A caribaea miehei and its use

Abstract

The application discloses a Meyerozyma caribbica and application thereof, and the application has screened a strain of Meyerozyma caribbica (Y-YGZ) with excellent fermentation performance. Meyerozyma caribbica The strain has outstanding advantages in fermenting phyllanthus emblica fruits, and the obtained phyllanthus emblica fruit fermentation product has good antioxidant, whitening and anti-inflammatory effects, and has a good application prospect in cosmetics.

Description

Technical Field

[0001] This invention relates to a novel type of Calibick Mayer's yeast ( Meyerozyma caribbica ), and also involves the Calibick Mayer's yeast ( Meyerozyma caribbica The application of amla fruit fermentation and the application of the resulting amla fruit fermentation products in cosmetics belong to the field of bio-fermentation technology. Technical Background

[0002] Phyllanthus emblica, a Chinese medicinal herb, is the fruit of the plant Phyllanthus emblica L., belonging to the Euphorbiaceae family. Rich in various vitamins, it is edible and can quench thirst, moisten the lungs and resolve phlegm, treat coughs, sore throats, and detoxify pufferfish poisoning. Initially, Phyllanthus emblica tastes sour and astringent, but after a while, it becomes sweet, hence the name "Emblica emblica" (meaning "sweet fruit"). Its roots and leaves are used medicinally to clear heat and detoxify, treating dermatitis, eczema, and rheumatic pain. Phyllanthus emblica has a long history of clinical use and is considered safe. It is recorded in many medical books, such as *Xinxiu Bencao* (Newly Revised Materia Medica): "Treats wind-heat deficiency"; *Bencao Shiyi* (Supplement to Materia Medica): "Primarily tonifies and strengthens qi"; *Haiyao Bencao* (Materia Medica of the Sea): "Treats lung damage from cinnabar and minerals, shortness of breath and cough. Long-term use lightens the body and prolongs life"; *Bencao Yanyi* (Extended Meaning of Materia Medica): "Detoxifies mineral poisoning; powdered and taken with decoction"; and *Fujian Zhongcaoyao* (Fujian Traditional Chinese Medicine): "Eliminates food stagnation and treats vomiting, abdominal pain, and diarrhea." Phyllanthus emblica is listed in the Chinese Pharmacopoeia and is a traditional Tibetan medicine, mainly used to treat diseases such as pelvic diseases, tripa diseases, blood disorders, and hypertension. Recent research indicates that Phyllanthus emblica possesses anti-inflammatory, antioxidant, anti-aging, and liver-protective effects.

[0003] Modern phytochemical studies have shown that Phyllanthus emblica mainly contains the following chemical components: polyphenols, organic acids, sugars, vitamins, amino acids, and trace elements such as Zn, Mn, Fe, Ca, and Se. A notable characteristic is its high content of polyphenols and abundant vitamin C. The main polyphenolic substances include: glucosinolates, gallic acid, galic acid, tannins, protochebulic acid, mucilage, and oleic acid. Modern pharmacological studies have shown that Phyllanthus emblica mainly possesses antioxidant, antimicrobial, antimutagenic, antiteratogenic, antitumor, lipid-lowering, and blood pressure-lowering effects.

[0004] Phyllanthus emblica is used both as food and medicine, and is listed in the "List of Items That Are Both Food and Medicine" published by the Ministry of Health in 2002. There is a tradition of eating fresh Phyllanthus emblica fruit raw. Currently, research on Phyllanthus emblica is increasing. Chinese patent CN108186456B discloses "A Method for Preparing Phyllanthus emblica Extract for Skin Whitening and Spot Removal," characterized by using Phyllanthus emblica fruit as raw material, and obtaining Phyllanthus emblica extract through low-temperature extraction, filtration, low-temperature concentration of the extract, solid-liquid separation, purification with macroporous adsorption resin, concentration of the eluent, and low-temperature drying. This extract has significant skin whitening and spot-removing activity and is non-irritating to the skin. Chinese patent CN111944870A discloses "Emblica emblica extract fermentation product and its preparation and application," characterized by first extracting emlica emblica to obtain an extract, then using Saccharomyces cerevisiae and Lactobacillus germinatus to carry out a first-step fermentation of the extract, followed by a second-step fermentation using acetic acid bacteria, resulting in a product containing at least one of several specific compounds, which can be added to pharmaceuticals, food, or skincare products, and has the uses of reducing body fat accumulation, whitening, and / or anti-aging. The literature "Analysis of the antioxidant capacity of emlica emblica extract and its effect on tyrosinase activity" mentions that the polyphenol and flavonoid contents in the aqueous extract of emlica emblica fruit are 22.95 mg / g DW and 34.61 mg / g DW, respectively, and the IC50 values ​​for scavenging DPPH, ABTS+, and hydroxyl radicals are 4.58 μg / mL, 9.61 μg / mL, and 1.99 mg / mL, respectively. The effect of Phyllanthus emblica fruit extract on tyrosinase is concentration-dependent. Extracts at concentrations of 0.2 and 0.6 mg / mL show some activating effect on tyrosinase, while extracts at 1.8 mg / mL exhibit an inhibitory effect. In summary, research on the application of Phyllanthus emblica extract in cosmetics primarily focuses on whitening, spot removal, anti-oxidation, and anti-aging.

[0005] Calibick Mayer's yeast is Mayer's yeast ( Mycoplasma These are microorganisms belonging to the genus *[]*, native to China, and primarily used in research on fruit wine fermentation. The literature "Study on the Diversity of Microorganisms in Natural Alcoholic Fermentation of Ginger and Kiwifruit" mentions that... MyerozymaIt is the dominant yeast community in the early and middle stages of natural alcoholic fermentation of ginger and kiwifruit. At the second academic event of the 2021-2022 academic year held by the National-Local Joint Engineering Research Center for Germplasm Innovation and Utilization of Southwest Chinese Medicinal Herbs, Liu Kunyi presented a report entitled "Fermentation of Coffea canephora inoculated with yeasts: Microbiological, chemical, and sensory characteristics," reporting that compared with traditional fermentation, inoculating coffee beans with *Coffea canephora* for enhanced fermentation helps control filamentous fungi during fermentation and obtains excellent sensory characteristics, making coffee inoculated with *Coffea canephora* have significant market potential. The literature "Research Progress on Non-Saccharifying Yeasts in Brewing Wines" mentions that there is a rich variety of non-saccharifying yeasts, including *Coffea canephora* (…). Mycoplasma These yeasts, such as *Callibac Myers*, are widely present in brewing raw materials, fermentation agents, and the brewing environment. Their metabolites and secreted enzymes have a significant impact on the aroma and flavor of brewed wines. As can be seen from the above literature, the application of non-brewing yeasts such as *Callibac Myers* in brewing raw materials is attracting increasing attention.

[0006] Different yeasts will inevitably have different fermentation states and metabolic products. Therefore, it is essential to screen out a yeast strain with excellent fermentation performance. Furthermore, yeast fermentation produces a relatively high concentration of active ingredients, and the complete cell breakdown after fermentation fully releases intracellular substances, making it highly promising for cosmetic applications. Effectively combining yeast with Phyllanthus emblica can yield a fermentation product richer in active ingredients, which will greatly promote the application of Phyllanthus emblica in various fields. Summary of the Invention

[0007] The purpose of this invention is to provide a novel *Callibik Mayer's yeast* strain with excellent fermentation performance, offering a new option for yeast strains. This strain exhibits superior fermentation performance, demonstrating significant advantages in fermenting *Amla emblica* fruit. When using this *Callibik Mayer's yeast* strain to prepare *Amla emblica* fruit extract, the production cycle is short, the product is stable, and it has a high content of active substances, ensuring safety and health, and meeting the requirements for cosmetic raw materials. The resulting fermented *Amla emblica* fruit extract possesses good antioxidant, whitening, and anti-inflammatory effects, showing promising application prospects in cosmetics.

[0008] This invention independently screened a high-performance strain of *Alpinia emblica* Mayer's yeast from fresh *Alpinia emblica* fruit. Meyerozyma caribbica The yeast was named *Calibic Mayer's yeast*. Meyerozyma caribbicaThe *Calibic Mayer's yeast* Y-YGZ was obtained by the inventor through screening and was deposited on September 23, 2022, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province, with accession number CCTCC NO: M 20221480. Meyerozyma caribbica The 26S rDNA gene sequence of ) is shown in SEQ ID NO: 1.

[0009] Furthermore, the present invention also provides a microbial agent containing the aforementioned *Calibic Mayer's yeast* (…). Meyerozyma caribbica Y-YGZ. This inoculum may contain only *Calibic Mayer's yeast* (Y-YGZ). Mycoplasma Caribbean Y-YGZ is a microorganism that may also contain other microorganisms.

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

[0011] The present invention also provides the above-mentioned Calibick Mayer's yeast ( Meyerozyma caribbica The application of Y-YGZ or the above-mentioned inoculants in the preparation of fermented products. In this application, *Calibic Mayer's yeast* (…) is generally used. Mycoplasma Caribbean Y-YGZ or the above-mentioned bacterial agents are added to the culture medium for fermentation to obtain fermentation products.

[0012] The fermentation uses of *Calibic Mayerii* yeast in various fields are disclosed in the prior art. This invention relates to *Calibic Mayerii* yeast (… Meyerozyma caribbica Y-YGZ can be used in these existing and publicly available fields to replace existing yeasts to obtain fermentation products.

[0013] Preferably, the present invention uses Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ or the above-mentioned bacterial agent is used to ferment and prepare Phyllanthus emblica fruit fermentation product, wherein the Phyllanthus emblica fruit fermentation product is prepared using the above-mentioned Caribik Mayer's yeast ( Meyerozyma caribbica The fermentation product obtained by fermenting Phyllanthus emblica fruit with Y-YGZ or the above-mentioned microbial agents can also be called Phyllanthus emblica fruit extract. During the fermentation process, Phyllanthus emblica fruit is generally ground into a pulp. Depending on the needs of fermentation, carbon sources, nitrogen sources, inorganic salts, and other components necessary for fermentation can be added to the pulp.

[0014] Furthermore, the Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ can be expanded using a culture medium to obtain the required number of strains, with an optimal culture temperature of 20℃-30℃. In a specific embodiment of this invention, *Calibic Myersella* (…) Meyerozyma caribbica The Y-YGZ strain was expanded using liquid seed culture medium. After expansion, the supernatant was removed by centrifugation to obtain *Calibic Mayer's yeast*. Meyerozyma caribbica Y-YGZ; Preferably, the seed culture medium formula is: 2-5 wt% carbon source, 1.5-3 wt% nitrogen source, 0.2-0.7 wt% inorganic salts, with water as the balance. The carbon source is a commonly used carbon source for microbial culture, such as fructose, sucrose, glucose, maltodextrin, etc.; the nitrogen source is a commonly used nitrogen source for microbial culture, such as peptone, yeast powder, soybean powder, etc.; and the inorganic salts are dipotassium hydrogen phosphate, magnesium sulfate, etc.

[0015] This invention provides an extract of Phyllanthus emblica fruit, which is derived from the aforementioned *Callibik Mayer's yeast* (…). Meyerozyma caribbica It is obtained by fermenting Phyllanthus emblica fruit with Y-YGZ.

[0016] This invention provides a method for preparing Phyllanthus emblica fruit extract, the method comprising using the above-mentioned Calibick Mayer's yeast ( Meyerozyma caribbica The steps of fermenting Phyllanthus emblica fruit using Y-YGZ.

[0017] Furthermore, the above method includes the following steps: (1) Pulp up the amla fruit, let the resulting pulp stand and ferment naturally until the pH no longer changes; (2) Mix the above-mentioned naturally fermented fruit pulp, nitrogen source, carbon source, inorganic salt and water to prepare a culture medium containing fruit pulp; (3) Add Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ was added to the above culture medium containing fruit pulp and fermented until the pH no longer changed, thus obtaining the fermentation broth; (4) Remove impurities and bacteria from the fermentation broth to obtain Phyllanthus emblica fruit extract.

[0018] Furthermore, in step (1), the whole amla fruit is used as raw material. Before pulping, the amla fruit can be washed, dried, and then pulped using a juicer or other equipment. The resulting pulp is then naturally fermented using its endophytic bacteria. The settling temperature is 15~25℃. Natural fermentation is stopped when the pH no longer changes, which generally takes 1~3 days.

[0019] In a specific embodiment of the present invention, the settling temperature is 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C.

[0020] In a specific embodiment of the present invention, the time required for natural fermentation is 1 day, 1.5 days, 2 days, 2.5 days, or 3 days.

[0021] Furthermore, in step (2), the naturally fermented fruit pulp is further mixed with nitrogen source, carbon source, inorganic salts, etc., to prepare a fruit pulp-containing culture medium for fermentation of *Calibic Mayer's yeast*. The mixing order of the naturally fermented fruit pulp, nitrogen source, carbon source, inorganic salts, and water in the culture medium is arbitrary. The naturally fermented fruit pulp can be mixed with water first, and then the nitrogen source, carbon source, and inorganic salts can be added. Alternatively, the nitrogen source, carbon source, and inorganic salts can be added to the water first, and then the naturally fermented fruit pulp can be added. Or, the naturally fermented fruit pulp, nitrogen source, carbon source, and inorganic salts can be added to the water simultaneously. Preferably, the nitrogen source, carbon source, and inorganic salts are added to the water first, sterilized, and then the naturally fermented fruit pulp is added. This protects the nutrients in the fruit pulp from damage, eliminates miscellaneous bacteria, reduces the probability of contamination, ensures pure culture fermentation, and stabilizes product quality. Sterilization can be performed using various feasible sterilization methods reported in the prior art, such as moist heat sterilization.

[0022] Furthermore, in step (2), the prepared culture medium containing fruit pulp contains 1-10% by mass of naturally fermented fruit pulp, 0.1-2% by mass of nitrogen source, 0.1-2% by mass of carbon source, 0.02-0.2% by mass of inorganic salts, and the remainder is water. The nitrogen source is a commonly used nitrogen source for microbial culture, such as peptone, yeast powder, soybean meal extract, etc.; the carbon source is a commonly used carbon source for microorganisms, such as glucose, sucrose, fructose, maltodextrin, etc.; and the inorganic salts are dipotassium hydrogen phosphate, magnesium sulfate, etc.

[0023] Furthermore, in step (3), *Calibic Mayer's yeast* Y-YGZ is added to the culture medium containing fruit pulp for further fermentation. *Calibic Mayer's yeast* ( Meyerozyma caribbica The inoculation amount of Y-YGZ strain (based on Calibick Mayer's yeast) Meyerozyma caribbica The Y-YGZ cell counter is 0.5-1 wt% of the culture medium containing fruit pulp. Calibick Mayer's yeast ( Meyerozyma caribbica Sufficient quantities of Y-YGZ strains can be obtained through the above-described expanded culture method.

[0024] Furthermore, in step (3), after inoculating the bacterial strain, fermentation is carried out at 20-30℃ until the pH remains constant. The fermentation time is generally 36-42 h.

[0025] In a specific embodiment of the present invention, the specific fermentation temperatures are 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, and 30℃; and the fermentation times are 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, and 42 h.

[0026] Furthermore, in step (4), the fermentation broth obtained from fermentation is purified and sterilized by means of filtration, centrifugation, etc.

[0027] Furthermore, according to the present invention, Calibick Mayer's yeast ( Meyerozyma caribbica The amla fruit extract prepared by Y-YGZ exhibits superior antioxidant, whitening, and anti-inflammatory effects compared to amla fruit extracts obtained through fermentation with other yeasts. It also demonstrates excellent inhibitory effects on three inflammatory factors: IL-6, TNF-α, and IL-1β. Therefore, the amla fruit extract obtained in this invention can be used in cosmetics as an antioxidant, whitening, and anti-inflammatory ingredient.

[0028] The present invention also provides the application of the above-mentioned Meyerozyma caribbica Y-YGZ, the above-mentioned inoculum, or the above-mentioned Phyllanthus emblica fruit extract in the preparation of cosmetics, as antioxidant, whitening, and anti-inflammatory ingredients in cosmetics.

[0029] The present invention also provides a composition comprising the above-described Phyllanthus emblica fruit extract of the present invention. The composition is preferably a cosmetic.

[0030] Furthermore, the cosmetic product is a cosmetic product that has at least one of the following effects: antioxidant, whitening, and anti-inflammatory.

[0031] The present invention has the following advantages: 1. This invention has independently screened and obtained a strain of *Calibic Mayer's yeast* with excellent fermentation performance (…). Mycoplasma Caribbean The strain Y-YGZ showed outstanding advantages in fermenting Phyllanthus emblica fruit, increasing the content of effective components in the fruit and producing fermentation products with great application value.

[0032] 2. The method for preparing Phyllanthus emblica fruit extract provided by this invention is an improvement on traditional fermentation technology. First, the insoluble proteins and other macromolecules in Phyllanthus emblica fruit are converted into soluble small-molecule amino acids through natural fermentation by endophytic bacteria. Then, fermentation is carried out using the special *Callibik Mayer's yeast* of this invention, which produces a variety of active ingredients. The resulting fermentation broth has better antioxidant, whitening, and anti-inflammatory effects. Unfermented Phyllanthus emblica fruit only inhibits the inflammatory factors IL-1β and TNF-α, but has no inhibitory effect on the inflammatory factor IL-6. After fermentation using this invention, the fermentation broth has a good inhibitory effect on the inflammatory factors IL-6, TNF-α, and IL-1β, and the inhibitory effect on IL-1β and TNF-α is greatly enhanced.

[0033] 3. This invention involves liquid fermentation of the entire amla fruit, with the fermentation extract as the final product. This fully utilizes both the amla fruit raw material and the amla fruit fermentation liquid. The process is simple, easy to operate, and low in cost, enabling large-scale production.

[0034] Preservation Information This invention is based on the yeast Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221480, on September 23, 2022. The deposit address is: Wuhan University, Luojia Mountain, Wuchang, Wuhan, Hubei Province. Attached Figure Description

[0035] Figure 1 It is Calibick Mayer's yeast ( Meyerozyma caribbica Photographs of the colony morphology of Y-YGZ CCTCC NO: M20221480.

[0036] Figure 2 It is Calibick Mayer's yeast ( Meyerozyma caribbica Photomicrograph of Y-YGZ CCTCC NO: M20221480 bacterial cells. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0038] Unless otherwise defined, the technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions in this specification shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not restrictive.

[0039] Example 1: Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ screening and identification process 1.1 Isolation of bacterial strains Fresh amla fruit, sea buckthorn fruit, cherry, old dough, and distiller's grains were used as raw materials. Under aseptic conditions, these materials were crushed, and 1 part by weight of sterile water and 0.1 g / L ampicillin sodium were added. The mixture was sealed and incubated at 22°C until bubbles were produced when shaken and a distinct alcoholic aroma was observed. The juice solution was then evenly spread onto yeast solid culture medium plates containing 0.1 mg / L ampicillin sodium using a spreader. The plates were incubated at 25°C for 1-2 days. Single colonies with round and smooth morphology were selected and inoculated onto yeast solid culture medium slant tubes. These slant colonies were incubated at 25°C for 1-2 days. After the cells had grown well, they were stored at 4°C. The selected strains were named Y-YGZ, Y-SJ, Y-YT, SM-5, CF-37, WJ-37, AN, Y-5-1, NJ-1, and SS-1, respectively. The yeast solid culture medium consisted of: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, 20 g / L agar, and the remainder was water.

[0040] 1.2 Fermentation Using an inoculation loop, a loopful of yeast cells was picked from each test tube and inoculated into the yeast seed culture medium, and incubated statically at 25°C overnight. The seed culture was then inoculated into the Phyllanthus emblica fermentation medium at an inoculation rate of 3 wt%, and incubated statically at 25°C for 20–24 h. After fermentation, the fermentation broth was filtered through a 0.22 μm mixed cellulose membrane, and the inhibition rate of the filtrate on tyrosinase activity was measured. The yeast seed culture medium consisted of: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and the remainder water. The Phyllanthus emblica fermentation medium consisted of: 50 g / L fresh Phyllanthus emblica juice, 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and the remainder water.

[0041] 1.3 Tyrosinase activity inhibition test 1.3.1 Solution Preparation Tyrosinase, activity ≥1000 units / mg solid; Levodopa, purity ≥98%; Tyrosinase solution: Prepared with PBS buffer (50mM, pH=6.8), 100 u / mL, prepare immediately before use; Levodopa solution: Prepared with PBS buffer (50 mM, pH = 6.8), 1 mg / mL, stored protected from light; Sample solution: Fermentation filtrate obtained from fermentation of different bacterial strains.

[0042] 1.3.2 Operating Procedures Referring to Table 1, use 10 mL test tubes to set up sample tubes (T), sample background (T0), enzyme reaction tubes (C), and solvent background (C0). For each sample, three parallel tubes should be set up for each test concentration of the sample tube (T), and three parallel tubes should also be set up for the enzyme reaction tube (C).

[0043] Add 1 mL of sample solution of the same concentration to both the sample tube (T) and the sample background (T0). Add 1 mL of PBS buffer (50 mM, pH 6.8) to both the enzyme reaction tube (C) and the solvent background (C0). Add 0.5 mL of tyrosinase solution to both the sample tube (T) and the enzyme reaction tube (C). Replace the sample background (T0) and solvent background (C0) with 0.5 mL of PBS buffer (50 mM, pH 6.8). Mix the sample and tyrosinase thoroughly and incubate in a 37°C water bath for 10 minutes. Add 2 mL of levodopa solution to each tube sequentially, controlling the reaction time for each tube to 10 minutes. Immediately transfer the reaction solutions from each tube to a cuvette and measure the absorbance at 475 nm.

[0044] 1.3.3 Calculation Method Calculate the tyrosinase inhibition rate: Inhibition rate (%) = [1 - (T - T0) / (C - C0)] * 100% In the formula: T-sample tube absorbance, which is the absorbance of the solution after the sample reacts with tyrosinase; T0 - Sample background absorbance; The absorbance of the C-enzyme reaction tube is the absorbance of the tyrosinase and dopa reaction when no sample is added. C0 - Solvent background absorbance.

[0045] 1.3.4 Results As can be seen from the results in Table 2, the fermentation broths of different strains after fermenting Phyllanthus emblica fruit showed different inhibitory effects on tyrosinase activity. Among them, the fermentation broth obtained by fermenting Y-YGZ, which was screened from fresh Phyllanthus emblica fruit, showed the best inhibitory effect on tyrosinase activity.

[0046] 1.4 Strain Identification The strain Y-YGZ, which exhibited the best tyrosinase inhibition effect, was selected as the target strain of this invention. Y-YGZ was sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for genome sequencing identification. The results showed that the strain was *Calibic Mayer's yeast*. Meyerozyma caribbica The 26S rDNA gene sequence of this strain is shown in SEQ ID NO: 1.

[0047] The Calibick Mayer's yeast ( Meyerozyma caribbica The morphological characteristics of ) are as follows: Calibick Mayer's yeast ( Meyerozyma caribbica On solid yeast culture plates, the colonies are uniform in texture, smooth and moist in surface, with neat edges, milky white in color, and opaque. Figure 1 As shown; the bacterial cell is a single cell, oval in shape, with a simple morphology, and reproduces by budding, as... Figure 2 As shown.

[0048] Example 2: Large-scale culture of the bacterial strain Calibick Mayer's yeast Y-YGZ was picked from a test tube and inoculated into 500 mL of sterile seed culture medium containing 2 wt% glucose, 2 wt% peptone, and 1 wt% yeast extract. It was then incubated statically at 23°C for 13 h until the logarithmic growth phase. The cells were then inoculated again and expanded into 5 L of sterile seed culture medium. After a second culture of 10 h until the logarithmic growth phase, the cells were centrifuged at 8000 rpm for 30 min and the supernatant was discarded to obtain fresh Calibick Mayer's yeast Y-YGZ cells.

[0049] Example 3 Fresh amla fruits were washed, dried, and then ground into a pulp using a grinder to obtain amla fruit pulp. The amla fruit pulp was allowed to stand at 20°C for 2 days to allow natural fermentation by its endophytic bacteria until the pH no longer changed. A culture medium containing 1 wt% peptone, 1 wt% sucrose, and 0.1 wt% dipotassium hydrogen phosphate was prepared and sterilized at high temperature. 100 L of the sterilized culture medium cooled to room temperature was mixed with 5 kg of the naturally fermented amla fruit pulp to obtain a culture medium containing the pulp. Yeast cells Y-YGZ from Example 2 were inoculated into the culture medium containing the pulp at an inoculum size of 1 wt%. Fermentation was carried out at 23°C for 40 h until the pH no longer changed, at which point the fermentation was complete, yielding the fermentation broth. The fermentation broth was centrifuged at 3000 rpm to remove impurities, and the supernatant was filtered through a 0.22 μm polyethersulfone filter to obtain fermented amla fruit extract S1.

[0050] Comparative Example 1 The whole amla fruits were washed, dried, and then ground into a pulp using a grinder to obtain amla fruit pulp. 0.5 kg of the pulp was allowed to stand at 20°C for 2 days to allow for natural fermentation by endophytic bacteria. Once the pH no longer changed, it was added to 10 L of sterile water cooled to room temperature and mixed thoroughly. The mixture was then placed at 23°C for 40 h to obtain an amla fruit solution. The amla fruit solution was centrifuged at 3000 rpm to remove impurities. The supernatant was then filtered through a 0.22 μm polyethersulfone filter to obtain unfermented amla fruit extract C1.

[0051] Comparative Example 2 Phyllanthus amla fruit extract was prepared according to the method in Example 3, except that the Saccharomyces cerevisiae Y-YGZ cells were replaced with Saccharomyces cerevisiae cells with preservation number CCTCC NO: M 2022135. The resulting Phyllanthus amla fruit extract was named Phyllanthus amla fruit extract C2.

[0052] Comparative Example 3 Phyllanthus amla fruit extract was prepared according to the method in Example 3, except that the Saccharomyces cerevisiae Y-YGZ cells were replaced with Saccharomyces cerevisiae cells with preservation number CCTCC NO: M 20211621. The resulting Phyllanthus amla fruit extract was named Phyllanthus amla fruit extract C3.

[0053] Experimental Example 1: Determination of Total Phenolic Content (1) Preparation of the standard curve of gallic acid Total phenol content was determined using gallic acid (GAE) as a reference standard. A precise 0.1 mg / mL gallic acid standard solution was prepared and stored protected from light. Accurately pipette 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the gallic acid standard solution into 10 mL volumetric flasks. Add 2.5 mL of 0.1 mol / L Folin-phenol reagent to each flask, shake well, and then add 2 mL of 15% sodium carbonate solution over 1–8 min. Dilute to the mark with distilled water and react at room temperature in the dark for 2 h. Measure the absorbance at 760 nm. Plot a standard curve with gallic acid concentration on the ordinate and absorbance on the abscissa, and calculate the corresponding regression equation.

[0054] (2) Determination of total phenols in Phyllanthus emblica fruit extract Take 100 μL of sample S1 from Example 3 and samples C1, C2, and C3 from Comparative Examples 1-3 into 10 mL volumetric flasks, and measure the absorbance of the samples according to the method described above (starting from the addition of 2.5 mL of 0.1 mol / L Folin-phenol reagent). Each group is repeated three times. Based on the regression equation and the dilution factor of the samples, the total phenol content in the fermentation broth is obtained using the following formula: Total phenolic equivalent per gram of extract (mg / g) = (91.205A - 1.6784) × W × 10 -3 / ρ Where: A - absorbance of the sample; Dilution factor of W-Phragmites australis fruit extract; ρ - Fermentation broth density (1.001 g / mL).

[0055] (3) The total phenol content is shown in Table 3 below: The results above show that the total phenol content in the extract after fermentation with *Calibic Myersia davidii* Y-YGZ is significantly higher than that in extracts obtained by traditional methods or fermentation with other yeasts.

[0056] Experimental Example 2: Detection of Amino Acid Content Using sample S1 from Example 3 and samples C1, C2, and C3 from Comparative Examples 1-3 as experimental samples, the content of free amino acids in the samples was determined by HPLC pre-column derivatization. The results are shown in Table 4.

[0057] As shown in the table above, the free amino acid content of the amla fruit extract fermented by the strain of this invention is increased compared with that obtained by the traditional extraction method, and the total amount of amino acids is also increased. Moreover, the amino acid content of amla fruit extracts fermented by different strains is also different. The free amino acid content of the strain obtained by this invention is significantly higher than that of other strains.

[0058] Experimental Example 3: Evaluation of safety, antioxidant, whitening, and anti-inflammatory effects This experiment aims to evaluate the safety, antioxidant, whitening, and anti-inflammatory effects of the samples prepared in the above embodiments and comparative examples.

[0059] Safety assessment Lamination: Human epidermal keratinocytes (HaCaT) in the logarithmic growth phase were used, at a concentration of 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 μL / mL in 96-well cell culture plates. The culture medium was DMEM high-glucose medium with 10% fetal bovine serum added. The seeded cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 24 h.

[0060] Sample solution preparation: Sample S1 prepared in Example 3 and samples C1-C3 prepared in Comparative Examples 1-3 were prepared into stock solutions of a certain concentration using serum-free culture medium and filtered through a 0.22 μm filter membrane for sterilization. When needed, they were diluted with serum-free culture medium to a concentration of 10%, 5%, 2%, and 1%.

[0061] Drug administration: After 24 h of routine culture, the old culture medium was discarded, and the experimental group was replaced with 100 μL of sample solution. The normal control group was given an equal volume of serum-free culture medium. There were 6 parallel wells in each level.

[0062] Detection: After culturing for another 24 h, the old culture medium was discarded, and 100 μL of 10% CCK-8 solution prepared with serum-free culture medium was added to each well. The wells were then incubated in a cell culture incubator for another 2 h. The absorbance was measured at 450 nm using a microplate reader. The relative growth rate (RGR) was the ratio of the absorbance of the experimental group to that of the normal control group. According to GB / T16886.5-2017, a RGR below 70% was considered cytotoxic. The results are shown in Table 5. The Phyllanthus emblica fruit extract samples before and after fermentation showed no potential cytotoxicity within the concentration range of 1%-10%.

[0063] Antioxidant activity evaluation 2.1 DPPH free radical scavenging test Preparation of 0.1 mM DPPH solution: Accurately weigh 4.0 mg DPPH and place it in a 100 mL brown volumetric flask. Dissolve and dilute to volume with 95% ethanol.

[0064] Preparation of sample solutions of different concentrations: Using purified water as a diluent, samples S1 in the examples and samples C1~C3 in the comparative examples were prepared into solutions with a volume concentration of 0.1~2.0%.

[0065] Accurately measure 5.0 mL of 0.1 mM DPPH solution and 5.0 mL of sample solutions of different concentrations into stoppered test tubes and mix well. Use an equal volume of water and 95% ethanol mixture as a blank control. Incubate at room temperature for 30 minutes and measure the absorbance of each solution at 523 nm. Set up a separate control group (Control) by accurately measuring 5.0 mL of DPPH solution and 5.0 mL of purified water, mixing them as above. The calculation method is as follows: The results are shown in Table 6.

[0066] The data in the table show that all samples have a high ability to scavenge DPPH free radicals under a concentration gradient of 0.1%-2.0%, but the S1 sample prepared by Y-TGZ shows better DPPH free radical scavenging ability.

[0067] Whitening activity evaluation 3.1 Melanoma cell proliferation detection Forskolin is a drug that stimulates melanocytes to produce tyrosinase and melanin. In this experiment, Forskolin was used as the model group. The cell culture medium for the model group was prepared by using serum-containing cell culture medium to prepare a Forskolin solution with a concentration of 40 μmol / L.

[0068] Preparation of sample solutions: Sample S1 prepared in Example 3 and samples C1-C3 prepared in Comparative Examples 1-3 were prepared with the above-mentioned Forskolin solution to a concentration of 1%, 2%, and 5%, respectively.

[0069] Melanoma cells from B16 mice in the logarithmic growth phase were collected and processed at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL in 96-well cells culture plates and incubated overnight at 37°C with 5% CO2. Samples were prepared using culture medium containing trichodin (trichodin concentration of 40 μmol / L). The old culture medium was discarded, and the experimental group was treated with the sample solution. The control group was treated with 100 μL of trichodin-containing cell culture medium per well. After culturing for 24 h, the relative cell proliferation rate was measured using the neutral red assay. The relative proliferation rate (RGR) was the ratio of the absorbance of the sample group to that of the control group.

[0070] As shown in Table 7, the cell proliferation rate of the samples before and after fermentation of Phyllanthus emblica at a concentration of 5% was lower than that of the model group (Forskolin group), indicating a certain inhibitory effect on the proliferation of B16 cells.

[0071] 3.2 Melanin content detection Sample solution preparation: Sample S1 prepared in Example 3 and samples C1-C3 prepared in Comparative Examples 1-3 were prepared into stock solutions of a certain concentration using serum-containing culture medium and filtered through a 0.22 μm filter membrane for sterilization. When needed, the solutions were diluted with serum-containing culture medium to a concentration of 1%, 2%, and 5%.

[0072] Take B16 cells in logarithmic growth phase, and use 5 × 10⁻⁶ cells. 4 Inoculate 3 mL of the culture medium per well into 6-well plates at a density of 1 / mL. Incubate at 37°C and 5% CO2 for 24 h. Discard the old culture medium. Add 3 mL of serum-containing culture medium to the normal control group and the model group. Add 3 mL of sample solution to the experimental group. Add 60 μL of 2 mM saliva extract solution to each well of the model group and the experimental group to stimulate melanin production. Continue to incubate for 72 h.

[0073] The method for determining intracellular melanin content is as follows: Discard the old culture medium, wash twice with PBS, add 500 μL of 1 mol / L (containing 10% DMSO) NaOH solution to each well to lyse the cells, heat at 80℃ for 60-90 min, centrifuge at 3000 rpm for 10 min, take the supernatant and add 100 μL / well to a 96-well plate, measure the absorbance at 450 nm to obtain the total melanin content. Taking the melanin content of the model group as 100%, the melanin inhibition rate of the experimental group is obtained. If the sample has a significant effect on the proliferation of B16 cells, the ratio of the relative value of melanin content to the corresponding B16 cell proliferation rate is the rate of change of melanin production per unit cell.

[0074] The method for calculating the total melanin content inhibition rate is as follows.

[0075] Considering the effect of the sample on B16 cell proliferation, the melanin inhibition rate was normalized to obtain the melanin inhibition rate per unit cell. The results are shown in Table 8. Compared with the model group, both fermentation and fermentation of Phyllanthus emblica fruit inhibited melanin production. Moreover, the inhibitory effect on melanin production increased with increasing sample concentration. However, the whitening effect of the fermented Phyllanthus emblica fruit extract was significantly better.

[0076] 3.3 Tyrosinase Activity Detection Within melanocytes, tyrosine is activated by tyrosinase to form dopa. Dopa then undergoes dehydrogenation to form dopaquinone, which rearranges to form 5,6-indole. This 5,6-indole polymerizes and binds to structural proteins within the melanocyte to form melanin. Tyrosinase is a pluripotent enzyme in this process; inhibiting its activity can suppress melanin formation. The experimental principle involves determining the relative activity of tyrosinase in a sample by measuring its ability to oxidize the substrate dopa to dopaquinone.

[0077] Sample solution preparation: Sample S1 prepared in Example 3 and samples C1-C3 prepared in Comparative Examples 1-3 were prepared into stock solutions of a certain concentration using serum-containing culture medium and filtered through a 0.22 μm filter membrane for sterilization. When needed, the solutions were diluted with serum-containing culture medium to a concentration of 1%, 2%, and 5%.

[0078] Take B16 cells in logarithmic growth phase, and use 5 × 10⁻⁶ cells. 4Inoculate 3 mL of the culture medium per well into 6-well plates at a density of 1 / mL. Incubate at 37°C and 5% CO2 for 24 h. Discard the old culture medium. Add 3 mL of serum-containing culture medium to the normal control group and the model group. Add 3 mL of sample solution to the experimental group. Add 60 μL of 2 mM saliva extract solution to each well of the model group and the experimental group to stimulate melanin production. Continue to incubate for 72 h.

[0079] Discard the old culture medium, wash the cells twice with PBS, then lyse the cells with Tirs-HCl (0.02 mol / L, pH 6.8) containing 0.1% Triton-100, sonicate for 10 min, add 0.1% L-DOPA (0.01 mol / L, pH 8.0) and incubate at 37℃ for 60 min. After centrifugation, collect the supernatant and add 100 μL / well of a 96-well plate. Measure the absorbance at 450 nm using a microplate reader. The ratio of absorbance between the experimental group and the model group is used as an indicator to evaluate the level of tyrosinase activity. If the sample has a significant effect on the proliferation of B16 cells, the tyrosinase activity should be normalized according to the proliferation rate.

[0080] As can be seen from the results in Table 9, the unfermented Phyllanthus emblica fruit extract sample C1 showed a very weak inhibitory effect on tyrosinase activity at a concentration of 1%-5%, which was almost negligible. However, the fermented Phyllanthus emblica fruit extract sample could significantly inhibit tyrosinase activity at a concentration of 1%-5%, and the inhibitory effect increased with increasing concentration. In particular, the inhibitory effect was significantly enhanced after fermentation with Y-YGZ provided by this invention.

[0081] Anti-inflammatory activity evaluation Using a mouse macrophage model, LPS was used to stimulate the production of inflammatory factors, and the results were examined to see if the sample could inhibit the secretion of inflammatory factors.

[0082] LPS solution: Prepare a stock solution with a concentration of 500,000 units / mL using serum-free 1640 culture medium, filter it through a 0.22 μm filter membrane for sterilization, store it in a -20℃ refrigerator, and dilute it to a working solution of 40,000 units / mL before use.

[0083] Sample solutions: Using LPS solution as a diluent, samples S1 in the examples and samples C1-C3 in the comparative examples were prepared into solutions with a volume concentration of 2%, and sterilized by filtration through a 0.22 µm filter membrane.

[0084] Blank control group: Raw264.7 cells were fed at a rate of 1×10⁻⁶. 5Inflammatory factors were detected by inoculating 1 / mL of the sample into 24-well plates and incubating at 37°C and 5% CO2 for 24 h. Serum-free medium was then added, and the plates were incubated for another 24 h. Inflammatory factors were then detected using an ELISA kit.

[0085] Model group: Raw264.7 cells were fed at a rate of 1×10⁻⁶. 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. Then, an equal volume of LPS solution was added to the serum-free medium used in the blank control group, and the plates were cultured for another 24 h. The inflammatory factors were then detected using an ELISA kit.

[0086] Experimental group: Raw264.7 cells were fed at a rate of 1×10⁻⁶. 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. Then, an equal volume of sample solution as the serum-free medium for the blank control group was added, and the plates were cultured for another 24 h. The inflammatory factors were then detected using an ELISA kit.

[0087] The inhibition rate of each sample against inflammatory factors was calculated using the following method: The test results are as follows: The results show that LPS stimulation increased the secretion of three pro-inflammatory factors, IL-6, TNF-α, and IL-1β, in mouse macrophages. After treatment with each sample, the secretion of IL-6, TNF-α, and IL-1β changed to varying degrees compared with the LPS model group without Phyllanthus emblica treatment, indicating that the samples had an inhibitory effect on IL-6, TNF-α, and IL-1β.

[0088] Among them, the unfermented Phyllanthus emblica fruit extract C1 only inhibited the secretion of TNF-α and IL-1β, and the inhibition rate was low. Although the inhibitory effect of Phyllanthus emblica fruit extract on inflammatory factors was improved after fermentation, the effects of different strains after fermentation were significantly different. Phyllanthus emblica fruit extract S1 after Y-YGZ fermentation inhibited the secretion of three pro-inflammatory factors, IL-6, TNF-α and IL-1β, and the inhibitory effect was significantly higher than that of other strains.

Claims

1. A type of Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ, characterized by: The accession number is CCTCC NO: M20221480.

2. A microbial agent, characterized in that: Contains the *Calibic Mayer's yeast* as described in claim 1 ( Meyerozyma caribbica )Y-YGZ.

3. The Calibick Mayer's yeast as described in claim 1 ( Meyerozyma caribbica The application of Y-YGZ or the microbial agent according to claim 2 in the preparation of fermentation products.

4. The application according to claim 3, characterized in that: The fermented product is a fermented product of Phyllanthus emblica fruit.

5. An extract of Phyllanthus emblica fruit, characterized in that: The Calibick Mayer's yeast as described in claim 1 ( Meyerozyma caribbica It is obtained by fermenting Phyllanthus emblica fruit with Y-YGZ.

6. A method for preparing an extract of Phyllanthus emblica fruit, characterized in that: Contains the *Calibic Mayer's yeast* as described in claim 1 ( Meyerozyma caribbica The steps of fermenting Phyllanthus emblica fruit using Y-YGZ.

7. The preparation method according to claim 6, characterized in that: Includes the following steps: (1) Pulp up the amla fruit, let the resulting pulp stand and ferment naturally until the pH no longer changes; (2) Mix the above-mentioned naturally fermented fruit pulp, nitrogen source, carbon source, inorganic salt and water to prepare a culture medium containing fruit pulp; (3) Add Calibick Mayer's yeast ( Meyerozyma caribbica Y-YGZ was added to the above culture medium containing fruit pulp and fermented until the pH no longer changed, thus obtaining the fermentation broth; (4) Remove impurities and bacteria from the fermentation broth to obtain Phyllanthus emblica fruit extract.

8. The use of Meyerozyma caribbica Y-YGZ as described in claim 1, or the inoculum as described in claim 2, or the Phyllanthus emblica fruit extract as described in claim 5, in the preparation of cosmetics.

9. The application according to claim 8, characterized in that: As an antioxidant, whitening, and anti-inflammatory ingredient in cosmetics.

10. A composition comprising the amla fruit extract of claim 5.

11. The composition according to claim 10, characterized in that: The composition is a cosmetic product.

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