandons the traditional "one-pot" fermentation process and purposefully performs two-stage fermentation with different fermentation times. In the first-stage fermentation, the substrates added to the basic fermentation medium include honey and royal jelly, and the fermentation time is relatively long, ranging from 36 to 40 hours. In the second-stage fermentation, the substrates added include honey, royal jelly, and propolis, and the fermentation time is relatively short, ranging from 18 to 20 hours. For the fermentation substrates honey and royal jelly, they are added in two separate stages during the first and second stages of fermentation. In the first stage of fermentation, some honey and royal jelly are used for rapid cell growth. After 36 to 40 hours of fermentation, the cell growth has reached a high density. At this point, the remaining honey and royal jelly are added, avoiding the potential inhibition of initial cell growth by high concentrations of honey and royal jelly, and also guiding the cells to produce a large amount of target metabolites during the stationary phase. For the fermentation substrate propolis, it is only added in the second stage of fermentation, and the fermentation time of the second stage is controlled not to exceed 20 hours. This is because propolis has a certain antibacterial property. If propolis is added to the fermentation system in the first stage of fermentation, it may lead to poor bacterial growth and affect the quality of fermentation products. In addition, the fermentation time of the second stage should not be too long. This ensures that the lipids (such as beeswax) in propolis are fully utilized, while avoiding the adverse effects of the antibacterial effect of propolis on the fermentation products.

[0029] As an example, the honey in the first-stage substrate is 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8%, 6% of the mass of the basal fermentation medium, or any range of the above values.

[0030] As an example, the royal jelly in the first-stage substrate is 1.5%, 1.8%, 2.0%, 2.2%, 2.4%, 2.5% of the mass of the basal fermentation medium, or any range of the above values.

[0031] As an example, the honey in the two-stage substrate is 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8%, 6% of the mass of the basal fermentation medium, or any range of the above values.

[0032] As an example, the royal jelly in the second-stage substrate is 1.5%, 1.8%, 2.0%, 2.2%, 2.4%, 2.5% of the mass of the basal fermentation medium, or any range of the above values.

[0033] As an example, the propolis in the second-stage substrate is 0.02%, 0.03%, 0.04%, 0.05% of the mass of the basal fermentation medium, or any range of the above values.

[0034] This application ensures the full realization of synergistic fermentation by controlling the addition of the substrates honey, royal jelly, and propolis. The amount of propolis, which has a certain antibacterial property, is controlled at a trace level that provides synergistic effects during fermentation without inhibiting the fermentation system. By combining a specific substrate ratio with a staged fermentation process, the three substrates can be efficiently and synergistically utilized by the two bacterial strains during fermentation. This directs metabolic flux towards the synthetic pathways of the target active ingredients, ultimately effectively enhancing the efficacy of the fermentation products in inhibiting inflammatory factors and promoting collagen synthesis at the molecular level.

[0035] As an example, based on the volume of the basal fermentation medium, the viable count of *Candida beescens* inoculated into the basal fermentation medium can be 4.5 × 10⁻⁶. 5 cfu / mL, 4.6×10 5 cfu / mL, 4.7×10 5 cfu / mL, 4.8×10 5 cfu / mL, 4.9×10 5 cfu / mL, 5.0×10 5 cfu / mL, 5.1×10 6 cfu / mL, 5.2×10 6 cfu / mL, 5.3×10 6 cfu / mL, 5.4×10 6 cfu / mL, 5.5×10 6 cfu / mL, or any range of the above values.

[0036] As an example, the ratio of viable cells of *Candida beeinae* and *Kluyveromyces martensii* inoculated into the basal fermentation medium can be 1:0.9, 1:1, 1:1.1, or any range of the above values.

[0037] As an example, based on the volume of the basal fermentation medium, the viable count of *Kluyveromyces martensii* inoculated into the basal fermentation medium can be 4.5 × 10⁻⁶. 5 cfu / mL, 4.6×10 5 cfu / mL, 4.7×10 5 cfu / mL, 4.8×10 5 cfu / mL, 4.9×10 5 cfu / mL, 5.0×10 5 cfu / mL, 5.1×10 6 cfu / mL, 5.2×10 6 cfu / mL, 5.3×10 6 cfu / mL, 5.4×10 6cfu / mL, 5.5×10 6 cfu / mL, or any range of the above values.

[0038] By controlling the viable cell counts of both Candida albicans and Kluyveromyces martensii seed cultures within the aforementioned specific high-density range, a sufficient number of starting microorganisms with highly consistent metabolic activity were provided for the dual-strain synergistic fermentation system. This ensured that the two strains could quickly adapt to the environment and efficiently utilize their respective preferred substrate components, thereby fully leveraging the synergistic effect in complementary metabolic pathways, promoting the synthesis and accumulation of target metabolites, and ultimately significantly improving the various properties of the fermentation products.

[0039] As a standard procedure in the field, inoculation with *Candida albicans* and *Kluyveromyces martensii* can be performed by pre-preparing *Candida albicans* and *Kluyveromyces martensii* seed cultures, and then inoculating these seed cultures into a basal fermentation medium. For example, the specific steps are as follows: *Candida albicans* and *Kluyveromyces martensii* are separately inoculated into YPD medium (yeast extract peptone glucose medium), activated at 28–32°C for 24–48 hours, and then transferred to seed culture medium at an inoculation rate of 2%–5% for expansion, yielding *Candida albicans* seed cultures and *Kluyveromyces martensii* seed cultures, respectively. As an example, the seed culture medium, by weight percentage, may include: 1% yeast extract, 0.5% peptone, 0.2% dipotassium hydrogen phosphate, 0.1% sodium chloride, 1% glucose, and the remainder being water, with a pH of 6.5.

[0040] In some embodiments, the basic fermentation medium comprises, by weight percentage: 0.3-0.6% yeast extract, 0.1-0.3% dipotassium hydrogen phosphate, 0.1-0.2% sodium chloride, 0.1-0.3% glucose, 0.1-0.3% L-phenylalanine, with the balance being water.

[0041] In some embodiments, the first-stage substrate also includes water, ethanol, or a mixture thereof; the second-stage substrate also includes water, ethanol, or a mixture thereof, that is, honey, royal jelly, and propolis are first diluted with an appropriate amount of water or ethanol as a solvent, and then added to the basic fermentation medium as substrates.

[0042] In some implementations, the fermentation temperature for both the first-stage and second-stage fermentation is 28°C to 32°C; the stirring speed is 200 rpm to 500 rpm; and the pH is 5.5 to 6.5.

[0043] As an example, in both the first-stage and second-stage fermentation, the fermentation temperature can be 28℃, 29℃, 30℃, 31℃, 32℃, or any range of the above values.

[0044] As an example, in both the first-stage and second-stage fermentation, the stirring speed can be 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, or any range of the above values.

[0045] As an example, in both the first-stage and second-stage fermentation, the pH can be 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or any range of the above values.

[0046] The above fermentation conditions not only ensured that *Candida albicans* and *Kluyveromyces martensii* maintained high activity and growth synchronicity throughout the entire staged fermentation cycle, thus enabling efficient synergistic utilization of the substrates honey, royal jelly, and propolis, but also optimized the metabolic flow and enzyme activity of the cells through stable dissolved oxygen and pH conditions. In particular, it promoted the conversion of phenylalanine in royal jelly to phenylethanol, ultimately strengthening the fermentation system's ability to accumulate the target active ingredients from the process control level and improving the overall efficacy of the resulting fermented composition.

[0047] In some embodiments, the second fermentation broth is further subjected to inactivation and cooling treatment before cell disruption. Inactivation includes heating the second fermentation broth to 80-100°C and maintaining it for 10-20 minutes. Preferably, inactivation includes heating the second fermentation broth to 90°C and maintaining it for 15 minutes.

[0048] In some embodiments, cell disruption of the second fermentation broth can be performed using a high-pressure microjet with a pressure of 1000-1500 bar to fully disrupt the yeast cells and release intracellular lysates.

[0049] As an example, the pressure of the high-pressure microjets can be 1000 bar, 1100 bar, 1200 bar, 1300 bar, 1400 bar, 1500 bar, or any range of the above values.

[0050] In some embodiments, post-processing includes solid-liquid separation of the second fermentation broth after cell disruption, and purification, sterilization, and preservation of the liquid portion.

[0051] In some embodiments, solid-liquid separation includes separating the bacterial culture using a ceramic membrane and collecting the supernatant. Optionally, the pore size of the ceramic membrane is 90 nm to 120 nm.

[0052] In some embodiments, based on the total mass of the supernatant, purification includes adding 0.3wt% to 0.7wt% activated carbon, stirring for 25 to 35 minutes, and then filtering with a 0.2μm to 0.25μm filter membrane to remove the activated carbon, yielding a clear, light yellow filtrate.

[0053] In some embodiments, sterilization includes reheating the purified filtrate to 102°C to 108°C for 9 to 12 minutes.

[0054] In some embodiments, the preservation treatment includes cooling the sterilized filtrate to 60°C, then adding 0.18% to 0.22% p-hydroxyacetophenone, 3% to 5% pentanediol, and 0.8% to 1.2% hexanediol by weight percentage of the total filtrate, stirring until completely dissolved and mixed evenly to obtain the fermentation composition.

[0055] The second aspect of this application provides a fermented composition of honey, royal jelly, and propolis, prepared according to the preparation method described in the first aspect.

[0056] The third aspect of this application provides the application of the fermentation composition prepared by the method of the first aspect or the fermentation composition of the second aspect in cosmetics, wherein the amount of fermentation composition added is 0.1% to 10% of the total mass of the cosmetic, for example 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any range of the above values.

[0057] In some embodiments, the dosage form of the cosmetic is any one of dressings, ointments, creams, emulsions, sprays, aerosols, ointments, liquids, gels, oils, patches, films, muds, powders, solutions, coatings, and powders.

[0058] Example

[0059] The following embodiments describe the disclosure of this application in more detail. These embodiments are for illustrative purposes only, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0060] The sources of some of the reagents used in Examples 1-4 and Comparative Examples 1-12 are as follows:

[0061] 1. Candida albicans ( Starmerella bombicola Purchased from Shanghai Titan Technology Co., Ltd., product number 041316279;

[0062] 2. Kluyveromycin ( Kluyveromyces marxianus Purchased from Shanghai Titan Technology Co., Ltd., product number 045637356;

[0063] 3. Lactobacillus plantarum ( Lactobacillus plantarum Purchased from Shanghai Titan Technology Co., Ltd., product number 041317476;

[0064] 4. Brewing yeast ( Saccharomyces cerevisiae Purchased from Shanghai Titan Technology Co., Ltd., product number 041321197;

[0065] 5. The honey was purchased from JD.com's Jingzao self-operated flagship store;

[0066] 6. Royal jelly and propolis were purchased from Beijing Mifengtang Biomedical Co., Ltd.

[0067] 7. YPD liquid culture medium was purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd.;

[0068] 8. Ethanol was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd.

[0069] 9. p-Hydroxyacetophenone was purchased from Kunshan Shuangyou Daily Chemical Co., Ltd.

[0070] 10. Pentylene glycol and hexanediol were purchased from Kunshan Shuangyou Daily Chemical Co., Ltd.

[0071] 11. β-Phenylenol standard was purchased from Shanghai Titan Technology Co., Ltd.

[0072] 12. Mouse macrophages RAW264.7 and mouse fibroblasts 3T3 were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0073] The basic fermentation medium and seed culture of each fermentation strain used in Examples 1-4 and Comparative Examples 1-12 were prepared in advance using the following methods:

[0074] 1. Prepare the basic fermentation medium according to the following mass percentage composition: 0.5% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.1% sodium chloride, 0.2% glucose, 0.2% L-phenylalanine, and the remainder is water. Adjust the pH to 7.0 and sterilize at 120℃ for 20 min to obtain the basic fermentation medium.

[0075] 2. Prepare the fermentation seed culture according to the following steps: Inoculate the fermentation strain into general YPD liquid medium for strain activation and activate it at 30℃ for 24 hours; then transfer 5% of the seed culture volume to the seed culture medium (60 mL) for expansion culture. The seed culture medium, based on its total mass, comprises the following components: 1% yeast extract, 0.5% peptone, 0.2% dipotassium hydrogen phosphate, 0.1% sodium chloride, 1% glucose, and the remainder is water, with a pH of 6.5. Then, incubate at 30℃ for 20 hours on a shaker at 200 rpm to obtain 1.0 × 10⁻⁶ microorganisms. 7 cfu / mL ~1.0×10 8 Seed cultures of each fermentation strain were prepared at CFU / mL. Subsequently, the seed cultures of each fermentation strain were inoculated according to the viable cell count requirements of the fermentation system.

[0076] Example 1

[0077] Example 1: A fermented composition of honey, royal jelly, and propolis was prepared by the following steps:

[0078] S1, first stage fermentation:

[0079] Add the first-stage substrate to the basal fermentation medium, which consists of 5% honey and 2% royal jelly (based on the mass of the basal fermentation medium). The honey and royal jelly are diluted with an appropriate amount of deionized water before addition. Then, inoculate with *Candida apigenin* seed culture and *Kluyveromyces martensii* seed culture, ensuring that the viable count of both *Candida apigenin* and *Kluyveromyces martensii* inoculated into the basal fermentation medium is 5 × 10⁻⁶. 6 The first fermentation broth was obtained by first-stage fermentation at cfu / mL (based on the volume of the basal fermentation medium) for 38 hours at 30°C, stirring speed of 400 rpm, pH of 6, and aeration of 1 vvm (3000 mL / min).

[0080] S2, second-stage fermentation:

[0081] The second-stage substrate, consisting of 5% honey, 2% royal jelly, and 0.04% propolis (based on the mass of the basal fermentation medium), was added to the first fermentation broth. The honey and royal jelly were diluted with an appropriate amount of deionized water before being added, and the propolis was diluted with an appropriate amount of ethanol before being added. The second-stage fermentation was carried out for 18 hours at 30°C, a stirring speed of 400 rpm, a pH of 6, and an aeration rate of 1 vvm (3000 mL / min) to obtain the second fermentation broth.

[0082] S3: Cell disruption and post-processing:

[0083] After fermentation, the second fermentation broth was heated to 90°C and maintained for 15 minutes to inactivate microorganisms, followed by cell disruption. The inactivated fermentation broth was cooled and treated using a high-pressure microfluidic device at a working pressure of 1500 bar, repeated three times. The cell-disrupted second fermentation broth underwent solid-liquid separation using a 100 nm ceramic membrane filtration system to remove bacterial cells and macromolecular impurities, collecting the clear filtrate. 0.5% (by weight) of activated carbon was added to the clear filtrate, and the mixture was stirred for 30 minutes. The mixture was then filtered through a 0.22 μm membrane to remove the activated carbon, yielding a clear, light yellow purified filtrate. The purified filtrate was heated to 105°C for sterilization for 10 minutes, cooled to 60°C, and then 0.2% (by weight) of p-hydroxyacetophenone, 4% (by weight) of pentanediol, and 1.0% (by weight) of hexanediol were added, stirring until completely dissolved and mixed thoroughly to obtain the fermentation composition.

[0084] Examples 2 to 4

[0085] The methods for preparing honey, royal jelly, and propolis fermentation compositions in Examples 2 to 4 are basically the same as those in Example 1, except that the viable cell counts of Candida albicans and Kluyveromyces martensii, the amount of each component added to the first-stage and second-stage substrates, and the fermentation times of the first and second stages have been adjusted within the reasonable range provided in this application.

[0086] Comparative Example 1

[0087] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 did not use staged fermentation, but instead added two kinds of yeast and three kinds of substrates, honey, royal jelly and propolis, to the fermentation system at the same time and carried out a "one-pot" fermentation for 56 hours.

[0088] Comparative Example 2

[0089] The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 omitted Kluyveromyces martensii and only inoculated 5 × 10⁻⁶ of it into the basal fermentation medium. 6 CFU / mL (based on the volume of the basal fermentation medium) of Candida albicans.

[0090] Comparative Example 3

[0091] The only difference between Comparative Example 3 and Example 1 is that Comparative Example 3 omitted *Candida bee-borne yeast* and only inoculated 5 × 10⁻⁶ of it into the basal fermentation medium. 6 Replace with CFU / mL (based on the volume of the basal fermentation medium) of Kluyveromyces maculae.

[0092] Comparative Example 4

[0093] The only difference between Comparative Example 4 and Example 1 is that Comparative Example 4 replaced *Candida bee-borne* with an equal amount of *Lactobacillus plantarum*, that is, the viable count of 5 × 10⁻⁶ cells was inoculated into the basal fermentation medium. 6 Lactobacillus plantarum CFU / mL (based on the volume of the basal fermentation medium), with a viable count of 5 × 10⁻⁶. 6 Kluyveromyces macrocarpa at cfu / mL (based on the volume of the basal fermentation medium).

[0094] Comparative Example 5

[0095] The only difference between Comparative Example 5 and Example 1 is that Comparative Example 5 replaced *Kluyveromyces martensii* with an equal amount of *Saccharomyces cerevisiae*, that is, the viable count was 5 × 10⁻⁶ cells / ml inoculated into the basal fermentation medium. 6 The viable count of *Candida albicans* was 5 × 10⁻¹⁰ CFU / mL (based on the volume of the basal fermentation medium). 6 Saccharomyces cerevisiae CFU / mL (based on the volume of the basal fermentation medium).

[0096] Comparative Example 6

[0097] The only difference between Comparative Example 6 and Example 1 is that royal jelly was omitted in both the first-stage and second-stage substrates, while the amount of honey was increased.

[0098] Comparative Example 7

[0099] The only difference between Comparative Example 7 and Example 1 is that honey was omitted from both the first-stage and second-stage substrates, while the amount of royal jelly was increased.

[0100] Comparative Example 8

[0101] The only difference between Comparative Example 8 and Example 1 is that propolis was omitted in the two-stage substrate, while the amount of honey and royal jelly was increased.

[0102] Comparative Example 9

[0103] The only difference between Comparative Example 9 and Example 1 is that 0.04% propolis (based on the mass of the basal fermentation medium) was added to the first-stage substrate, while propolis was omitted from the second-stage substrate.

[0104] Comparative Example 10

[0105] The only difference between Comparative Example 10 and Example 1 is that 0.02% propolis (based on the mass of the basal fermentation medium) was added to both the first-stage substrate and the second-stage substrate.

[0106] Comparative Example 11

[0107] The only difference between Comparative Example 11 and Example 1 is that, without changing the total fermentation time, the fermentation time of the first stage was shortened and the fermentation time of the second stage was extended.

[0108] Comparative Example 12

[0109] The only difference between Comparative Example 12 and Example 1 is that the second fermentation broth was not subjected to cell disruption.

[0110] Table 1 shows the key parameters of Examples 1 to 4 and Comparative Examples 1 to 12. Parameters not shown in Table 1 are the same as those in Example 1.

[0111] Table 1 Key parameters of Examples 1-4 and Comparative Examples 1-12

[0112]

[0113] Detection

[0114] The fermentation compositions prepared in Examples 1-4 and Comparative Examples 1-12 were tested as follows:

[0115] 1. Appearance:

[0116] Observe the color of the prepared fermentation composition and whether it is clear and transparent.

[0117] 2. Phenylacetyl alcohol content:

[0118] The content of phenylethanol in the sample was determined by high performance liquid chromatography (HPLC). The test method is as follows:

[0119] (1) Conditions for determination of phenylethanol: The chromatographic column was a Waters XBridge C18 column (4.6 × 250 mm, 5 μm); the mobile phase was acetonitrile-water (volume ratio 30:70); the flow rate was 1.0 mL / min; the detection wavelength was 260 nm; and the column temperature was 30℃.

[0120] (2) Preparation of reference standards: The standard 2-phenylethanol was diluted with methanol to concentrations of 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, and 1000 mg / L, respectively. After filtration through a 0.22 μm membrane, 20 μL samples were injected for detection. A standard curve was plotted with peak area 'a' as the abscissa and sample concentration 'y' as the ordinate, and the calculation formula was obtained:

[0121]

[0122] (3) Sample preparation of fermentation composition: Take 1 ml of the prepared fermentation composition, add 1 ml of methanol, mix thoroughly, filter through a 0.22 μm membrane, and inject 20 μl of the sample to obtain the peak area b. Substitute this into the following formula to calculate the concentration of phenylethanol in the fermentation composition (mg / L):

[0123]

[0124] Where: b is the peak area of ​​the sample; y is the concentration of the sample.

[0125] 3. Aroma detection:

[0126] Take the prepared fermentation composition, apply it to the wrist of a professionally trained odor tester, place it in front of the nose to identify the odor, determine whether the fermentation composition has a rose fragrance and the intensity of the fragrance, and score it. " / " represents no rose fragrance, and "+", "++", "+++" represent rose fragrance in that order, with the intensity of the fragrance increasing in that order.

[0127] 4. Inhibition rate of inflammatory factors (IL-1β, IL-6):

[0128] An inflammation model was established using RT-qPCR to stimulate RAW264.7 macrophages with lipopolysaccharide (LPS). After administration of the fermented compositions prepared according to the proportions of each example, changes in the mRNA expression of relevant inflammatory factors were detected to characterize the inhibitory effect of inflammatory factors on the test samples, thereby evaluating the anti-inflammatory and soothing efficacy of the fermented compositions. The experimental steps are as follows:

[0129] (1) Concentration of fermentation sample (%, V / V): 0.1%.

[0130] (2) Cell seeding: RAW264.7 cells were seeded into 6-well plates in a certain amount and incubated overnight at 37 ℃ in a 5% CO2 incubator.

[0131] (3) Experimental groups: NC solvent control group, LPS induction group, positive control group (dexamethasone) and sample group were set up respectively, with 3 replicates.

[0132] (4) RNA extraction: The Beyotime RNAeasy™ Animal RNA Extraction Kit (centrifuge column type) was used and the operation was carried out in accordance with the kit instructions.

[0133] (5) Reverse transcription PCR:

[0134] System (20 μL): 10×buffer 2 µL, 2.5 mM dNTP 2 µL, DNA polymerase (Taq 0.5 µL), primer 2 μL, template (cDNA obtained from reverse transcription diluted 10 times as template) 2-10 μL, ultrapure water to make up to 20 μL.

[0135] Reaction conditions: Pre-denaturation 94℃ for 5 min; 30 cycles: denaturation 94℃ for 30 s, annealing 60℃ for 30 s, extension 72℃ for 30 s; post-extension 72℃ for 10 min.

[0136] (6) Quantitative Real-Time PCR (qPCR) Experiment:

[0137] System (20 μL): 10 µL of 2×SYBR, 2-5 µL of template (cDNA obtained by reverse transcription diluted as template), 1-2 µL of primer, and ultrapure water to make up to 20 µL.

[0138] Reaction conditions: Pre-denaturation 94℃, 5 min; 40 cycles: denaturation 94℃, 30 s, annealing 60℃, 30 s, extension 72℃, 30 s (real-time fluorescence imaging); melting curve 94℃, 30 s; 60℃, 30 s, 72℃, 1 s, with real-time fluorescence imaging during the heating process. CT values ​​(representing the number of amplification cycles required for the fluorescence signal to reach the set threshold - nucleic acid quantity) were obtained for the NC solvent control group and the LPS-induced group. The relative expression level of the target gene and the inhibition rate were calculated using the following formula:

[0139]

[0140] Inhibition rate R(%) = [1-(E 样品组 -E 诱导组 ) / (E 对照组 -E 诱导组 )]*100%

[0141] in,

[0142] CTm: Fluorescence intensity value of the target gene in the sample group;

[0143] CTn: Fluorescence intensity value of the internal reference gene in the sample group;

[0144] CTmnc: Fluorescence intensity of the target gene in the solvent control group;

[0145] CTnnc: Fluorescence intensity value of the internal reference gene in the solvent control group;

[0146] E: Relative expression level of mRNA;

[0147] R: Sample inhibition rate.

[0148] 5. Increase in collagen COL-I and COL-III levels:

[0149] The anti-aging efficacy of the test samples was evaluated by detecting changes in the expression levels of Collagen-I and Collagen-III gene mRNA in mouse fibroblast 3T3 cells after adding the fermentation compositions (test samples) prepared in the comparative proportions of each example using RT-qPCR. The experimental steps are as follows:

[0150] (1) Cell seeding: Mouse fibroblasts 3T3 were seeded at a rate of 3 × 10⁻⁶. 5 Inoculate 1 cell / well into a 6-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0151] (2) Experimental groups: control group and sample group were set up respectively.

[0152] (3) Sample feeding: When the cell layering rate of the 6-well plate reaches 60-70%, discard the culture medium. Add fresh culture medium to the control group and add fresh culture medium containing the fermentation composition to the sample group. Make the final concentration of the fermentation composition 0.1%. After the sample feeding is completed, place the 6-well plate in an incubator (37℃, 5% CO2) for culture.

[0153] (4) After the incubation period, discard the supernatant; wash twice with pre-cooled sterile PBS.

[0154] (5) Total RNA extraction: The Beyotime RNAeasy™ Animal RNA Extraction Kit (centrifuge column type) was used and the operation was performed according to the kit instructions.

[0155] (6) Reverse transcription PCR

[0156] System (20 μl): 5×Primescript buffer: 4 µl; Primescript RT Emzyme Mix: 1 µl; 100 μM Random 6mers: 2 µl; template (cDNA obtained from reverse transcription diluted 10 times as template) 2-10 µl, and ultrapure water to make up the system to 20 µl.

[0157] Reaction conditions: Pre-denaturation 94℃ for 5 min; 30 cycles: denaturation 94℃ for 30 s, annealing 60℃ for 30 s, extension 72℃ for 30 s; post-extension 72℃ for 10 min.

[0158] (7) Quantitative Real-Time PCR (qPCR) Experiment

[0159] System (10 μl): 2×SYBR 5 µL, template (cDNA obtained from reverse transcription diluted 10 times as template) 2 μL, primer 0.5 μL, and ultrapure water to make up to 10 μL.

[0160] Reaction conditions: Pre-denaturation 94℃, 5 min; 40 cycles: denaturation 94℃, 30 s; annealing 60℃, 30 s; extension 72℃, 30 s (real-time fluorescence imaging); melting curve 94℃, 30 s; 60℃, 30 s; 72℃, 1 s. Real-time fluorescence imaging was performed during the heating process to obtain the CT values ​​(representing the number of amplification cycles required for the fluorescence signal to reach the set threshold - nucleic acid quantity) for the control group and sample group. The relative expression level of the target gene and the enhancement rate were calculated using the following formula:

[0161]

[0162] Improvement rate R (%) = (E 样品组 / E 对照组 -1) *100%

[0163] in,

[0164] CTm: The fluorescence intensity value of the target gene in the sample group;

[0165] CTn: The fluorescence intensity value of the internal reference gene in the sample group;

[0166] CTmnc: The fluorescence intensity value of the target gene in the solvent control group;

[0167] CTnnc: The fluorescence intensity value of the internal reference gene in the solvent control group;

[0168] E: Relative expression level of mRNA;

[0169] R: Sample lift rate.

[0170] 6. Stability test

[0171] The fermentation compositions prepared in Examples 1-4 and Comparative Examples 1-12 were respectively packaged into transparent plastic bottles and placed under light, room temperature (about 25°C), 4°C and high and low temperature cycling (-20°C to 48°C, once every 24 hours) conditions for 2 months to evaluate their stability.

[0172] The performance test results of the fermentation compositions prepared in Examples 1-4 and Comparative Examples 1-12 are shown in Table 2:

[0173] Table 2 Performance test results of Examples 1-4 and Comparative Examples 1-12

[0174]

[0175] The detection results of Examples 1 to 4 and Comparative Examples 1 to 12 show that:

[0176] Examples 1-4 employed *Candida benzii* and *Kluyveromyces martensii* to conduct staged synergistic fermentation of honey, royal jelly, and propolis as triple fermentation substrates. The types and amounts of substrates added in the first and second stages of fermentation, as well as the fermentation time, were carefully controlled. After fermentation, cells were disrupted using high-pressure microfluidics to release yeast lysates, resulting in a fermentation composition that was clear and transparent in appearance, possessed a rich rose aroma, and exhibited significant abilities to inhibit inflammatory factors and promote collagen synthesis. Stability tests showed that the fermentation composition demonstrated good stability under various storage conditions.

[0177] Comparative Example 1 did not use staged fermentation, but instead fermented two types of yeast and a triple substrate of honey, royal jelly and propolis in one pot. Since propolis has a certain antibacterial property, the excessively high concentration of triple substrates will also inhibit the growth of yeast, resulting in poor cell growth and a single metabolite profile, which seriously affects the quality of fermentation products.

[0178] The comparison between Comparative Examples 2-5 and Examples 1-4 demonstrates the synergistic effect of *Candida albicans* and *Kluyveromyces martensii* in the fermentation process of triple substrates. Comparative Examples 2 and 3 used only a single yeast (either *Candida albicans* or *Kluyveromyces martensii*) to ferment the triple substrates under the same conditions in stages. Because the microbial enzyme system of a single yeast is relatively simple and its metabolic pathways are limited, it cannot simultaneously and efficiently utilize various substances in the substrate to produce rich and balanced active ingredients. Therefore, the fermentation compositions prepared in Comparative Examples 2 and 3 are significantly inferior in efficacy to those in Examples 1-4. Furthermore, a comparison of efficacy test data shows that the fermentation composition prepared by the dual-strain synergistic fermentation in Example 1 exhibits superior performance in inhibiting inflammatory factors and promoting collagen synthesis compared to the sum of the corresponding performances in Comparative Examples 2 and 3. This fully demonstrates the synergistic effect of *Candida albicans* and *Kluyveromyces martensii* in the fermentation of triple substrates in this application. Comparative Examples 4 and 5 replaced one of *Candida albicans* and *Kluyveromyces martensii* in equal amounts with *Lactobacillus plantarum* or *Saccharomyces cerevisiae*, which are commonly used in the art, and carried out staged fermentation of the triple substrate under the same conditions. However, they could not produce a fermentation composition that was clear and transparent in appearance, had a rich rose aroma, and was effective and stable. Therefore, it can be seen that the selection of *Candida albicans* and *Kluyveromyces martensii* in this application is not a conventional technique in the art. The combined use of *Candida albicans* and *Kluyveromyces martensii* has a synergistic effect, thereby enabling the preparation of a fermentation composition that is clear and transparent in appearance, has a rich rose aroma, and is effective and stable.

[0179] The comparison between Comparative Examples 6-10 and Examples 1-4 illustrates the impact of the triple substrate and its timing of addition on the technical effects of this application. In Comparative Example 6, royal jelly was omitted from the substrate, and the amount of honey was increased; in Comparative Example 7, honey was omitted from the substrate, and the amount of royal jelly was increased; in Comparative Example 8, propolis was omitted from the substrate, and the amounts of honey and royal jelly were increased. Because the complementary and synergistic effects of honey, royal jelly, and propolis during fermentation were not fully utilized, the efficacy of the final product was not comprehensive or significant. Comparative Examples 9 and 10 demonstrate the importance of intentionally adding trace amounts of propolis during the two-stage fermentation process in this application. Comparative Example 9 added a trace amount of propolis to the fermentation system during the first-stage fermentation process. Comparative Example 10 added a trace amount of propolis to the fermentation system twice, during the first-stage and second-stage fermentation processes, respectively. Because propolis has a certain antibacterial property, it has an adverse effect on the growth of microorganisms, affecting the quality and efficacy of the fermentation product. Under the condition that the growth of microorganisms is inhibited, the beeswax contained in propolis cannot be fully utilized by yeast to produce a sufficient amount of glycolipids, thus affecting the effect of improving the stability of the fermentation product.

[0180] Comparative Example 11 illustrates the impact of setting the fermentation time for the two stages on the technical effect of this application.

[0181] In the first stage of fermentation, some honey and royal jelly are used for rapid bacterial growth. After 36-40 hours of fermentation, the bacterial growth reaches a high density. Adding the remaining honey and royal jelly at this point avoids the potential inhibition of early bacterial growth caused by high concentrations of honey and guides the bacteria to produce a large amount of target metabolites during the stable phase. Adding honey, royal jelly, and propolis to the fermentation system at this stage, while controlling the second-stage fermentation time to no more than 20 hours, ensures that the inhibition of bacteria by propolis is within a controllable range, while allowing the fermentation system to continue producing a large amount of target metabolites. It also effectively utilizes the glycolipids produced during metabolism to improve the solubility of royal jelly acid in water and the stability of the fermentation products. Comparative Example 11 shortened the first-stage fermentation time and prolonged the second-stage fermentation time, failing to achieve the above effects. Consequently, the efficacy and stability of the fermentation products decreased, and no significant synergistic effect was obtained.

[0182] Comparative Example 12 illustrates the effect of the cell rupture step after fermentation on the technical effect of this application:

[0183] Comparative Example 12 did not use high-pressure microfluidic jet to break down cells and release yeast lysates. The fermentation composition prepared contained only the components metabolized by yeast cells into the extracellular space. The ability to inhibit inflammatory factors and promote collagen synthesis was lower than that of Example 1.

[0184] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working process described above can be referred to the corresponding process in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. A method for preparing a fermented composition of honey, royal jelly, and propolis, characterized in that, include: Add the first-stage substrate to the basic fermentation medium, inoculate with Candida albicans and Kluyveromyces martensii, and carry out the first-stage fermentation for 36 to 40 hours to obtain the first fermentation broth; A second-stage substrate is added to the first fermentation broth, and a second-stage fermentation is carried out for 18 to 20 hours to obtain a second fermentation broth; the second fermentation broth is subjected to cell disruption and post-treatment to obtain the honey, royal jelly, and propolis fermentation composition. The first-stage substrate, based on the mass of the basic fermentation medium, includes 4% to 6% honey and 1.5% to 2.5% royal jelly, while the second-stage substrate includes 4% to 6% honey, 1.5% to 2.5% royal jelly, and 0.02% to 0.05% propolis. Based on the volume of the basal fermentation medium, the viable count of the *Candida albicans* was 4.5 × 10⁻⁶. 5 cfu / mL ~5.5×10 6 The ratio of viable counts of *Candida albicans* to *Kluyveromyces martensii* is 1:(0.9~1.1). The basic fermentation medium comprises, by weight percentage: 0.3-0.6% yeast extract, 0.1-0.3% dipotassium hydrogen phosphate, 0.1-0.2% sodium chloride, 0.1-0.3% glucose, 0.1-0.3% L-phenylalanine, with the remainder being water; The first-stage fermentation was carried out at 28-32°C, a stirring speed of 200-500 rpm, and a pH of 5.5-6.

5. The two-stage fermentation was carried out at 28-32°C, a stirring speed of 200-500 rpm, and a pH of 5.5-6.

5.

2. The preparation method according to claim 1, characterized in that, The first-stage substrate also includes water and / or ethanol; the second-stage substrate also includes water and / or ethanol.

3. The preparation method according to claim 1, characterized in that, The cell disruption is performed using a high-pressure microjet, with a pressure of 1000~1500 bar.

4. The preparation method according to claim 1, characterized in that, The post-processing includes solid-liquid separation of the second fermentation broth after cell disruption, and purification, sterilization and preservation of the liquid portion.

5. A fermented composition of honey, royal jelly, and propolis, characterized in that, Prepared by the method according to any one of claims 1-4.

6. The application of the fermented composition of honey, royal jelly, and propolis prepared by any one of claims 1-4 in the preparation of cosmetics.

7. The application according to claim 6, characterized in that, The amount of the honey, royal jelly, and propolis fermentation composition added is 0.1% to 10% of the total mass of the cosmetic.

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