Method for preparing mannosyl-erythritol lipids, mannosyl-erythritol lipids and use thereof

Abstract

The application discloses a preparation method of a mannose erythritol lipid, the mannose erythritol lipid and application of the mannose erythritol lipid. The preparation method of the mannose erythritol lipid comprises the following steps: adding a substrate into an initial fermentation medium, inoculating a Candida bombicola seed liquid, and then performing fermentation; heating and inactivating the fermentation liquor, cooling and collecting a product layer, dissolving and decoloring, and then obtaining a decolorized clear liquid; and performing vacuum concentration and cooling on the decolorized clear liquid to obtain the mannose erythritol lipid. The substrate is one of C8-C25 fatty acids or esters thereof; and the adding amount of the substrate is 10%-30% of the mass of the initial fermentation medium. The inoculated viable cell number of the Candida bombicola is 4.5*10 5 cfu / mL-5.5*10 6 cfu / mL. During the fermentation, the carbon-nitrogen ratio of the fermentation system is maintained as (5-10):1, and the pH value is maintained as 4.0-6.5. The mannose erythritol lipid prepared by the method has a single fatty acid structure, high substrate conversion rate and product purity, a clear melting point range and HLB value, and improved application value.

Description

Technical Field

[0001] This application belongs to the field of surfactant technology, specifically relating to a method for preparing mannose erythritol ester, mannose erythritol ester and its application. Background Technology

[0002] Mannosyl erythritol lipids (MELs) are glycolipid biosurfactants. Besides their excellent emulsifying and surface activity, they also possess unique metabolic and structural characteristics, leading to various functions such as gene transfection, broad-spectrum antibacterial activity, and skin repair. In recent years, MELs have shown broad application prospects in the food, pharmaceutical, and cosmetic fields, and are widely recognized as one of the most promising biosurfactants. The following are some references on the application of mannose erythritol lipids in the fields of food, medicine and cosmetics: (1) Qiu Siyuan, Xu Jingxue, Duan Yuyang, et al. Research progress on production and application of mannose erythritol lipids [J]. Progress in Biotechnology, 2023, 13(2):210-219. (2) Liu Xiayu. Study on the antibacterial activity and mechanism of mannose erythritol lipids against Listeria monocytogenes [D]. Zhejiang University, 2023. (3) Yang Yanxiao, Niu Yongwu, Niu Ben, et al. Effect of mannose erythritol lipids on the quality of frozen cooked noodles [J]. Food Science, 2024, 45(14):37-42.

[0003] Existing reported MELs production technologies use vegetable oils such as soybean oil, olive oil, and sunflower oil as substrates, and utilize aphid-borne yeasts (… Pseudozyma aphidis ) or Candida antarcticis ( Candida antarctica Microorganisms such as α and β are used for fermentation culture. However, in these existing technologies, because vegetable oils are complex mixtures composed of multiple fatty acids, microorganisms utilize multiple fatty acids simultaneously during fermentation, resulting in mixed glycolipids (MELs) products. This means that a single MEL product group contains molecules with multiple different fatty acid chain structures. This leads to at least the following drawbacks in the MELs prepared using these technologies: (1) The fermentation products of vegetable oil are a mixture of MELs molecules with different fatty acid chain structures. The products are randomly distributed, have low purity, and appear as brownish-black viscous oil.

[0004] (2) Mixed MELs have diverse molecular structures and similar polarity and physicochemical properties, resulting in low separation and purification efficiency and high cost. Furthermore, the final products are mostly viscous oily substances, which limits their application in high-end cosmetics. In addition, oily products are prone to incompatibility with other components when added to the formulation, affecting the stability of the formulation.

[0005] (3) Due to the high uncertainty of fatty acid composition and poor batch-to-batch consistency in mixed MELs products, key performance indicators such as surface activity parameters (such as HLB value), melting point range, and antibacterial activity of the products fluctuate significantly, making it difficult to achieve customized functional design and limiting their application value. Summary of the Invention

[0006] To address the aforementioned issues, this application provides a method for preparing mannose erythritol esters, the mannose erythritol esters themselves, and their applications. The mannose erythritol esters prepared by the method provided in this application have a single fatty acid structure, high product purity, and are in crystalline or paste form. They possess a well-defined melting point range and HLB value, thus enhancing their practical application value.

[0007] In a first aspect, embodiments of this application provide a method for preparing mannose erythritol ester, comprising: adding a substrate to an initial fermentation medium and inoculating with Candida albicans (… Starmerella bombicola The seed culture was fermented at 26℃~32℃ for 96 to 144 hours with stirring to obtain the fermentation broth. The fermentation broth was heated to inactivate the bacteria, cooled to room temperature, centrifuged, and the aqueous phase and cell layer were removed. The product layer was collected, dissolved in ethanol with stirring, and then decolorized with activated carbon. The activated carbon was removed by filtration, yielding a decolorized supernatant. The decolorized supernatant was concentrated under reduced pressure, and the concentrate was cooled to room temperature to obtain mannoerythritol ester. The substrate is one of C8~C25 fatty acids or their esters. The amount of substrate added was 10% to 30% of the total mass of the initial fermentation medium. The viable count of *Candida beeiensis* inoculated with the initial fermentation medium was 4.5 × 10⁻⁶ cells / day. 5 cfu / mL ~5.5×10 6 cfu / mL. During fermentation, maintain the carbon-to-nitrogen ratio of the fermentation medium at (5~10):1 and the pH value at 4.0~6.5.

[0008] According to the embodiments of this application, mannoerythritol esters are prepared by fermentation of pure fatty acids or their esters using *Candida beemannii* yeast. During the preparation process, fatty acids or their esters with carbon chain lengths in the C8-C25 range are selected as substrates to ensure the activity of key enzymes in the fermentation synthesis of mannoerythritol esters by *Candida beemannii*. Simultaneously, by controlling the carbon-nitrogen ratio and pH of the fermentation system, the metabolic flux distribution of the fatty acid substrate and the selectivity of acyltransferases are affected, directing the metabolic flow towards the MELs synthesis pathway and promoting the synthesis of the target product, diacyl MELs, while reducing the accumulation of byproducts such as monoacyl MELs and dimerized MELs, thereby improving the yield and stability of the target product. The MELs prepared by the method of this application have a single fatty acid structure, high product purity, and are in crystalline or paste form with a defined melting point range and HLB value, thus improving their practical application value.

[0009] In some alternative embodiments, the initial fermentation medium comprises, by weight percentage: 0.5% to 0.7% yeast extract, 0.5% to 0.7% peptone, 2% to 3.2% glucose, 0.3% to 0.6% ammonium sulfate, 1% to 3% dipotassium hydrogen phosphate, 0.5% to 1.5% potassium dihydrogen phosphate, 0.2% to 0.5% glycerol, with the balance being water.

[0010] In some optional implementations, the carbon-to-nitrogen ratio of the fermentation medium is monitored during fermentation. When the carbon-to-nitrogen ratio is below 5:1, a carbon source is added to the fermentation medium, selected from one or more of glucose, sucrose, maltose, and fructose. When the carbon-to-nitrogen ratio is above 10:1, a nitrogen source is added to the fermentation medium, selected from one or more of yeast extract, peptone, or ammonium sulfate.

[0011] In some alternative implementations, the pH value of the fermentation medium is monitored during fermentation. When the pH value of the fermentation medium is below 4.0, sodium hydroxide or ammonia is added to the fermentation medium. When the pH value of the fermentation medium is above 6.5, hydrochloric acid is added to the fermentation medium.

[0012] In some optional embodiments, the C8-C25 fatty acid or its ester is a C8-C25 fatty acid or a C8-C25 fatty acid ester. The C8-C25 fatty acid is selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, docosahexaenoic acid, and docosapentaenoic acid. The C8-C25 fatty acid ester is selected from any one of methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl caprylate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

[0013] In some alternative embodiments, the substrate is selected from C8-C25 fatty acids or their esters with an iodine value of 0-350 g I2 / 100 g.

[0014] In some optional embodiments, the C8-C25 fatty acids or their esters with an iodine value of 0-350 I2 / 100g are selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl caprylate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

[0015] By selecting fatty acids or their esters with iodine values ​​ranging from 0 to 350 g I2 / 100 g as substrates, membrane fluidity can be optimized during fermentation, the controllability of oxidation rate can be improved, and more carbon metabolic flux can be directed to diacytized MELs, thereby further improving substrate conversion and the purity of the main product diacytized MELs.

[0016] Secondly, embodiments of this application provide a mannoerythritol ester, which is prepared by the preparation method of the first aspect of this application.

[0017] Thirdly, embodiments of this application provide the use of mannose erythritol ester prepared according to the preparation method of the first aspect of this application or mannose erythritol ester according to the second aspect of this application in the preparation of cosmetics, food or pharmaceuticals.

[0018] Fourthly, embodiments of this application provide a mannose erythritol ester composition comprising at least two types of mannose erythritol esters prepared according to the preparation method of the first aspect of this application or mannose erythritol esters according to the second aspect of this application. Attached Figure Description

[0019] Figure 1 The image shows a comparison of TLC thin-plate chromatography results of the product of Example 1 and the product of Comparative Example 9.

[0020] Figure 2 The figure shown is an HPLC chromatogram of the product of Example 2.

[0021] Figure 3 The figure shows the HPLC chromatogram of the product of Comparative Example 9.

[0022] Figure 4 The image shown is a comparison of the appearance of the product of Example 2 and the product of Comparative Example 9. Detailed Implementation

[0023] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the embodiments described in this specification are merely for explaining this application and are not intended to limit it.

[0024] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0025] In the description of this application, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0026] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0027] Mannoerythritol esters (MELs) are glycolipid biosurfactants with excellent emulsifying and surface activity, broad-spectrum antibacterial properties, and skin repair capabilities. In recent years, MELs have shown broad application prospects in the food, pharmaceutical, and cosmetic fields and are widely recognized as one of the most promising biosurfactants.

[0028] This application provides a method for preparing mannose erythritol esters, the mannose erythritol esters themselves, and their applications. The mannose erythritol esters prepared by the method provided in this application have a single fatty acid structure, high product purity, and are in crystalline or paste form. They have a well-defined melting point range and HLB value, thus improving their utilization value.

[0029] The preparation method of the mannose erythritol ester, the mannose erythritol ester and its application will be introduced below with reference to the embodiments of this application.

[0030] Preparation method of mannose erythritol ester This application provides a method for preparing mannose erythritol ester, which includes: After adding substrate to the initial fermentation medium, inoculate with Candida albicans seed liquid and ferment at 26℃~32℃ for 96 hours~144 hours to obtain fermentation broth.

[0031] The fermentation broth was heated to inactivate it, cooled to room temperature, centrifuged, and the aqueous phase and cell layer were removed. The product layer was collected, ethanol was added and stirred to dissolve it, then activated carbon was added for decolorization, and the activated carbon was removed by filtration to obtain the decolorized clear liquid.

[0032] The decolorized clear liquid was concentrated under reduced pressure, and the concentrate was cooled to room temperature to obtain mannoerythritol ester.

[0033] The substrate is a C8-C25 fatty acid or its ester. It should be noted that in this application, "C8-C25" refers to a fatty acid molecule having 8 to 25 carbon atoms in its carbon chain; or, a fatty acid ester is obtained by reacting a fatty acid with 8 to 25 carbon atoms in its carbon chain with an alcohol of any number of carbon atoms. As an example, the number of carbon atoms in a fatty acid molecule can be 8, 10, 12, 15, 18, 20, 22, 25, or any range of the above values.

[0034] The amount of substrate added is 10% to 30% of the total mass of the initial fermentation medium, based on the mass of the fermentation medium. As an example, the amount of substrate added can be 10%, 12%, 14%, 16%, 18%, 20%, 22%, 25%, 28%, 30% of the total mass of the initial fermentation medium, or any range of the above values.

[0035] Based on the initial fermentation medium volume, the viable count of *Candida beescens* inoculated was 4.5 × 10⁻⁶. 5 cfu / mL ~5.5×10 6 cfu / mL. As an example, the viable count of *Candida benzii* inoculated can be 4.5 × 10⁻⁶. 5 cfu / mL, 5×10 5 cfu / mL, 6×10 5 cfu / mL, 7×10 5 cfu / mL, 8×10 5 cfu / mL, 9×10 5 cfu / mL, 10 6 cfu / mL, 2×10 6 cfu / mL, 3×10 6 cfu / mL, 4×10 6 cfu / mL, 5×10 6 cfu / mL, 5.5×10 6 cfu / mL, or any range of the above values.

[0036] During fermentation, the carbon-to-nitrogen ratio of the fermentation medium is maintained at (5~10):1, and the pH value is maintained at 4.0~6.5. As an example, the C / N ratio of the fermentation system can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any range of the above values. The pH value can be 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, or any range of the above values.

[0037] During fermentation, the carbon-to-nitrogen ratio and pH value of the fermentation medium change as the carbon and nitrogen sources are consumed and fermentation products are generated. In the embodiments of this application, the carbon-to-nitrogen ratio and pH value of the fermentation medium are measured during the fermentation process. As a well-known and routine operation in the art, the carbon-to-nitrogen ratio (C / N ratio) of the fermentation medium is the ratio of the total number of moles of carbon to the total number of moles of nitrogen in the medium; it can be obtained by determining the total organic carbon (TOC) content of the medium using an elemental analyzer or by determining the total nitrogen (TN) using the Kjeldahl method. The pH value of the fermentation medium can be obtained using an online or offline pH meter.

[0038] It should be noted that during the fermentation process at 26℃~32℃ for 96 to 144 hours with stirring, the stirring speed can be 150 rpm~800 rpm, and the aeration rate can be 0.5 vvm~2.0 vvm. Furthermore, after centrifugation of the sterilized and cooled fermentation broth, the layering relationship between the aqueous phase, cell layer, and product layer varies depending on the C-chain length of the substrate fatty acids. For example, if the C-chain of the fatty acid or its ester in the substrate is longer, the product layer is located below the aqueous phase and cell layer, with the order from top to bottom being aqueous phase, cell layer, and product layer; if the C-chain of the fatty acid or its ester in the substrate is shorter, the product layer is located above the aqueous phase and cell layer, with the order from top to bottom being product layer, aqueous phase, and cell layer. The product layer is light brown or brown in color, while the aqueous phase and cell layer are milky white or grayish white.

[0039] According to embodiments of this application, mannoerythritol esters are prepared by fermentation of pure fatty acids or their esters using Candida albicans. The MELs prepared by the method of this application have a single fatty acid structure, high product purity, and are in crystalline or paste form, with a well-defined melting point range and HLB value.

[0040] In the fermentation synthesis of mannoerythritol esters by *Candida albicans*, Mac1 acyltransferase, as the core enzyme in the MEL synthesis pathway, is specifically responsible for adding short-chain or medium-chain fatty acids to the core structural region of MELs. Mac1 exhibits substrate preference, and the length of the fatty acid carbon chain has a significant impact on the metabolic synthesis of MELs. In this application, the C-chain length of the fatty acid or its ester used as the substrate must be within the range of C8 to C25 to ensure that *Candida albicans* can utilize fatty acids to metabolize and synthesize MELs with good conversion rates. When the C-chain of the fatty acid is too short (less than C8), the Mac1 enzyme activity is low, and it cannot utilize fatty acids to synthesize MELs; when the C-chain is too long (greater than C25), the molecular structure is too large, and the enzyme's active structural center cannot accommodate molecular structures above C25, resulting in almost no metabolic synthesis of related MELs or extremely low yields.

[0041] The preparation method described in this application produces MELs with two fatty acid chains simultaneously linked to the 4' and 6' positions of mannose, forming diacylated MELs. Compared to monoacylated MELs, diacylated MELs exhibit higher surface activity, emulsifying properties, and cosmetic value. Acyltransferases in *Candida benjamina* can catalyze the sequential linking of fatty acids to mannose. However, these enzymes exhibit substrate selectivity, meaning their efficiency in continuing linking to a substrate already linked with one fatty acid varies, making them susceptible to the influence of culture conditions. This leads to the accumulation of monoesters as intermediates, resulting in byproducts such as monoacylated or dimerized MELs. Furthermore, esterases produced during *Candida benjamina* metabolism may also enzymatically hydrolyze the products, altering their structure. The purpose of this application is to prepare single-structure MELs; the presence of byproducts obviously affects the purity of the MELs.

[0042] Building upon this, this application further influences the distribution of metabolic flux of fatty acid substrates by the microbial cells through the regulation of fermentation conditions, thereby affecting the selectivity of acyltransferases. Specifically, this includes regulating the carbon-to-nitrogen ratio (C / N ratio) and pH of the fermentation system: controlling the C / N ratio within the range of (5~10):1 directs metabolic flow towards the MEL synthesis pathway and promotes the synthesis of the target product, diacyl MELs, while reducing the accumulation of monoacyl MELs and dimerized MELs. Simultaneously, maintaining the pH of the fermentation system within the range of 4.0~6.5 can increase the target yield while inhibiting esterase activation and maintaining the stability of the product structure. This results in the final product achieving a purity and substrate conversion rate of over 85% for both the target product, diacylated MELs.

[0043] When the C / N ratio is below 5:1, the microorganisms initially prioritize cell growth, but in the later stages, due to a lack of C source, cell metabolism and the synthesis of key enzymes such as acyltransferases are limited, affecting substrate conversion, increasing byproducts, and decreasing the purity of the target product. When the C / N ratio is above 10:1, the C source concentration is too high relative to the N source concentration, resulting in a glucose effect, slowing cell growth, and failing to produce sufficient enzyme activity, which is detrimental to the accumulation of the target product, thus reducing substrate conversion. When the pH value is below 4.0, microbial growth is slow, the activity of multiple enzymes is limited, and the target product cannot be synthesized in large quantities, resulting in a decrease in both purity and conversion rate. When the pH value is above 6.5, the growth of *Candida beeiensis* is slightly limited, but the yield of MELs synthesized in the later stages is low, exceeding the optimal enzyme activity pH for its lipase and acyltransferase, increasing byproducts, and hindering the accumulation of the target product.

[0044] In some embodiments, the initial fermentation medium comprises, by weight percentage: 0.5% to 0.7% yeast extract, 0.5% to 0.7% peptone, 2% to 3.2% glucose, 0.3% to 0.6% ammonium sulfate, 1% to 3% dipotassium hydrogen phosphate, 0.5% to 1.5% potassium dihydrogen phosphate, 0.2% to 0.5% glycerol, with the balance being water.

[0045] As an example, the mass percentage of yeast extract in the initial fermentation medium can be 0.5%, 0.52%, 0.54%, 0.58%, 0.60%, 0.62%, 0.64%, 0.68%, 0.70%, or any range of the above values. The mass percentage of peptone in the initial fermentation medium can be 0.5%, 0.52%, 0.54%, 0.58%, 0.60%, 0.62%, 0.64%, 0.68%, 0.70%, or any range of the above values. The mass percentage of glucose in the initial fermentation medium can be 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, or any range of the above values. The mass percentage of ammonium sulfate in the initial fermentation medium can be 0.3%, 0.32%, 0.35%, 0.38%, 0.4%, 0.42%, 0.45%, 0.48%, 0.5%, 0.52%, 0.55%, 0.58%, 0.6%, or any combination of the above values. The mass percentage of dipotassium hydrogen phosphate in the initial fermentation medium can be 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, or any combination of the above values. The mass percentage of potassium dihydrogen phosphate in the initial fermentation medium can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, or any combination of the above values. The mass percentage of glycerol in the initial fermentation medium can be 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or any combination of the above values.

[0046] In some embodiments, the carbon-to-nitrogen ratio of the fermentation medium is monitored during fermentation. When the carbon-to-nitrogen ratio is below 5:1, a carbon source is added to the fermentation medium, selected from one or more of glucose, sucrose, maltose, and fructose. When the carbon-to-nitrogen ratio is above 10:1, a nitrogen source is added to the fermentation medium, selected from one or more of yeast extract, peptone, or ammonium sulfate.

[0047] In some embodiments, the carbon-to-nitrogen ratio of the fermentation medium is monitored during fermentation. When the pH of the fermentation medium is below 4.0, sodium hydroxide (e.g., a 10%–15% aqueous solution of sodium hydroxide by mass) or ammonia (e.g., a 25%–28% aqueous solution of ammonia by mass) is added to the fermentation medium. When the pH of the fermentation system is above 6.5, hydrochloric acid (e.g., a 1%–5% aqueous solution of hydrochloric acid by mass) is added to the fermentation medium.

[0048] As an example, when the pH of the fermentation system is below 4.0, a 10% sodium hydroxide aqueous solution, a 15% sodium hydroxide aqueous solution, or a 25% ammonia solution can be added. When the pH of the fermentation system is above 6.5, a 1% hydrochloric acid aqueous solution, a 3% hydrochloric acid aqueous solution, or a 5% hydrochloric acid aqueous solution can be added to the fermentation medium.

[0049] In some embodiments, the substrate C8-C25 fatty acid or its ester is a C8-C25 fatty acid or a C8-C25 fatty acid ester. The C8-C25 fatty acid is selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, docosahexaenoic acid, and docosapentaenoic acid.

[0050] In some embodiments, the C8-C25 fatty acid esters are selected from any one of methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl octanoate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

[0051] In some embodiments, the substrate is selected from C8-C25 fatty acids or their esters with an iodine value of 0 to 350 g I2 / 100g. As an example, the iodine value of the substrate can be 0, 50 g I2 / 100g, 100 g I2 / 100g, 150 g I2 / 100g, 200 g I2 / 100g, 250 g I2 / 100g, 300 g I2 / 100g, 350 g I2 / 100g, or any range of the above values.

[0052] According to embodiments of this application, the iodine value (IV) of fatty acids or their esters reflects the number of carbon-carbon double bonds in the fatty acid chain. A lower iodine value indicates a higher degree of saturation and a tendency towards hydrophobicity in the fatty acid or its ester; a higher iodine value indicates a higher degree of unsaturation and a tendency towards hydrophilicity in the fatty acid or its ester. In some embodiments of this application, fatty acids or their esters with iodine values ​​ranging from 0 to 350 g I2 / 100 g are selected as substrates. During fermentation, this optimizes membrane fluidity, improves the controllability of the oxidation rate, directs more carbon metabolic flux towards diacytized MELs, and further improves substrate conversion rate and the purity of the main product diacytized MELs, both reaching over 90%.

[0053] In some embodiments, the C8-C25 fatty acids or their esters with an iodine value of 0-350 I2 / 100g are selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl caprylate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

[0054] Mannose erythritol ester This application provides a mannoerythritol ester, which is prepared by the first aspect of the preparation method.

[0055] In some embodiments, the HLB value of mannose erythritol ester can be 2.5 to 13.0. As an example, the HLB value of mannose erythritol ester can be 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or any range of the above values.

[0056] As an example, mannose erythritol palmitate is a milky white paste with a melting point of 100°C to 105°C and an HLB value of 9.0 to 9.5. Mannoose erythritol oleate is also a milky white paste with a melting point of 103°C to 108°C and an HLB value of 7.5 to 8.0.

[0057] Applications of mannose erythritol ester This application provides a first aspect of the application of mannoerythritol ester prepared by the preparation method or a second aspect of the application of mannoerythritol ester in the preparation of cosmetics.

[0058] Mannose-erythritol ester composition This application provides a mannose erythritol ester composition comprising at least two mannose erythritol esters prepared in the second aspect.

[0059] According to the embodiments of this application, the HLB value of the mannose erythritol ester composition can be precisely controlled by selecting different mannose erythritol esters and their ratios, making it suitable for various cosmetic systems such as emulsions, serums, creams, lotions, and makeup removers. After reasonably controlling the HLB value, this glycolipid surfactant can achieve differentiated functions such as stable emulsification, solubilization and dispersion, wetting and spreading, and gentle foaming or foam suppression according to the formulation requirements. At the same time, the mannose erythritol ester composition with targeted optimization of the HLB ratio can balance the interfacial tension of the formulation, improve the spreadability and skin feel of the product, and avoid defects such as stickiness, dryness, and pilling. Moreover, relying on the natural and mild properties of this glycolipid, while adapting to a wide range of HLB adjustments, the addition of chemically synthesized emulsifiers and solubilizers can be reduced, thereby reducing the irritation of the formulation and taking into account both the gentle skincare requirements of cosmetics and the long-term stability of the system, further improving the compatibility and application value of the product.

[0060] Example The following embodiments describe the disclosure of this invention in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing. The instruments used in the embodiments are also commercially available. Among them, *Candida benzi* (… Starmerella bombicola There are no particular limitations. As an example, in the embodiments of this application, *Candida bee-borne yeast* (…) Starmerella bombicola It can be purchased from Shanghai Titan Technology Co., Ltd., product number 041316279, or you can use Lip of Candida benzi from Shanghai Zhizhen Zhichen Technology Co., Ltd. DYH (CGMCC No. 25797).

[0061] Example 1 A mannoerythritol palmitate is prepared by the following method: Lip DYH ( Starmerella bombicola The yeast extract was inoculated into YPD medium and activated at 30°C for 24 hours. Then, it was transferred to seed culture medium at an inoculation rate of 2.5% for expansion culture for 20 hours to obtain *Candida benzi* seed culture. The seed culture medium (by weight percentage) consisted of: 0.5% yeast extract, 0.4% peptone, 1% glucose, 0.2% ammonium sulfate, 0.1% sodium chloride, 0.5% glycerol, and the remainder being water, pH=6.8.

[0062] The initial fermentation medium was sterilized at 121℃ for 20 min. After cooling, methyl palmitate was added to the initial fermentation medium, and *Candida benzi* seed culture was inoculated into the initial fermentation medium. The composition of the initial fermentation medium was as follows (by weight percentage): yeast extract 0.6%, peptone 0.6%, glucose 3%, ammonium sulfate 0.4%, dipotassium hydrogen phosphate 2%, potassium dihydrogen phosphate 1%, glycerol 0.4%; the C / N ratio of the initial fermentation medium was 8:1; the pH of the initial fermentation medium was adjusted to 5.5 using dilute hydrochloric acid or sodium hydroxide solution. The amount of methyl palmitate added was 18% of the mass of the initial fermentation medium; the inoculum size of *Candida benzi* was 5.0 × 10⁻⁶. 6 cfu / mL.

[0063] Fermentation was carried out at 28℃~30℃, aeration rate of 1.5 vvm, and stirring speed of 600 rpm for 120 h. During fermentation, the C / N ratio and pH of the fermentation medium were monitored every 6 hours, and feed was added as needed based on the monitoring results. When the C / N ratio of the fermentation medium was detected to be within the range of (5~10):1 and the pH value was detected to be within the range of 4.0~6.5, no feed was added. When the C / N ratio of the fermentation medium was detected to be lower than 5:1, an appropriate amount of glucose was added to the fermentation medium to make the C / N ratio of the fermentation medium 8:1; when the C / N ratio of the fermentation medium was detected to be higher than 10:1, an appropriate amount of peptone was added to the fermentation medium to make the C / N ratio of the fermentation medium 8:1. When the pH value of the fermentation medium is detected to be lower than 4.0, add an appropriate amount of 10% sodium hydroxide aqueous solution to the fermentation medium to make the pH value of the fermentation medium 5.5; when the pH value of the fermentation medium is detected to be higher than 6.5, add an appropriate amount of 1% hydrochloric acid aqueous solution to the fermentation medium to make the pH value of the fermentation medium 5.5.

[0064] After fermentation, the fermentation broth was heated to 80℃~100℃ for 20 min to inactivate the bacteria. After cooling to room temperature, the inactivated fermentation broth was centrifuged at 8000 rpm for 20 min to remove the aqueous phase and bacterial layer. The brown layer was collected, and an equal volume of 95% ethanol was added and stirred to dissolve it. Then, 3% (w / v) activated carbon was added, and the mixture was stirred at 40℃ for decolorization for 90 min. The activated carbon was then removed by filtration. The decolorized supernatant was concentrated under reduced pressure at 60℃ to 1 / 2~1 / 3 of its original volume to obtain a concentrated solution with a moisture content of ≤5%. The concentrated solution was cooled to room temperature to obtain a milky white paste-like product.

[0065] The purity and substrate conversion of the diacylated MELs were determined by the following methods: The product was analyzed by high performance liquid chromatography (HPLC). The purity of the product was confirmed to be 90.5% by comparison with the standard curve of diacytylmannoerythritol-palmitate.

[0066] Substrate conversion rate is obtained using the following formula:

[0067] The substrate conversion rate of Example 1 was calculated to be 93.0%.

[0068] Example 2 The difference between Example 2 and Example 1 is that the initial fermentation medium composition was: 0.5% yeast extract, 0.5% peptone, 2.63% glucose, 0.3% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.5% glycerol; the C / N ratio of the initial fermentation medium was 9:1; and the pH of the initial fermentation medium was adjusted to 4.5 using dilute hydrochloric acid or sodium hydroxide solution. The substrate was oleic acid, added at 16% of the initial fermentation medium mass; and the inoculum size of *Candida beescens* was 5.5 × 10⁻⁶. 5 cfu / mL. The temperature, aeration rate, stirring speed, and fermentation time during the fermentation process are shown in Table 1.

[0069] Following the method of Example 1, during fermentation, the C / N ratio of the fermentation medium was checked every 6 hours to ensure it was within the range of (5~10):1 and the pH value was within the range of 4.0~6.5. Based on the test results, feed was added as needed to achieve a C / N ratio of 9:1 and a pH value of 4.5 in the fermentation medium after feeding. The remaining preparation process and parameters were the same as in Example 1, resulting in a light yellow paste-like product.

[0070] The substrate conversion and diacylated MEL purity of the product were determined: The product was analyzed by high performance liquid chromatography (HPLC). Comparison with the standard curve of diacytylmannoerythritol-oleate confirmed that the purity of diacytylmannoerythritol-oleate in the product was 93.0%. The substrate conversion rate of Example 2 was calculated to be 90.3%.

[0071] Example 3 The difference between Example 3 and Example 1 is that the initial fermentation medium composition is as follows: 0.5% yeast extract, 0.5% peptone, 3.1% glucose, 0.3% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.5% glycerol; the C / N ratio of the initial fermentation medium is 10:1; and the pH of the initial fermentation medium is adjusted to 6.0 using dilute hydrochloric acid or sodium hydroxide solution. Ethyl laurate is used as the substrate, and the amount of ethyl laurate added is 12% of the mass of the initial fermentation medium; the inoculum size of *Candida beescens* is 6 × 10⁻⁶. 5 cfu / mL. The temperature, aeration rate, stirring speed, and fermentation time during the fermentation process are shown in Table 1.

[0072] Following the method of Example 1, during fermentation, the C / N ratio of the fermentation medium was checked every 6 hours to ensure it was within the range of (5~10):1 and the pH value was within the range of 4.0~6.5. Based on the test results, feed was added as needed to ensure that the C / N ratio of the fermentation medium after feeding was 10:1 and the pH value was 6.0. The remaining preparation process and parameters were the same as in Example 1, resulting in a milky white paste-like product.

[0073] The substrate conversion and diacylated MEL purity of the product were determined: The product was analyzed by high performance liquid chromatography (HPLC). Comparison with the standard curve of diacytylmannoerythritol-laurate confirmed that the purity of diacytylmannoerythritol-laurate in the product was 90.8%. The substrate conversion rate of Example 3 was calculated to be 92.4%.

[0074] Example 4 The difference between Example 4 and Example 1 is that the initial fermentation medium composition is as follows: 0.7% yeast extract, 0.7% peptone, 2% glucose, 0.6% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.2% glycerol; the C / N ratio of the initial fermentation medium is 5:1; the pH of the initial fermentation medium is adjusted to 4.0 using dilute hydrochloric acid or sodium hydroxide solution. Linoleic acid is used as the substrate, and its addition amount is 30% of the initial fermentation medium mass; the inoculum size of *Candida beescens* is 5 × 10⁻⁶. 5 cfu / mL. The temperature, aeration rate, stirring speed, and fermentation time during the fermentation process are shown in Table 1.

[0075] Following the method of Example 1, during fermentation, the C / N ratio of the fermentation medium was checked every 6 hours to ensure it was within the range of (5~10):1 and the pH value was within the range of 4.0~6.5. Based on the test results, feed was added as needed to maintain a C / N ratio of 5:1 and a pH value of 4.0 in the fermentation medium after feeding. The remaining preparation process and parameters were the same as in Example 1, resulting in a yellow paste-like product.

[0076] The substrate conversion and diacylated MEL purity of the product were determined: The product was analyzed by high performance liquid chromatography (HPLC). Comparison with the standard curve of diacytylmannoerythritol-linoleate confirmed that the purity of diacytylmannoerythritol-linoleate in the product was 92.2%. The substrate conversion rate of Example 4 was calculated to be 91.5%.

[0077] Example 5 The difference between Example 5 and Example 1 is that the initial fermentation medium composition is as follows: 0.5% yeast extract, 0.5% peptone, 3.1% glucose, 0.3% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.5% glycerol; the C / N ratio of the initial fermentation medium is 10:1; the pH of the initial fermentation medium is adjusted to 6.2 using dilute hydrochloric acid or sodium hydroxide solution. Ethyl octanoate is used as the substrate, and the amount of ethyl octanoate added is 15% of the mass of the initial fermentation medium; the inoculum size of *Candida beeiensis* is 4.5 × 10⁻⁶. 5 cfu / mL. The temperature, aeration rate, stirring speed, and fermentation time during the fermentation process are shown in Table 1.

[0078] Following the method of Example 1, during fermentation, the C / N ratio of the fermentation medium was checked every 6 hours to ensure it was within the range of (5~10):1 and the pH value was within the range of 4.0~6.5. Based on the test results, feed was added as needed to ensure that the C / N ratio of the fermentation medium after feeding was 10:1 and the pH value was 6.2. The remaining preparation process and parameters were the same as in Example 1, resulting in a milky white paste-like product.

[0079] The substrate conversion and diacylated MEL purity of the product were determined: The product was analyzed by high performance liquid chromatography (HPLC). Comparison with the standard curve of diaylated mannoerythritol-octanoate confirmed that the purity of diaylated mannoerythritol-octanoate in the product was 92.5%. The substrate conversion rate was calculated to be 90.1%.

[0080] Example 6 The difference between Example 6 and Example 1 is that the *Candida bee-like yeast* used was Titan Technology (041316279). The initial fermentation medium consisted of: 0.6% yeast extract, 0.6% peptone, 3% glucose, 0.4% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.4% glycerol; the C / N ratio of the initial fermentation medium was 8:1; and the pH of the initial fermentation medium was adjusted to 5.0 using dilute hydrochloric acid or sodium hydroxide solution. Arachidonic acid was used as the substrate, added at 10% of the initial fermentation medium mass, and the inoculum size of *Candida bee-like yeast* was 5.5 × 10⁻⁶.6 cfu / mL. The temperature, aeration rate, stirring speed, and fermentation time during the fermentation process are shown in Table 1.

[0081] Following the method of Example 1, during fermentation, the C / N ratio of the fermentation medium was checked every 6 hours to ensure it was within the range of (5~10):1 and the pH value was within the range of 4.0~6.5. Based on the test results, feed was added as needed to achieve a C / N ratio of 8:1 and a pH value of 5.0 in the fermentation medium after feeding. The remaining preparation process and parameters were the same as in Example 1, resulting in a light brown paste-like product.

[0082] The substrate conversion and diacylated MEL purity of the product were determined: The product was analyzed by high performance liquid chromatography (HPLC). Comparison with the standard curve of diacytylmannoerythritol-arachidonic acid confirmed that the purity of diacytylmannoerythritol-arachidonic acid in the product was 90.1%. The substrate conversion rate was calculated to be 90.5%.

[0083] Example 7 The only difference between Example 7 and Example 1 is that the substrate used is eicosapentaenoic acid (EPA), and mannose erythritol-EPA is prepared. See Table 1 for details. The purity of the diacylated mannose erythritol-EPA in the product is 87.5%. The calculated substrate conversion rate is 88.0%.

[0084] Example 8 The difference between Example 8 and Example 1 is that docosahexaenoic acid was used as the substrate to prepare mannose erythritol-docosahexaenoic acid ester. See Table 1 for details. The purity of the diacylated mannose erythritol-docosahexaenoic acid ester in the product was 86.9%. The calculated substrate conversion rate was 85.0%.

[0085] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that hexanoic acid was used as the substrate. All other operating steps and parameters were the same as in Example 1, as detailed in Table 1.

[0086] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that n-heptanoic acid was used as the substrate. All other operating steps and parameters were the same as in Example 1, as detailed in Table 1.

[0087] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the substrate used is waxy acid. All other operating steps and parameters are the same as in Example 1, as detailed in Table 1.

[0088] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the substrate used is hexadecenoic acid. All other operating steps and parameters are the same as in Example 1, as detailed in Table 1.

[0089] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the initial fermentation medium composition was: 0.6% yeast extract, 0.6% peptone, 0.63% glucose, 0.6% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.1% glycerol; the C / N ratio of the initial fermentation medium was 3:1, and the pH of the initial fermentation medium was adjusted to 5.5 using dilute hydrochloric acid or sodium hydroxide solution. During fermentation, the C / N ratio and pH of the fermentation medium were not monitored or replenished. See Table 1 for details.

[0090] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that the initial fermentation medium composition was: 0.7% yeast extract, 0.7% peptone, 6.83% glucose, 0.55% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.35% glycerol; the C / N ratio of the initial fermentation medium was 12:1, and the pH of the initial fermentation medium was adjusted to 5.5 using dilute hydrochloric acid or sodium hydroxide solution. During fermentation, the C / N ratio and pH of the fermentation medium were not monitored or replenished. See Table 1 for details.

[0091] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the initial fermentation medium composition was: 0.6% yeast extract, 0.6% peptone, 3% glucose, 0.4% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.4% glycerol; the C / N ratio of the initial fermentation medium was 8:1; dilute hydrochloric acid was added to make the pH of the initial fermentation medium 3.0; during the fermentation process, the C / N ratio and pH of the fermentation medium were not monitored or replenished. See Table 1 for details.

[0092] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that the initial fermentation medium consisted of 0.6% yeast extract, 0.6% peptone, 3% glucose, 0.4% ammonium sulfate, 2% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 0.4% glycerol; the C / N ratio of the initial fermentation medium was 8:1; sodium hydroxide solution was added to adjust the pH of the initial fermentation medium to 7.5; and during fermentation, the C / N ratio and pH of the fermentation medium were not monitored or replenished. See Table 1 for details.

[0093] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that soybean oil was used as the substrate. All other operating steps and parameters were the same as in Example 1, as detailed in Table 1.

[0094] The preparation process parameters and conditions of Examples 1-8 and Comparative Examples 1-9 are shown in Table 1 below.

[0095] Table 1. Preparation parameters for Examples 1-8 and Comparative Examples 1-9

[0096] The results of the product appearance, substrate conversion rate, and diacylated MEL purity of Examples 1-8 and Comparative Examples 1-9 are detailed in Table 2.

[0097] Table 2. Detection results of products from Examples 1-8 and Comparative Examples 1-9

[0098] As shown in Table 2, Examples 1-8 used pure fatty acids or their esters as substrates to prepare mannoerythritol esters via fermentation using *Candida beemannii*. During the preparation process, fatty acids or their esters with carbon chain lengths in the C8-C25 range were selected as substrates to ensure the activity of key enzymes in the fermentation synthesis of mannoerythritol esters by *Candida beemannii*. Simultaneously, real-time monitoring and feeding maintained the carbon-to-nitrogen ratio of the fermentation system within the range of (5-10):1 and the pH value within the range of 4-6.5. This controlled the metabolic flux distribution of the fatty acid substrates and the selectivity of acyltransferases, directing the metabolic flow towards the MELs synthesis pathway and promoting the synthesis of the target product, diacyl MELs, while reducing the accumulation of byproducts such as monoacyl MELs and dimerized MELs, resulting in a target product yield and purity exceeding 85%.

[0099] Furthermore, in Examples 1-8, the substrates of Examples 1-6, namely C8-C25 fatty acids or their esters, all met the requirement of iodine values ​​ranging from 0 to 350 g I2 / 100 g. Compared to Examples 7-8, Examples 1-6 exhibited better membrane fluidity, controllable oxidation rate, and a greater carbon metabolic flux directed towards diacytized MELs during fermentation, further improving substrate conversion and the purity of the main product diacytized MELs to over 90%.

[0100] The comparison between Example 1 and Comparative Examples 1-4 illustrates the effect of the C-chain length of the substrate fatty acid or its ester on the fermentation production of MELs. When the fatty acid C-chain is too short (less than C8), the Mac1 enzyme activity is low, making it impossible to utilize the fatty acid to synthesize MELs; when the C-chain is too long (greater than C25), the molecular structure is too large, and the enzyme's active structural center cannot accommodate molecular structures above C25, resulting in almost no metabolism and synthesis of related MELs or extremely low yields. Therefore, the substrate conversion rates of Comparative Examples 1-4 are extremely low.

[0101] The comparison between Example 1 and Comparative Examples 5-6 illustrates the effect of controlling the C / N ratio of the fermentation system on the production of MELs during fermentation. When the C / N ratio is below 5:1, the microorganisms tend to grow predominantly in the early stages, but in the later stages, due to the lack of C source, cell metabolism and the synthesis of key enzymes such as acyltransferases are limited, affecting substrate conversion, increasing byproducts, and decreasing the purity of the target product. When the C / N ratio is above 10:1, the C source concentration is too high relative to the N source concentration, resulting in a glucose effect, slow cell growth, insufficient enzyme activity, and also hindering the accumulation of the target product and substrate conversion.

[0102] The comparison between Example 1 and Comparative Examples 7-8 illustrates the effect of controlling the pH value of the fermentation system on the production of MELs during fermentation. When the pH value is below 4.0, microbial growth is slow, the activity of various enzymes is limited, and the target product cannot be synthesized in large quantities, resulting in a decrease in both purity and conversion rate. When the pH value is above 6.5, the growth of *Candida beeiensis* is slightly restricted, but the yield of MELs in the later stage of synthesis is low, exceeding the optimal enzyme activity pH of its lipase and acyltransferase, and the amount of by-products increases, which is not conducive to the accumulation of the target product. Therefore, the substrate conversion rate and the purity of the main product, acylated MELs, in Comparative Examples 5-8 are significantly lower than those in Examples 1-8.

[0103] Comparative Example 9 used soybean oil as a substrate. Soybean oil is a mixture of fatty acids with different chain lengths and degrees of unsaturation. During fermentation, Candida albicans utilizes multiple fatty acids simultaneously, resulting in MELs produced by fermentation being mixed glycolipids with low purity and difficult separation and purification. Consequently, the product performance is unclear, and functional development is limited.

[0104] Please see Figures 1 to 3 The MELs prepared using the method described in this application exhibited significantly higher purity than those prepared using vegetable oil, as determined by both TLC thin-plate chromatography and HPLC. Please refer to [link to relevant documentation]. Figure 4 The MELs prepared in Example 2 were light yellow pastes with a well-defined melting point range and HLB value.

[0105] Example 9 Mannoerythritol-palmitate from Example 1 (with an HLB value of 9.5 as determined by the emulsification method described in GB / T 5559-2010) and mannoerythritol-oleate from Example 2 (with an HLB value of 7.7 as determined by the emulsification method described in GB / T 5559-2010) were mixed at a mass ratio of 1:1 to obtain a MEL composition with an HLB value of 8.6.

[0106] After obtaining the mannose erythritol esters prepared according to the embodiments of this application, mannose erythritol esters with target HLB values ​​can be obtained by selecting different mannose erythritol esters and mixing them in different proportions. Therefore, the HLB value of the mannose erythritol ester composition can be precisely controlled to suit the various needs of multiple cosmetic systems such as lotions, serums, creams, lotions, and makeup removers.

[0107] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing mannose erythritol ester, characterized in that, include: Add substrate to the initial fermentation medium, inoculate with Candida albicans seed liquid, and stir and ferment at 26℃~32℃ for 96 hours~144 hours to obtain fermentation broth; The fermentation broth was heated to inactivate it, cooled to room temperature, centrifuged, and the aqueous phase and cell layer were removed. The product layer was collected, ethanol was added and stirred to dissolve it, then activated carbon was added for decolorization, and the activated carbon was removed by filtration to obtain the decolorized clear liquid. The decolorized clear liquid was concentrated under reduced pressure, and the concentrate was cooled to room temperature to obtain mannoerythritol ester. The substrate is one of C8-C25 fatty acids or their esters; The amount of substrate added is 10% to 30% of the total mass of the initial fermentation medium, based on the mass of the initial fermentation medium. Based on the volume of the initial fermentation medium, the viable count of the inoculated *Candida albicans* is 4.5 × 10⁻⁶. 5 cfu / mL ~5.5×10 6 cfu / mL; During the fermentation process, the carbon-to-nitrogen ratio of the fermentation medium is maintained at (5~10):1, and the pH value is 4.0~6.

5.

2. The method for preparing mannose erythritol ester according to claim 1, characterized in that, The initial fermentation medium, by weight percentage, comprises: 0.5% to 0.7% yeast extract, 0.5% to 0.7% peptone, 2% to 3.2% glucose, 0.3% to 0.6% ammonium sulfate, 1% to 3% dipotassium hydrogen phosphate, 0.5% to 1.5% potassium dihydrogen phosphate, 0.2% to 0.5% glycerol, with the balance being water.

3. The method for preparing mannose erythritol ester according to claim 1, characterized in that, During the fermentation process, the carbon-to-nitrogen ratio of the fermentation medium is measured. When the carbon-to-nitrogen ratio of the fermentation medium is less than 5:1, a carbon source is added to the fermentation medium. The carbon source is selected from one or more of glucose, sucrose, maltose, and fructose. When the carbon-to-nitrogen ratio of the fermentation medium is higher than 10:1, a nitrogen source is added to the fermentation medium. The nitrogen source is selected from one or more of yeast extract, peptone, or ammonium sulfate.

4. The method for preparing mannose erythritol ester according to claim 1, characterized in that, During the fermentation process, the pH value of the fermentation medium is detected; When the pH value of the fermentation medium is lower than 4.0, sodium hydroxide or ammonia water is added to the fermentation medium. When the pH of the fermentation medium is higher than 6.5, hydrochloric acid is added to the fermentation medium.

5. The method for preparing mannose erythritol ester according to claim 1, characterized in that, The C8-C25 fatty acid or its ester is a C8-C25 fatty acid or a C8-C25 fatty acid ester; The C8~C25 fatty acids are selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, docosahexaenoic acid, and docosapentaenoic acid. The C8~C25 fatty acid esters are selected from any one of methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl octanoate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

6. The method for preparing mannose erythritol ester according to claim 1, characterized in that, The substrate is selected from C8-C25 fatty acids or their esters with an iodine value of 0-350 g I2 / 100 g.

7. The method for preparing mannose erythritol ester according to claim 6, characterized in that, The C8-C25 fatty acids or their esters with an iodine value of 0-350 I2 / 100g are selected from any one of palmitic acid, caprylic acid, capric acid, oleic acid, linoleic acid, linolenic acid, lauric acid, myristoleic acid, nervonic acid, arachidonic acid, methyl palmitate, ethyl palmitate, vinyl palmitate, ethyl caprylate, methyl decanoate, methyl oleate, ethyl oleate, vinyl oleate, and ethyl laurate.

8. A mannoerythritol ester, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the mannoerythritol ester prepared by the preparation method according to any one of claims 1 to 7 or the mannoerythritol ester according to claim 8 in the preparation of cosmetics, food or pharmaceuticals.

10. A mannose-erythritol ester composition, characterized in that, It includes mannoerythritol ester prepared by at least two of the preparation methods according to any one of claims 1-7 or mannoerythritol ester according to claim 8.

Images