Meychekia pulchella and application thereof

Abstract

The application discloses a kind of Mei Ji Mei Chong yeast and application, by screening, a kind of suitable for the yeast of Cistanche Fermentation-Beauty extreme Mei Chong yeast, the Beauty extreme Mei Chong yeast is fermented to Cistanche, reduce the release or formation of harmful ingredients in Cistanche, the natural active substance in Cistanche is kept to the greatest extent, the obtained Cistanche fermentation filtrate has lower cytotoxicity, higher safety, has more superior anti-inflammatory, antioxidant, enhances the nonspecific immune performance of skin, so as to improve its safety and addition amount used in daily chemical product, so that Cistanche fermentation filtrate can be added in daily chemical product at high concentration, has higher performance.

Description

Technical Field

[0001] This invention relates to a yeast strain, specifically to a type of Saccharomyces cerevisiae (Megami), Metschnikowia most beautiful The invention also relates to the application of the yeast in the fermentation preparation of Cistanche deserticola products, and to a Cistanche deserticola fermentation filtrate and its application in the field of daily chemical products. Background Technology

[0002] Daily chemical products, commonly known as daily chemical supplies, include shampoos, shower gels, skincare products, haircare products, cosmetics, and so on. While pursuing efficacy, modern daily chemical product raw materials increasingly emphasize safety, making it the primary factor to consider when developing such materials. Natural plant extracts, especially traditional Chinese herbal extracts, are generally considered safe and non-toxic. However, due to the complex composition of plants and varying extraction processes, the components of plant extracts differ, making component analysis difficult and posing a safety concern.

[0003] Non-specific immunity, also known as innate immunity or inherent immunity, refers to the normal physiological defense function that the body possesses by nature. It can make corresponding immune responses to the invasion of various pathogenic microorganisms and foreign substances and is an important part of the human immune system. The raw materials of daily chemical products that can promote non-specific immunity can enhance the skin's barrier and protective function and reduce the inflammatory response caused by external stimuli such as bacteria, chemicals, and dust.

[0004] Cistanche deserticola, also known as golden bamboo shoot, earth spirit, cistanche, and dayun, is the dried, fleshy stem with scaly leaves of the plant Cistanche deserticola or Cistanche tubulosa, belonging to the Orobanchaceae family. It is mainly produced in Inner Mongolia, Gansu, Xinjiang, and Qinghai. Cistanche deserticola is warm in nature and sweet and salty in taste, and is a key medicine for tonifying the kidneys and strengthening yang, as well as moistening the intestines and relieving constipation; it is known as "desert ginseng." There are several species of Cistanche deserticola in my country, including desert Cistanche deserticola, white-flowered salt Cistanche deserticola, salt-grown Cistanche deserticola, Cistanche tubulosa, and sand Cistanche deserticola.

[0005] Currently, research on the efficacy and active ingredients of Cistanche deserticola mainly focuses on Cistanche deserticola extracts such as polysaccharides, total glycosides, and total oligosaccharides. Efficacy studies are concentrated on edible and medicinal applications, with limited research on its application in daily chemical products. Patents CN201410502922.2 and CN201410502895.9 disclose a method for degrading Cistanche deserticola bulb polysaccharides under strong acid and 70-200℃ conditions to produce an oligosaccharide mixture. These patents propose that the prepared Cistanche deserticola bulb extract and its purified and enriched oligosaccharide products have moisturizing and antioxidant effects and applications in daily chemical products. However, these patents only utilize the polysaccharide components of Cistanche deserticola, lacking utilization of the main component—phenylethanol glycosides—and employ a strong acid high-temperature hydrolysis method. This results in significant damage to the sugar chain groups and structures of the natural polysaccharides and the resulting oligosaccharides, affecting the bioactivity of the active substances.

[0006] There are few studies on enhancing the safety of Cistanche deserticola extract in daily chemical products through fermentation in existing technologies. Some reports mention that fermentation can enhance the non-specific immunity of Cistanche deserticola extract. For example, the study "Macrophage Activation Effect of Cistanche tubulosa Phenylephrine and its Synergistic Effect with Angelica sinensis and Astragalus membranaceus in Immunomodulation" mentions that Cistanche tubulosa phenylethanoid glycoside extract (Cistanche tubulosa extracted twice with 75% ethanol solution, concentrated and dried, containing echinacoside + verbascoside: 21.3%, total glycosides: 41%) can significantly enhance the phagocytic activity of macrophages when added at a concentration of 50 g / L to 125 g / L. The study "Immunomodulatory Activity and Absorption Characteristics of Cistanche deserticola Polysaccharide" mentions that Cistanche deserticola polysaccharide (Cistanche deserticola extract, polysaccharide content 87%) has a certain enhancing effect on the phagocytic function of mouse primary peritoneal macrophages. At a dose of 6.25 g / L, it can significantly promote their phagocytic function, and the effect increases with increasing dose. While the Cistanche deserticola extract in these existing technologies can effectively enhance the phagocytic activity of macrophages and improve non-specific immunity, the required amount is very high, making it unsuitable for direct application in daily chemical products. Summary of the Invention

[0007] To address the shortcomings of existing fermented Cistanche deserticola products in the application of daily chemical products, this invention provides a newly screened strain of Maggimidy yeast (… Metschnikowia pulcherrima The fermentation product obtained by fermenting Cistanche deserticola with the Magemycin yeast has low cytotoxicity and high safety. It also has excellent anti-inflammatory and antioxidant effects, and has better application prospects in daily chemical products.

[0008] The Magemycin yeast provided by this invention ( Metschnikowia pulcherrima This strain was isolated in the laboratory from orchard soil (Jinan, Shandong). It was deposited on May 23, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC NO:24953. This *Megamiprid* strain (…) Metschnikowia most beautiful The gene sequence of the 18S rDNA is shown in SEQ ID NO: 1.

[0009] The present invention also provides a microbial agent containing the aforementioned *Megamich.* yeast. This microbial agent may contain only *Megamich.* yeast, or it may contain other yeasts.

[0010] Furthermore, the bacterial agent can be either a solid formulation or a liquid formulation.

[0011] The present invention also provides the above-mentioned Magemycosis yeast ( Metschnikowia pulcherrimaThe application of the above-mentioned microbial agents in the preparation of fermented products. In this application, *Saccharomyces cerevisiae* (Megamiprid) is generally used. Metschnikowia most beautiful Alternatively, the above-mentioned microbial agents can be added to the substrate for fermentation to obtain fermented products.

[0012] The fermentation uses of yeast in various fields are disclosed in the prior art. The present invention is based on the yeast strain Magemycin (…). Metschnikowia pulcherrima Alternatively, the aforementioned microbial agents can be used in these existing and publicly available fields, and specific fermentation methods can be referred to existing technologies.

[0013] Preferably, the present invention uses the Magemyc yeast ( Metschnikowia pulcherrima The above-mentioned microbial agents are used to prepare Cistanche deserticola fermented products. The Cistanche deserticola fermented products are prepared using *Megamiprid* (or the above-mentioned microbial agents). Metschnikowia most beautiful The product obtained by fermenting the slurry of Cistanche deserticola raw material with the above-mentioned microbial agents.

[0014] This invention also provides a method for preparing a fermented product of Cistanche deserticola—Cistanche deserticola fermentation filtrate, the method comprising using *Megamiprid* (… Metschnikowia pulcherrima The steps for fermenting Cistanche deserticola.

[0015] Furthermore, the above preparation method specifically includes the following steps:

[0016] (1) Cut open the bulb of Cistanche deserticola, then dry, crush and sieve to obtain Cistanche deserticola powder;

[0017] (2) Dissolve the Cistanche deserticola powder, pre-treat it with ultrasound, sterilize it, and then inoculate it with Saccharomyces cerevisiae (Saccharomyces cerevisiae). Metschnikowia pulcherrima The mixture is fermented to obtain fermented Cistanche deserticola extract.

[0018] (3) Remove insoluble matter from the processed Cistanche slurry to obtain Cistanche fermentation filtrate.

[0019] Furthermore, the Cistanche deserticola used in this invention can be fresh or dried, and various varieties of Cistanche deserticola can be selected, with Cistanche tubulosa being the preferred variety.

[0020] Furthermore, in step (2) above, the content of Cistanche deserticola powder in water is 2.5-15 wt%. Preferably, after adding Cistanche deserticola powder to water, it is left to stand for 1-2 hours to allow the Cistanche deserticola powder to fully absorb water and become moist.

[0021] Furthermore, in step (2) above, the purpose of ultrasound is twofold: first, to better disperse the Cistanche deserticola powder in water, and second, to initially destroy the lignified structure of the Cistanche deserticola powder. Ultrasound can be performed using commonly used laboratory ultrasound equipment, and the ultrasound time is generally 10 to 30 minutes.

[0022] Furthermore, in step (2) above, after ultrasonic pretreatment, the slurry is sterilized for later fermentation. Sterilization can be carried out using conventional high-temperature sterilization methods, such as high-pressure sterilization at 121°C for about 20 minutes.

[0023] Furthermore, in step (2) above, the sterilized Cistanche deserticola slurry is cooled to the fermentation temperature, and inoculum seed liquid is added for fermentation. The fermentation temperature is 28℃-37℃, and the fermentation time is 24h-48h.

[0024] Furthermore, in step (2) above, the inoculum seed solution is formed by resuspending the inoculum in water, and the OD in the seed solution... 600 Approximately 4.0-5.0, the inoculum size for the seed culture is generally 1-2%. The strain can be expanded by adding it to a liquid culture medium to obtain the required amount of strain for the seed culture. The liquid culture medium consists of: yeast extract 5-15 g / L, peptone 15-25 g / L, and glucose 15-25 g / L. The expansion culture temperature is 28℃-37℃.

[0025] Furthermore, in step (3) above, the fermented Cistanche deserticola pulp is first centrifuged and then filtered to remove solid residues, bacterial cells, and other insoluble substances. Preferably, the centrifugation conditions are 5000-8000 rpm and the centrifugation time is 5-20 min.

[0026] This invention also provides a Cistanche deserticola fermentation filtrate prepared according to the above method. This Cistanche deserticola fermentation filtrate is a simple yeast fermentation product and can be used in food and daily chemical products. It retains the natural active substances in Cistanche deserticola to the greatest extent, without the addition of other ingredients or irritating byproducts. The yeast fermentation metabolism reduces the irritating and cytotoxic substances in Cistanche deserticola, making it more suitable for use in daily chemical products. This Cistanche deserticola fermentation filtrate has superior anti-inflammatory and antioxidant effects, can significantly enhance non-specific skin immunity, and can be added to daily chemical products at high concentrations. Even at high concentrations, it is safe and non-toxic, showing better application prospects in the field of daily chemical products.

[0027] Furthermore, the present invention also provides a daily chemical product comprising the above-mentioned Cistanche fermentation filtrate of the present invention, which can be used in the daily chemical product as an anti-inflammatory, antioxidant, and skin non-specific immune-enhancing component.

[0028] Furthermore, the amount of Cistanche deserticola fermentation filtrate added to daily chemical products according to the present invention can be 0.04%~10% (w / w), preferably 0.8%~4% (w / w). Experimental verification has shown that it is safe and non-toxic within this range.

[0029] Furthermore, the daily chemical products can be of various types, such as shampoo, hair care products, cosmetics, etc.

[0030] Furthermore, the cosmetics can be toners, lotions, creams, serums, eye creams, face masks, soaps, facial cleansers, shower gels, aerosols, sprays, etc.

[0031] The present invention has the following advantages:

[0032] 1. This invention has obtained a suitable yeast strain for fermenting Cistanche deserticola—Megmycin yeast—through screening. After fermentation of Cistanche deserticola, this yeast reduces the release or formation of harmful components in Cistanche deserticola. The resulting Cistanche deserticola fermentation filtrate has lower cytotoxicity and higher safety, thus improving its safety and dosage in daily chemical products. This allows Cistanche deserticola fermentation filtrate to be added at high concentrations in daily chemical products, resulting in higher performance.

[0033] 2. The Cistanche deserticola fermentation filtrate obtained by this invention is a simple yeast fermentation product of unprocessed Cistanche deserticola substrate, which retains the natural active substances in Cistanche deserticola to the greatest extent, without the addition of other ingredients or irritating byproducts. The fermentation and metabolism of Cistanche deserticola by Cistanche deserticola reduces the irritating and cytotoxic substances in Cistanche deserticola.

[0034] 3. The Cistanche deserticola fermentation filtrate obtained by this invention has superior anti-inflammatory, antioxidant, and skin non-specific immune-enhancing properties, among which the self-selected Methylmechi yeast ( Metschnikowia pulcherrima The best results are achieved with this fermented Cistanche deserticola filtrate. It significantly enhances the skin's non-specific immunity and macrophage phagocytic capacity, thus making it a safe non-specific immune-enhancing ingredient for use in daily chemical products. This fermented Cistanche deserticola filtrate also exhibits significant anti-inflammatory and antioxidant effects, making it suitable for daily chemical products requiring anti-inflammatory, anti-aging, and antioxidant properties.

[0035] Preservation Information

[0036] The Magemycin yeast strain described in this invention ( Metschnikowia pulcherrima The sample has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO:24953, deposited on May 23, 2022, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen Road, Chaoyang District, Beijing. Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments, wherein the Cistanche deserticola used in the experiment is Cistanche tubulosa (produced in Xinjiang).

[0038] Unless otherwise specified, all concentrations mentioned in the following embodiments are mass percentage concentrations.

[0039] Example 1: Screening and identification process of Saccharomyces cerevisiae

[0040] Soil samples were collected from orchards (Jinan, Shandong Province). The samples were then diluted and isolated using a standard 10-fold dilution method. Single colonies were obtained by streaking the samples onto plates and inoculated onto slant agar plates for microscopic examination. This process of dilution and isolation was repeated until a single bacterial strain was obtained. After initial and secondary screening (fermentation culture of Cistanche deserticola was conducted to assess the cytotoxicity of the fermentation filtrate and its effect on macrophage phagocytosis), strains exhibiting no significant cytotoxicity within a 10% addition range and enhancing macrophage phagocytosis were selected for strain preservation and identification.

[0041] The physiological, biochemical, and genetic characteristics of the final selected bacterial strains are as follows:

[0042] (1) Cell characteristics: The cells are oval, spherical or nearly spherical, and reproduce by budding;

[0043] (2) Colony characteristics: Colonies are white, flat, smooth, opaque, creamy, and have smooth edges;

[0044] (3) Genetic characteristics: The genome of the strain was sequenced and identified by Beijing Liuhe Huada Gene Technology Co., Ltd. The results showed that the strain was *Saccharomyces cerevisiae*. Metschnikowia pulcherrima Its 18S rDNA gene sequence is shown in SEQ ID NO: 1.

[0045] Example 2: Preparation of Cistanche deserticola yeast fermentation filtrate

[0046] Fresh Cistanche tubulosa bulbs were sliced, dried at 40°C until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, and purified water was added to make up to a final volume to prepare a Cistanche tubulosa slurry with a dry matter content of 10wt%.

[0047] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes and set aside for later use.

[0048] A single colony of *Megamiprid* was inoculated into 100 mL of YPD liquid medium. The medium consisted of 10 g yeast extract, 20 g peptone, 20 g glucose, and 1 L purified water. After inoculation, the culture was incubated in a shake flask at 200 rpm and 30 °C for 24 h. Then, the culture was centrifuged at 5000 rpm for 10 min, the precipitated cells were collected, the supernatant was discarded, and the cells were resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0;

[0049] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 1% of the slurry volume. The mixture was then shaken at 200 rpm and 28°C for 24 hours to obtain Cistanche deserticola yeast fermentation broth.

[0050] The Cistanche deserticola yeast fermentation broth was centrifuged at 8000 rpm for 20 min, then filtered to remove solid residue and bacterial cells. The resulting supernatant was the Cistanche deserticola fermentation filtrate. The Cistanche deserticola fermentation filtrate was diluted 4 times with purified water (so that the dry matter extraction ratio of Cistanche deserticola was 2.5 wt%) for subsequent experiments.

[0051] Example 3: Preparation of Cistanche deserticola yeast fermentation filtrate

[0052] Take dried slices of Cistanche deserticola bulbs, dry them at 40℃ until the moisture content is <4%, then pulverize them, sieve them, and collect the Cistanche deserticola bulb powder. Weigh the Cistanche deserticola powder, add purified water to make up to a final volume, and prepare a Cistanche deserticola slurry with a dry matter content of 15wt%.

[0053] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 10 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes for later use.

[0054] A single colony of *Megamiprid* was inoculated into 100 mL of YPD liquid medium. The medium consisted of 10 g yeast extract, 20 g peptone, 20 g glucose, and 1 L purified water. After inoculation, the culture was incubated in a shake flask at 200 rpm and 30 °C for 24 h. Then, the culture was centrifuged at 5000 rpm for 10 min, the precipitated cells were collected, the supernatant was discarded, and the culture was resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0;

[0055] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 1% of the slurry volume. The mixture was then shaken at 200 rpm and 37°C for 24 hours to obtain Cistanche deserticola yeast fermentation broth.

[0056] The Cistanche deserticola yeast fermentation broth was centrifuged at 5000 rpm for 5 min, then filtered to remove solid residue and bacterial cells. The resulting supernatant was the Cistanche deserticola fermentation filtrate. The Cistanche deserticola fermentation filtrate was diluted 6 times with purified water (so that the dry matter extraction ratio of Cistanche deserticola was 2.5%) for subsequent experiments.

[0057] Example 4: Preparation of Cistanche deserticola yeast fermentation filtrate

[0058] Fresh Cistanche tubulosa bulbs were sliced, dried at 40℃ until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, purified water was added to make up to a final volume, and a Cistanche tubulosa slurry with a dry matter content of 2.5 wt% was prepared.

[0059] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes and set aside for later use.

[0060] A single colony of *Megamiprid* was inoculated into 100 mL of YPD liquid medium. The medium consisted of 10 g yeast extract, 20 g peptone, 20 g glucose, and 1 L purified water. After inoculation, the culture was incubated in a shake flask at 200 rpm and 30 °C for 24 h. Then, the culture was centrifuged at 5000 rpm for 10 min, the precipitated cells were collected, the supernatant was discarded, and the cells were resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0;

[0061] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 2% of the slurry volume. The mixture was then shaken at 200 rpm and 30°C for 48 hours to obtain Cistanche deserticola yeast fermentation broth.

[0062] The fermentation broth of Cistanche deserticola yeast was centrifuged at 5000 rpm for 10 min, and then filtered to remove solid residues and bacterial cells. The resulting supernatant was the fermentation filtrate of Cistanche deserticola, which was used for subsequent experiments.

[0063] Comparative Example 1: Preparation of Cistanche deserticola aqueous extract filtrate

[0064] Fresh Cistanche tubulosa bulbs were sliced, dried at 40℃ until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, purified water was added to make up to a final volume, and a Cistanche tubulosa slurry with a dry matter content of 2.5 wt% was prepared.

[0065] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes for later use.

[0066] The sterilized Cistanche slurry was added to a shake flask and cultured at 30°C for 48 hours to obtain Cistanche extract.

[0067] The extract of Cistanche deserticola was centrifuged at 5000 rpm for 10 min, and then filtered to remove solid residue. The resulting supernatant was the filtrate of the Cistanche deserticola extract (the dry matter extraction ratio of Cistanche deserticola was 2.5%).

[0068] Comparative Example 2: Preparation of Cistanche deserticola lactic acid bacteria fermentation filtrate

[0069] Fresh Cistanche tubulosa bulbs were sliced, dried at 40℃ until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, purified water was added to make up to a final volume, and a Cistanche tubulosa slurry with a dry matter content of 2.5 wt% was prepared.

[0070] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes for later use.

[0071] A single colony of lactic acid bacteria (Lactobacillus paracasei, ATCC11578) was inoculated into 100 mL of MRS liquid medium. The medium formulation was as follows: 10.0 g peptone, 8.0 g beef meal, 4.0 g yeast extract, 20.0 g glucose, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 5.0 g sodium acetate, 2.0 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 1000 mL distilled water. The culture was carried out in a shake flask at 200 rpm and 30 °C for 24 h. After centrifugation at 5000 rpm for 10 min, the precipitated bacterial cells were discarded, the supernatant was discarded, and the culture was resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0;

[0072] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 1% of the slurry volume. The mixture was then shaken at 200 rpm and 30°C for 48 hours to obtain Cistanche deserticola lactic acid bacteria fermentation broth.

[0073] The fermentation broth of Cistanche deserticola with lactic acid bacteria was centrifuged at 5000 rpm for 10 min, and then filtered to remove solid residues and bacteria. The supernatant was the fermentation filtrate of Cistanche deserticola (the dry matter extraction ratio of Cistanche deserticola was 2.5%).

[0074] Comparative Example 3: Preparation of Cistanche deserticola yeast fermentation filtrate

[0075] Fresh Cistanche tubulosa bulbs were sliced, dried at 40℃ until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, purified water was added to make up to a final volume, and a Cistanche tubulosa slurry with a dry matter content of 2.5 wt% was prepared.

[0076] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes for later use.

[0077] A single colony of *Cytomyces coccidioides* (CICC1019) was inoculated into 100 mL of YPD liquid medium. The medium composition was: 10 g yeast extract, 20 g peptone, 20 g glucose, and 1 L purified water. After inoculation, the culture was carried out in shake flasks at 200 rpm and 30 °C for 24 h. Then, the cells were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0;

[0078] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 1% of the slurry volume. The mixture was then cultured in a shake flask at 200 rpm and 30°C for 48 hours to obtain Cistanche deserticola yeast fermentation broth.

[0079] The fermentation broth of Cistanche deserticola yeast was centrifuged at 5000 rpm for 10 min, and then filtered to remove solid residues and bacterial cells. The resulting supernatant was the fermentation filtrate of Cistanche deserticola (the dry matter extraction ratio of Cistanche deserticola was 2.5%).

[0080] Comparative Example 4: Preparation of Cistanche deserticola yeast fermentation filtrate

[0081] Fresh Cistanche tubulosa bulbs were sliced, dried at 40℃ until the moisture content was <4%, then pulverized, sieved, and the Cistanche tubulosa bulb powder was collected. The Cistanche tubulosa powder was weighed, purified water was added to make up to a final volume, and a Cistanche tubulosa slurry with a dry matter content of 2.5 wt% was prepared.

[0082] After allowing the Cistanche deserticola slurry to stand for 1 hour to allow the Cistanche deserticola powder to fully absorb water, the slurry was then ultrasonically pretreated for 30 minutes to initially disrupt the lignified structure of the powder. The ultrasonically treated slurry was then autoclaved at 121℃ for 20 minutes for later use.

[0083] A single colony of *Saccharomyces cerevisiae* (CICC1053) was inoculated into 100 mL of YPD liquid medium. The medium composition was: 10 g yeast extract, 20 g peptone, 20 g glucose, and 1 L purified water. After inoculation, the culture was carried out in shake flasks at 200 rpm and 30 °C for 24 h. Then, the cells were centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in purified water to obtain the seed culture. Its OD... 600 Approximately 4.0.

[0084] Under aseptic conditions, the seed liquid was inoculated into sterilized Cistanche deserticola slurry at an inoculation volume of 1% of the slurry volume. The mixture was then cultured in a shake flask at 200 rpm and 30°C for 48 hours to obtain Cistanche deserticola yeast fermentation broth.

[0085] The fermentation broth of Cistanche deserticola yeast was centrifuged at 5000 rpm for 10 min, and then filtered to remove solid residues and bacterial cells. The resulting supernatant was the fermentation filtrate of Cistanche deserticola (the dry matter extraction ratio of Cistanche deserticola was 2.5%).

[0086] Experimental Example 1: Cytotoxicity Evaluation of Cistanche deserticola Fermentation Filtrate

[0087] The cytotoxicity evaluation experiments were conducted in accordance with GB / T16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests (ISO 10993-5:2009, IDT) and the "Entry-Exit Inspection and Quarantine Industry Standard of the People's Republic of China" SNT 2328-2009 Keratinocyte Test for Acute Toxicity of Cosmetics, to evaluate the cytotoxicity of the products prepared in the above examples and comparative examples. The detection index was cell proliferation rate, and the detection method was the WST-1 method.

[0088] 1. Experimental Materials

[0089] Experimental samples: Cistanche fermentation filtrate or diluted fermentation filtrate prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in each case; Cistanche water extract filtrate prepared in Comparative Example 1; Cistanche fermentation filtrate prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in each fermentation filtrate was 2.5%.

[0090] 2. Instruments and Equipment

[0091] Inverted microscope (OLYMPUS, CKX41), ultra-clean workbench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., SCB-1520), carbon dioxide incubator (SANYO), digital display constant temperature water bath (Jintan Zhongda Instrument Factory), constant temperature microplate rapid shaker (Haimen Qilin Medical Instrument Factory), microplate reader (BIO-RAD), etc.

[0092] 3. Experimental Procedure and Evaluation Criteria

[0093] Logarithmic growth phase mouse embryonic fibroblasts BALB / 3T3 were harvested at 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 100 μL / well in 96-well plates using DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS). Each experimental sample was diluted with FBS-containing medium to prepare sample solutions with concentrations of 0.04%, 0.8%, 2%, 4%, and 10%, respectively, and then filtered through a 0.22 µm filter for sterilization. After 24 hours of conventional incubation at 37°C and 5% CO2, the culture medium was discarded and replaced with 100 μL of sample solution. The serum-containing medium served as a control. Five concentration levels were established for each sample, with six parallel wells per level. Cells were cultured for another 48 hours, and the relative proliferation rate (RGR) was determined using the WST-1 assay. RGR was the ratio of the absorbance of the sample group to that of the normal control group. According to GB / T16886.5-2017, a RGR below 70% was considered cytotoxic.

[0094] 4. Experimental Results

[0095]

[0096] A relative growth rate (RGR) below 70% is considered cytotoxic. As shown in Table 1, under the same extraction ratio of Cistanche deserticola dry matter, the Cistanche deserticola fermentation filtrate from Examples 2-4 showed no significant cytotoxicity when added to daily chemical products at concentrations of 0.04%-10%. Furthermore, even at concentrations as high as 10%, the relative growth rate of fibroblasts remained above 90%, indicating that the prepared Cistanche deserticola fermentation filtrate was not only non-toxic but also highly safe. Comparative Example 1 data showed that the cytotoxicity of the unfermented Cistanche deserticola aqueous extract increased with increasing concentration within the range of 0.04%-10%, exhibiting certain cytotoxicity at a concentration of 2%. Concentrations of 2% and above are not suitable for addition to daily chemical products. Comparison of data from Examples 2-4 and Comparative Example 2 shows that although the Cistanche deserticola lactic acid bacteria fermentation filtrate showed no significant cytotoxicity at addition levels of 0.04%-10%, the Cistanche deserticola fermentation filtrate prepared by yeast fermentation was significantly safer than that prepared by lactic acid bacteria fermentation. Therefore, the Cistanche deserticola fermentation filtrate prepared in this invention improves the safety and usage concentration of Cistanche deserticola in daily chemical products compared to the aqueous extract, allowing for high-concentration addition. The presumably, this is because the yeast decomposes and utilizes some harmful and allergenic components in Cistanche deserticola.

[0097] Experimental Example 2: The Enhancing Effect of Cistanche deserticola Fermentation Filtrate on Non-specific Immunity—Effect on Macrophage Proliferation

[0098] The effect of Cistanche fermentation filtrate on macrophage proliferation was evaluated to assess its non-specific immune-enhancing effect.

[0099] Macrophages are immune cells distributed throughout the body and are among the first cells to respond to microbial infections. They play a crucial role in the immune system by engulfing pathogens, presenting antigens, and releasing cytokines, thus participating in all stages of the immune response.

[0100] 1. Experimental Materials

[0101] Experimental samples: Cistanche fermentation filtrate or diluted solution prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in all samples; water extract filtrate of Cistanche prepared in Comparative Example 1; fermentation filtrate of Cistanche prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in the fermentation filtrate was 2.5% in all samples.

[0102] 2. Instruments and Equipment

[0103] Inverted microscope (OLYMPUS, CKX41), ultra-clean workbench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., SCB-1520), carbon dioxide incubator (SANYO), digital display constant temperature water bath (Jintan Zhongda Instrument Factory), microplate reader (Spark, Tecan).

[0104] 3. Experimental Methods

[0105] 3.1. Cell Culture and Sample Processing

[0106] Experimental samples were sterilized by filtration through a 0.22µm filter membrane and then diluted with serum-free 1640 medium to prepare test samples (containing experimental samples with different additive amounts). Test samples were prepared and used immediately. Frozen mouse macrophage cell line RAW264.7 was revived and cultured in 100mm diameter dishes until 80% confluence. RAW cells passaged to the third generation were digested with trypsin and the cell density was adjusted to 2×10⁶ cells / year. 4 Cells / mL were seeded into 96-well cell culture plates, with 100 μL of cell suspension in each well. After 24 h of routine culture in a CO2 incubator at 37 °C and 5% CO2, the culture medium was discarded and replaced with 100 μL of test sample (containing experimental samples with different amounts of added serum). The negative control was added with only serum-free culture medium and cultured in a CO2 incubator at 37 °C and 5% CO2 for 24 h.

[0107] 3.2. Detection and Data Processing

[0108] After culturing for 24 hours, discard the test solution, add 10% WST-1 solution (prepared with PBS) to each well, and incubate in a carbon dioxide incubator at 37°C and 5% CO2 in the dark for 2 hours. Measure the absorbance at 450 nm using an ELISA reader and calculate the relative proliferation rate (RGR) according to formula (1).

[0109] RGR = A / A0 × 100% (1)

[0110] In the formula:

[0111] RGR – Relative growth rate, %

[0112] A – Absorbance of the test sample group, minus the blank;

[0113] A0 – Absorbance of the negative control group, minus the blank.

[0114] 4. Experimental Results

[0115]

[0116] As shown in Table 2, when the fermented filtrate or diluted solution of Cistanche deserticola in Examples 2-4, the aqueous extract of Cistanche deserticola in Comparative Example 1, and the fermented filtrate of Cistanche deserticola in Comparative Examples 2-4 were added to daily chemical products at a concentration of 0.04%-10%, none of them showed significant cytotoxicity to macrophages.

[0117] Experimental Example 3: The Enhancing Effect of Cistanche deserticola Fermentation Filtrate on Non-specific Immunity—Effect on Macrophage Phagocytic Capacity

[0118] The effect of Cistanche deserticola fermentation filtrate on macrophage phagocytosis was evaluated to assess its non-specific immune-enhancing effect. The macrophage phagocytosis capacity was detected by a neutral red uptake assay.

[0119] 1. Experimental Materials

[0120] Experimental samples: Cistanche fermentation filtrate or diluted solution prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in all samples; water extract filtrate of Cistanche prepared in Comparative Example 1; fermentation filtrate of Cistanche prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in the fermentation filtrate was 2.5% in all samples.

[0121] 2. Instruments and Equipment

[0122] Inverted microscope (OLYMPUS, CKX41), ultra-clean workbench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., SCB-1520), carbon dioxide incubator (SANYO), digital display constant temperature water bath (Jintan Zhongda Instrument Factory), microplate reader (Spark, Tecan).

[0123] 3. Experimental Methods

[0124] Using RAW macrophages as model cells, the cells were digested with trypsin and then placed in 96-well plates with 5 × 10⁻⁶ cells / well. 4 Cells were cultured overnight for 24 hours in 100 μL of cell suspension per ml. Serum-free 1640 medium containing 30 µg / mL LPS served as a positive control. Test sample solution (experimental samples were sterilized by filtration through a 0.22 µm filter and then diluted with serum-free 1640 medium to form test samples; test samples contained different amounts of experimental samples and were prepared fresh for each use) was added. After culturing for another 24 hours, cells were washed twice with PBS before staining, and then treated with 1.0% neutral red staining solution (PBS) for 30 minutes. Neutral red was aspirated, and cells were washed three times with PBS. The absorbance was measured at 570 nm with lysis buffer. Phagocytic capacity was expressed as the absorbance of the test sample / the absorbance of the control group × 100.

[0125] 4. Experimental Data

[0126]

[0127] As shown in Table 3, the fermented filtrate of Cistanche deserticola from Examples 2-4, when added to daily chemical products at concentrations of 0.04%-10%, all exhibited a certain degree of enhancement in the phagocytic ability of macrophages, with the optimal addition concentration being 0.8%-4%. In Comparative Example 1, the Cistanche deserticola aqueous extract, when added at concentrations of 0.04%-10%, showed an increasing inhibitory effect on the phagocytic ability of macrophages, with a significant reduction in macrophage phagocytosis occurring at a concentration of 2%. In Comparative Example 2, the Cistanche deserticola lactic acid bacteria fermented filtrate, when added at concentrations of 0.04%-10%, did not affect the phagocytic ability of macrophages and did not induce any increase in macrophage phagocytic ability. The fermented filtrates of Cistanche deserticola from Comparative Examples 3 and 4 showed a slight improvement in the phagocytic ability of macrophages, but the increase was minimal and significantly lower than that of the fermented filtrates from Examples 2-4. Therefore, it can be seen that the fermented filtrate of Cistanche deserticola prepared by fermentation with Maggi Megi yeast not only overcomes the drawback of Cistanche deserticola water extract reducing the phagocytic ability of macrophages, but also does the opposite, significantly improving the phagocytic ability of macrophages and significantly enhancing non-specific immunity. It is suitable for the preparation of daily chemical products with effects such as enhancing skin immune function and enhancing skin resistance.

[0128] Experimental Example 4: Anti-inflammatory effect of Cistanche deserticola fermentation filtrate

[0129] The anti-inflammatory effect of Cistanche deserticola fermentation filtrate was assessed using the Cyprotex anti-inflammatory efficacy evaluation protocol. Balbc3T3 mouse embryonic fibroblasts were used as model cells. LPS stimulation induced the secretion of inflammatory factors. Samples were then treated, and the expression of inflammatory factors in the culture supernatant was quantitatively detected to investigate the inhibitory effect of different samples on the release of inflammatory factors. The factors investigated included IL-1β and IL-6.

[0130] 1. Experimental Materials

[0131] Experimental samples: Cistanche fermentation filtrate or diluted solution prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in all samples; water extract filtrate of Cistanche prepared in Comparative Example 1; fermentation filtrate of Cistanche prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in the fermentation filtrate was 2.5% in all samples.

[0132] 3T3 mouse embryonic fibroblasts, Mouse IL-6 ELISA Kit (Beijing Sizhengbai Biotechnology Co., Ltd.), Mouse IL-1β ELISA Kit (Beijing Sizhengbai Biotechnology Co., Ltd.), 1640 culture medium (Gibco), fetal bovine serum (Gibco), NaOH (Sinopharm Group).

[0133] 2. Instruments and Equipment

[0134] Inverted microscope (OLYMPUS, CKX41), ultra-clean workbench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd., SCB-1520), carbon dioxide incubator (SANYO), digital display constant temperature water bath (Jintan Zhongda Instrument Factory), constant temperature microplate rapid shaker (Haimen Qilin Medical Instrument Factory), microplate reader (BIO-RAD).

[0135] 3. Experimental methods: Dosing treatment and detection

[0136] 3T3 cells were fed at a rate of 1×10 5 Inflammatory factors were inoculated at a rate of 1 / ml into 24-well plates and cultured at 37°C and 5% CO2 for 24 h. Experimental samples were then added (2% of the reaction mixture), with LPS used as a model control. The plates were cultured for another 24 h, and inflammatory factors were detected using an ELISA kit. The procedure was performed according to the kit instructions.

[0137] 4 Experimental Data

[0138]

[0139] Table 4 shows that the fermented filtrate of Cistanche deserticola in Examples 2-4 has significant anti-inflammatory effects, effectively clearing inflammatory factor IL-1β (clearance rate higher than 75%) and partially clearing inflammatory factor IL-6 (clearance rate higher than 34%). At the same time, the anti-inflammatory effect of the fermented filtrate of Cistanche deserticola in Examples 2-4 is better than that of the samples in Comparative Examples 1-4.

[0140] Experimental Example 5: Antioxidant effect of Cistanche deserticola fermentation filtrate

[0141] The antioxidant effect of Cistanche deserticola fermentation filtrate was evaluated by the scavenging rate of organic free radicals (DPPH•).

[0142] 1. Experimental Materials

[0143] Experimental samples: Cistanche fermentation filtrate or diluted solution prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in all samples; water extract filtrate of Cistanche prepared in Comparative Example 1; fermentation filtrate of Cistanche prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in the fermentation filtrate was 2.5% in all samples.

[0144] Diphenylpicrylhydrazine radical (DPPH) (sigma).

[0145] 2. Instruments and Equipment

[0146] Incubators, UV spectrophotometers, microplate readers (Spark, Tecan), etc.

[0147] 3. Experimental Methods

[0148] 3.1 Preparation of test sample solution

[0149] All experimental samples were diluted 200 times with purified water before use.

[0150] 3.2 Reaction System, Conditions, and Measurement Methods:

[0151] Mix 1 mL of the test sample solution with 2 mL of DPPH solution, react in the dark at 25ºC for 40 min, and then measure the absorbance at 525 nm. During the measurement, zero the sample using an equal volume of distilled water and 50% ethanol mixture. Use 1 mL of distilled water as a blank control instead of the sample, and add 1 mL of sample at different concentrations using 2 mL of 50% ethanol instead of DPPH solution to measure the background absorbance of the sample. Perform three replicates for each sample and take the average value.

[0152] 3.3 Calculation of Results

[0153] The formula for calculating the DPPH• scavenging rate (D) of the sample is as follows:

[0154] D (%) = [1 – (Ai – Aj) / A0] × 100

[0155] Where Ai is the absorbance of the sample; Aj is the absorbance of the sample at the background absorption; and A0 is the absorbance of the blank control.

[0156] 4. Experimental Data

[0157]

[0158] Table 5 shows that all experimental samples have strong antioxidant capacity. Even when diluted 200 times, the scavenging rate (%) of organic free radicals (DPPH•) can reach 64.7% to 75.1%, with the Mickey Mouse yeast fermentation filtrate showing the best effect.

[0159] Experimental Example 6: Composition Analysis of Cistanche deserticola Fermentation Filtrate

[0160] 1. Experimental Materials

[0161] Experimental samples: Cistanche fermentation filtrate or diluted solution prepared in Examples 2-4, wherein the extraction ratio of Cistanche dry matter was 2.5% in all samples; water extract filtrate of Cistanche prepared in Comparative Example 1; fermentation filtrate of Cistanche prepared in Comparative Examples 2-4, wherein the extraction ratio of Cistanche dry matter in the fermentation filtrate was 2.5% in all samples.

[0162] 2. Instruments and Equipment

[0163] Ultraviolet spectrophotometer, microplate reader (Spark, Tecan), HPLC (Agilent, G7111A), biochemical analyzer, multi-angle laser light scattering instrument (WYATT TECHNOLOGY, DAWN HELEOS II).

[0164] 3. Experimental Methods

[0165] The content of echinacoside in Cistanche deserticola was determined according to the method for determining echinacoside in the Chinese Pharmacopoeia (2010 edition). The total sugar content was determined by the phenol-sulfuric acid method. The flavonoid content was determined by the method in "NY / T 2010-2011 Determination of Total Flavonoid Content in Citrus Fruits and Products". The glucose content was determined by a biochemical analyzer. The molecular weight distribution of polysaccharides in the samples was determined by liquid chromatography-multi-angle laser light scattering.

[0166] 4. Experimental Data

[0167]

[0168] As shown in Table 6, after fermentation with *Saccharomyces cerevisiae*, the echinacoside content in the fermentation filtrate of Examples 2-4 increased slightly, the total sugar content was significantly higher than that of Comparative Example 1, and the proportion of high molecular weight polysaccharides increased. This indicates that during the preparation of the fermentation filtrate, the yeast metabolized glucose and other components to synthesize some high molecular weight polysaccharides. It is speculated that this may be the reason why the fermentation filtrate of *Cistanche deserticola* has increased safety and enhanced non-specific immune effects.

[0169] Compared to Comparative Example 2, Examples 2-4 showed a significant increase in total sugar content and the proportion of high-molecular-weight polysaccharides in the fermentation filtrate. This is presumably because *Megrichinosaccharide* is more effective than lactic acid bacteria in fermenting *Cistanche deserticola* to form sugars, and some of the resulting polysaccharides, especially high-molecular-weight polysaccharides, enhance non-specific immunity. Compared to Comparative Examples 3-4, Examples 2-4 primarily contained low-molecular-weight polysaccharides, with a lower content of high-molecular-weight polysaccharides. This indicates that *Megrichinosaccharide* has a stronger ability to ferment *Cistanche deserticola* to form polysaccharides, especially high-molecular-weight polysaccharides, compared to *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae*. The experiment showed that the filtrate obtained solely from *Megrichinosaccharide* fermentation significantly enhanced non-specific immunity. This is presumably because the fermentation process produced some polysaccharides (especially high-molecular-weight polysaccharides), which enhanced non-specific immunity, or because other components were produced during fermentation or due to other reasons.

Claims

1. A strain of the species Meyerozyma guilliermondii (formerly known as Williopsis zeae) characterized by: Metschnikowia pulcherrima Its accession number is CGMCC NO:24953. ​ 2. M. extremoglutinis according to claim 1, characterized in that: Metschnikowia pulcherrima Its 18S rDNA gene sequence is shown in SEQ ID NO:

1. ​ 3. An inoculant characterized by: The M. pulcherrima yeast of claim 1, Metschnikowia pulcherrima ).

4. The use of the Mei-mecheng Saccharomyces cerevisiae (Saccharomyces boulardii) of claim 1 or 2 or the microbial agent of claim 3 in the preparation of a fermentation product of Cistanche deserticola Y.C.Ma. Metschnikowia pulcherrima .

5. A method for preparing a Cistanche Fermentation Filtrate, characterized in that: including the step of fermenting Cistanche with the M. extremum of claim 1. Metschnikowia pulcherrima ) 6. The method of claim 5, wherein the specific Includes the following steps: ​ (1) Cut open the bulb of Cistanche deserticola, then dry, crush and sieve to obtain Cistanche deserticola powder; (2) Dissolve the Cistanche deserticola powder, pre-treat it with ultrasound, sterilize it, and then inoculate it with Saccharomyces cerevisiae (Saccharomyces cerevisiae). Metschnikowia pulcherrima The mixture is fermented to obtain fermented Cistanche deserticola extract. (3) Remove insoluble matter from the processed Cistanche slurry to obtain Cistanche fermentation filtrate.

7. A fermention filtrate of Cistanche, characterized in that, The filtrate of Cistanche deserticola is prepared according to the method described in claim 5 or 6.

8. The application of the Cistanche deserticola fermentation filtrate according to claim 7 in the preparation of daily chemical products.

9. Use according to claim 8, characterized in that: The fermented filtrate of Cistanche deserticola is used as an anti-inflammatory, antioxidant, or skin-enhancing non-specific immune component in daily chemical products.

10. A daily care product characterized in that: Includes the Cistanche deserticola fermentation filtrate as described in claim 7.

11. The daily care product according to claim 10, characterized in that This includes shampoos, hair care products, shower gels, toners, lotions, creams, serums, eye creams, face masks, soaps, facial cleansers, shower gels, aerosols, and sprays.

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