Saccharomyces mirabilis fermentation liquor as well as preparation method and application thereof

Abstract

The invention discloses a Saccharomyces mirabilis fermentation broth and a preparation method and application thereof, and the preparation method comprises the following steps: crushing or homogenizing coconut solid endosperm to prepare a fermentation medium; inoculating saccharomyces cerevisiae to the fermentation culture medium for fermentation; and after the fermentation is finished, carrying out separation treatment to obtain the saccharomyces cerevisiae fermentation liquor. Compared with the prior art, the saccharomyces mirabilis is selected as a fermentation strain, the coconut solid endosperm is taken as a main fermentation substrate, the metabolic characteristics of the strain are fully exerted under a mild fermentation condition, and the mannan in the coconut solid endosperm is efficiently converted into mannan oligosaccharide; the obtained saccharomyces cerevisiae fermentation liquor has excellent moisturizing and repairing effects and the like, and has a good application prospect in cosmetics.

Description

Technical Field

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

[0002] With the continuous development of the cosmetics industry, consumers are increasingly demanding higher standards for product safety, natural origin, and efficacy. Microbial fermentation technology, due to its ability to enrich or generate various active ingredients with skin-care benefits through biotransformation, has gained widespread attention and application in the field of cosmetic raw material development. Among these, yeast fermentation products, rich in polysaccharides, amino acids, nucleosides, and nucleotides, possess skin-care benefits such as moisturizing, repairing, and soothing, and have become one of the important research directions for functional cosmetic raw materials.

[0003] In existing technologies, the yeast fermentation raw materials used in cosmetics are mostly brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae The main fermentation strains are *Schizosaccharomyces* or *Candida*, and common fermentation substrates include glucose, sucrose, corn syrup, and plant extracts. While these substrates are beneficial for microbial growth, they still have limitations in the targeted enrichment of specific functional components, especially oligosaccharides. Furthermore, the polysaccharide components obtained in existing fermentation systems are mostly in high molecular weight form, resulting in relatively low skin permeability and bioavailability, making it difficult to fully realize their skincare benefits.

[0004] Mannooligosaccharides, as functional ingredients with good moisturizing properties, biocompatibility, and skin barrier regulation, have broad application prospects in the cosmetics field. However, existing mannooligosaccharide preparation methods mainly involve chemical degradation or enzymatic hydrolysis, which often suffer from complex steps, high costs, or numerous byproducts, hindering their large-scale and stable application as cosmetic raw materials. Therefore, developing a bio-fermentation method that can efficiently obtain mannooligosaccharides under mild conditions is of significant practical importance.

[0005] Coconut solid endosperm, as a natural plant-derived raw material, is rich in mannan, polysaccharides, and various nutrients, exhibiting good safety and sustainability. However, in current technologies, coconut solid endosperm is mainly used as a food or simple extraction raw material, and its research and application as a fermentation substrate for the targeted conversion of functional oligosaccharides and nucleosides remains relatively limited.

[0006] Miracle brewer's yeast ( Saccharomyces paradoxus As a natural yeast strain in the genus *Saccharomyces*, its metabolic characteristics and biosynthetic capabilities have not yet been fully explored in the field of cosmetic raw material fermentation.

[0007] Therefore, there is an urgent need to provide a new microbial fermentation method that, by rationally selecting fermentation strains and substrates, can fully utilize mannan in coconut solid endosperm to convert it into active ingredient mannan oligosaccharides, while ensuring the naturalness and safety of the raw materials. This will yield fermentation broth raw materials with both moisturizing and repairing effects, suitable for the cosmetics industry, thus overcoming the shortcomings of existing technologies. Summary of the Invention

[0008] In view of the problems of low mannan oligosaccharide content, complex preparation methods, limited types of functional components, and insufficient utilization efficiency of coconut solid endosperm resources in existing cosmetic yeast fermentation raw materials, the purpose of this invention is to provide a method for preparing a unique brewing yeast fermentation liquid and its application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a fermentation broth of *Saccharomyces cerevisiae*, comprising the following steps: S1. Crush or homogenize the solid coconut endosperm to prepare a fermentation medium; S2. Inoculate the *Saccharomyces cerevisiae* into the fermentation medium and carry out fermentation; S3. After fermentation, the fermentation liquid of the exotic brewing yeast is obtained through separation.

[0010] The coconut solid endosperm mentioned above is the white solid endosperm of the coconut fruit, i.e., coconut meat.

[0011] In a preferred embodiment, the Saccharomyces cerevisiae is one or more of Saccharomyces cerevisiae CBS 406, ATCC96886, or CBS 432.

[0012] In a preferred embodiment, the fermentation culture contains 10-30 g / L of the coconut solid endosperm.

[0013] In a preferred embodiment, the fermentation medium further includes a nitrogen source of 10-30 g / L, a carbon source of 5-20 g / L, and the remainder is water.

[0014] In a preferred embodiment, the nitrogen source is one or more of yeast extract powder, potato extract powder, and peptone.

[0015] In a preferred embodiment, the carbon source is one or more of glucose, sucrose, and maltose.

[0016] As a preferred embodiment, the fermentation medium is autoclaved at 121°C for at least 20 minutes before use.

[0017] As a preferred embodiment, the inoculation amount of Saccharomyces cerevisiae is 1-5%.

[0018] As a preferred implementation method, the fermentation temperature is 25-30℃.

[0019] As a preferred embodiment, the stirring speed is 50-300 rpm.

[0020] As a preferred implementation method, the fermentation time is 48-96 hours.

[0021] In a preferred embodiment, the separation process includes centrifugation and filtration steps.

[0022] In a preferred embodiment, the mannan oligosaccharide content in the *Saccharomyces cerevisiae* fermentation broth is 1.81~2.28 mg / mL.

[0023] Secondly, the present invention provides a unique brewing yeast fermentation broth prepared according to the method described above.

[0024] Thirdly, the present invention provides the application of the aforementioned Saccharomyces cerevisiae fermentation broth in cosmetics.

[0025] Compared with existing technologies, this invention selects one or more of the following yeast strains: Saccharomyces cerevisiae CBS 406, ATCC 96886, or CBS 432, as the fermentation strain, and uses coconut solid endosperm as the main fermentation substrate. Under mild fermentation conditions, the metabolic characteristics of this strain are fully utilized to achieve efficient conversion of mannan in coconut solid endosperm into mannooligosaccharides. The resulting Saccharomyces cerevisiae fermentation broth has excellent moisturizing and repairing effects and has good application prospects in cosmetics. Attached Figure Description

[0026] Figure 1 The mRNA expression level of aquaporin AQP3 is shown.

[0027] Figure 2 The mRNA expression level of filaggrin FLG is shown.

[0028] Figure 3 The cell viability level is shown. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0030] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0033] Potato extract powder is a raw material for commercially available conventional culture media.

[0034] Miracle brewer's yeast ( Saccharomyces paradoxus CBS 406 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. (which is an agent for the Dutch fungal culture collection Center Centraalbureau voor Schimmelcultures, CBS), and the unique brewer's yeast ( Saccharomyces paradoxus ATCC 96886 and Saccharomyces cerevisiae ( ) Saccharomyces paradoxus CBS 432 comes from Wuhan Gray Algae Biotechnology Co., Ltd. (which is an agent of the American Type Culture Collection (ATCC) and the Central Bureauvoor Schimmelcultures (CBS) in the Netherlands).

[0035] brewing yeast ( Saccharomyces cerevisiae CICC 1445 and *Saccharomyces cerevisiae* ( Saccharomycopsis fibuligera CICC 33077 was purchased from the China Industrial Microbial Culture Collection Center.

[0036] "Coconut solid endosperm" refers to the white, solid endosperm of the coconut fruit, i.e., the coconut meat. "Coconut liquid endosperm" refers to the liquid endosperm of the coconut fruit, i.e., coconut water.

[0037] Preparation of fermentation medium: 10-30 g / L coconut solid endosperm (crushed or homogenized), 10-30 g / L nitrogen source, 5-20 g / L carbon source, with the balance being water. The nitrogen source is one or more of yeast extract, potato extract, and peptone. The carbon source is one or more of glucose, sucrose, and maltose. The medium is autoclaved at 121℃ for 20 min before use.

[0038] Examples A1-A4 The preparation steps of the unique brewing yeast fermentation broth of the present invention are as follows: 1. Crush or homogenize the solid coconut endosperm and pass it through a sieve with a mesh size of 50 or smaller to prepare a fermentation medium.

[0039] 2. Activate the *Saccharomyces cerevisiae* strain using PDB medium (containing 4 g / L potato extract powder and 20 g / L glucose), and then culture at 30℃ and 200 rpm for 24 h with shaking to obtain the seed culture.

[0040] 3. Inoculate one or more of the following yeasts, CBS 406, ATCC 96886, or CBS 432, into the fermentation medium for fermentation.

[0041] 3. After fermentation, use a disc centrifuge at 12000g to initially remove residual fermentation solid waste and yeast cells. Then, filter through a 50nm ceramic membrane to further remove large particulate solid impurities to obtain a clear yeast fermentation broth.

[0042] The composition of the fermentation medium for Examples 1A-4A is shown in Table 1.

[0043] Table 1. Fermentation medium composition for Examples A1-A4

[0044] The inoculum size and fermentation conditions of *Saccharomyces cerevisiae* in Examples A1-A4 are shown in Table 2.

[0045] Table 2. Inoculum size and fermentation conditions for Examples A1-A4

[0046] Comparative Examples B1-B3, C1-C3, D1-D4 The fermentation medium for comparative examples B1-B3, C1-C3, and D1-D4 consisted of 20 g / L of fermentation substrate (coconut solid endosperm, or coconut liquid endosperm, or potato extract powder), 10 g / L of nitrogen source (potato extract powder), 20 g / L of carbon source (glucose), and the remainder water.

[0047] The inoculum size was 2%, the fermentation temperature was 30℃, the fermentation speed was 200 rpm, and the fermentation time was 72 h.

[0048] Except for the different fermentation medium composition and yeast strain, the preparation steps and process conditions of comparative examples B1-B3, C1-C3, and D1-D4 are the same as those of example A1.

[0049] The composition of the fermentation medium for each comparative example is shown in Table 3.

[0050] Table 3. Components of the fermentation medium in the comparative example

[0051] The content of mannan oligosaccharides in the yeast fermentation broth obtained after fermentation in each example and comparative example was determined.

[0052] The fermentation strains, fermentation substrates, and mannan oligosaccharide content in the yeast fermentation broth used in the examples and comparative examples are shown in Table 4.

[0053] Table 4. Mannan oligosaccharide content in fermentation strains, fermentation substrates, and post-fermentation yeast fermentation broth

[0054] Table 4 shows that the fermentation combination of Saccharomyces kiwifruit and coconut solid endosperm resulted in a significantly higher content of mannan oligosaccharides in the fermentation broth compared to Saccharomyces kiwifruit fermentation of other types of substrates, as well as significantly higher content compared to other yeast strains fermenting coconut solid endosperm or other fermentation substrates. This indicates that coconut solid endosperm contains a high content of mannan, and that Saccharomyces kiwifruit can convert mannan into mannan oligosaccharides to a high degree.

[0055] Efficacy Evaluation 1. Expression of AQP3 and FLG, genes related to skin hydration and barrier repair. Aquaporin 3 (AQP3) is mainly expressed in keratinocytes (Hacat) and fibroblasts in the skin, participating in skin hydration and barrier function. Increased AQP3 expression can directly or indirectly improve skin moisturizing effects. Filagrin (FLG) in the epidermis can break down to form a small molecule complex of natural moisturizing factor (NMF) with water-absorbing properties. This complex effectively absorbs and locks in moisture, working in conjunction with the protective structure of keratinocytes to strengthen the water-locking barrier.

[0056] This experiment used real-time quantitative PCR to detect changes in the expression levels of AQP3 and FLG proteins mRNA in Hacat cells cultured in vitro, and evaluated the efficacy of the samples in moisturizing and improving skin barrier function.

[0057] Hacat cells were seeded in 6-well plates (70%-80% confluence) and cultured for 24 h. The culture medium in each well was aspirated, and different concentrations (0%, 0.5%, 1%) of the test substance (3000 μL / well) were added. For the negative control, 100% culture medium was added. After another 24 h of culture, intracellular RNA was extracted using an RNA extraction kit (FastPureCell / Tissue Total RNA Isolation Kit V2 (RC112), Vazyme), and RNA purity and concentration were measured. cDNA was reverse transcribed from the RNA using the TaKaRa PrimeScript reverse transcription kit. TM RT reagent Kit). RT-qPCR was performed using a standard reaction system, which mainly consisted of cDNA template, specific primers, a dNTP mixture, Taq DNA polymerase, and SYBR Green reagent (from Vazyme). PCR conditions were: 95℃ for 30 s, 95℃ for 10 s, and 60℃ for 30 s, for a total of 40 cycles.

[0058] Using GAPDH as the housekeeping gene, and with the relative expression level of the negative control set at 1, the relative gene expression level was calculated as 2 - ΔΔCT. The calculation formula is as follows: △CT = CT value 靶标基因 - CT value 内参基因 .

[0059] △△CT = △CT 样品组 - △CT 对照组 .

[0060] Relative expression level = 2 - △△CT.

[0061] Upregulation rate % = (relative expression level of sample group - relative expression level of NC group) × 100%.

[0062] Results are expressed as mean ± standard deviation. Differences between groups were statistically analyzed using one-way ANOVA (α=0.05).

[0063] Table 5. Primer sequence information

[0064] The expression of skin moisturizing and barrier-related genes AQP3 and FLG is as follows: Figure 1 and Figure 2As shown in the figure, Examples A1-A4 significantly promoted the expression of AQP3 and FLG genes, demonstrating excellent efficacy. In contrast, the expression levels of both genes in Control Examples B1, C1, D1, and D2 were significantly lower than in the three Examples. This indicates that the method of using *Saccharomyces cerevisiae* to ferment coconut solid endosperm can significantly enhance the moisturizing and barrier effects of the samples.

[0065] 2. Skin repair efficacy indicator – Hacat cell vitality level Hacat cell vitality affects skin barrier function; higher vitality indicates a stronger skin barrier stability. Therefore, the repair efficacy of a sample can be evaluated by testing its effect on keratinocyte vitality.

[0066] This experiment uses the CCK-8 assay to detect cell viability. The more cells proliferate, the greater the cell viability, and the darker the color.

[0067] Hacat cells were seeded in 96-well plates (50%-70% confluence) and incubated in a CO2 incubator for 24 h. The culture medium in each well was aspirated, and culture medium containing different volume concentrations (0%, 0.5%) of the test substance was added. For the negative control, 100% culture medium was added. After 24 h of incubation, the old culture medium was removed, and the cells were washed once with PBS. CCK-8 working solution diluted with the culture medium was then added. The cells were incubated in a CO2 incubator for 1 h, and the absorbance at 450 nm was measured using a microplate reader.

[0068] The results are as follows Figure 3 As shown, Examples A1-A4 demonstrated excellent effects in promoting Hacat cell viability, significantly outperforming Comparative Examples B1, C1, D1, and D2. This indicates that the method of using Saccharomyces cerevisiae to ferment coconut solid endosperm can significantly enhance the skin repair efficacy of the samples.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing a unique brewing yeast fermentation broth, characterized in that, Includes the following steps: S1. Crush or homogenize the solid coconut endosperm to prepare a fermentation medium; S2. Inoculate the *Saccharomyces cerevisiae* into the fermentation medium and carry out fermentation; S3. After fermentation, the fermentation liquid of the exotic brewing yeast is obtained through separation.

2. The method according to claim 1, characterized in that, The Saccharomyces cerevisiae is one or more of Saccharomyces cerevisiae CBS406, ATCC 96886, or CBS 432.

3. The method according to claim 1, characterized in that, The fermentation culture contains 10-30 g / L of the coconut solid endosperm.

4. The method according to claim 1, characterized in that, The fermentation medium also includes 10-30 g / L of nitrogen source, 5-20 g / L of carbon source, and the remainder is water.

5. The method according to claim 1, characterized in that, The nitrogen source is one or more of yeast extract powder, potato extract powder, and peptone.

6. The method according to claim 1, characterized in that, The carbon source is one or more of glucose, sucrose, and maltose.

7. The method according to claim 1, characterized in that, The inoculation amount of Saccharomyces cerevisiae is 1-5%.

8. The method according to claim 1, characterized in that, The fermentation temperature is 25-30℃, the stirring speed is 50-300 rpm, and the fermentation time is 48-96 hours.

9. The *Saccharomyces cerevisiae* fermentation broth prepared by any one of claims 1 to 8.

10. The application of the unique brewer's yeast fermentation broth as described in claim 9 in cosmetics.

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