Bumblebee Saccharomyces cerevisiae and its application in the preparation of glycolipids with anti-aging and / or moisturizing functions

Abstract

This invention relates to the field of microbial technology, and more particularly to *Saccharomyces simulans* and its application in the preparation of glycolipids with anti-aging and / or moisturizing functions. This invention provides *Saccharomyces simulans* strain with accession number CCTCC NO:M 2025061, and a method for preparing glycolipids. The preparation of glycolipids using this strain has the advantages of short fermentation time, high oil utilization rate, and high glycolipid yield. Furthermore, the glycolipids obtained by this method are a mixture of sophorolipids, glycerol glycolipids, and glucose glycolipids, which can be widely used in pharmaceuticals, food, agriculture, environmental protection, cosmetics, and other fields, and can significantly reduce the production cost of glycolipids.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to *Bombyx mori* and its application in the preparation of glycolipids. Background Technology

[0002] Glycolipids (GLs) are a class of important compounds widely found in living organisms, composed of sugars linked to lipids via glycosidic bonds. They play an indispensable role in cellular physiological processes, participating in many key physiological activities such as cell recognition, intercellular communication, signal transduction, and maintaining the structure and function of biological membranes. Based on their unique chemical structure and physiological activity, glycolipids demonstrate enormous application potential in numerous fields, including medicine, food, cosmetics, and industry.

[0003] Glycolipids consist of two parts: a glycosyl group and a lipid group. The glycosyl group can contain monosaccharides, oligosaccharides, or polysaccharides, while the lipid group is mostly composed of fatty acids, glycerides, or sphingosine. Glyceryl glycolipids use glycerol as their lipid backbone, with fatty acids and glycosyl groups linked by ester and glycosidic bonds, respectively. Glucose lipips, on the other hand, have a glucose glycosyl group that is linked to the lipid group to form a specific structure. Sophorolipids have a sophorose glycosyl group, with the lipid group linked to the sophorose glycosyl group.

[0004] Glycolipids play multiple roles in living organisms. They constitute the glycocalyx on cell surfaces, aiding in cell recognition and communication; as components of biological membranes, glycolipids regulate membrane fluidity, stability, and permeability, participating in processes such as membrane fusion; and they can also act as signaling molecules, regulating physiological activities such as cell growth and differentiation. In industry and other fields, due to their excellent surface activity, glycolipids, such as sophorolipids, can be used as biosurfactants in industries such as oil extraction and cosmetics; furthermore, thanks to their antioxidant and antibacterial properties, glycolipids can also be used as food additives and emulsifiers; in the pharmaceutical field, glycolipids can be used to prepare drug carriers such as liposomes, improving drug targeting and bioavailability.

[0005] There are three main methods for synthesizing glycolipids: chemical synthesis, enzymatic synthesis, and microbial fermentation. Chemical synthesis connects glycosyl groups and lipids through organic reactions, allowing for precise control of glycolipid structure and meeting the needs of industrial production. However, it is cumbersome, costly, requires specialized equipment, and is prone to environmental pollution, affecting product purity and activity. Enzymatic synthesis utilizes the high efficiency and specificity of enzymes to synthesize glycolipids under mild conditions, offering high selectivity and low waste. However, enzymes are expensive, have poor stability, are limited by substrate concentration and reaction rate, and are complex to obtain and prepare, hindering large-scale production. Therefore, microbial fermentation is currently the most important method for glycolipid synthesis. This method uses microorganisms to synthesize glycolipids under mild reaction conditions, low energy consumption, and produces bioactive and environmentally friendly products. However, most existing strains can only synthesize a single type of glycolipid, and the oil utilization rate and glycolipid yield are low, with long fermentation times, resulting in high production costs and limiting glycolipid production and applications. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide bumblebee yeast and its application in the preparation of glycolipids.

[0007] This invention provides Starmerella bombicola, a bumblebee yeast with accession number CCTCC NO: M 2025061.

[0008] Existing methods for preparing glycolipids all suffer from drawbacks such as limited glycolipid types, low oil utilization and glycolipid yield, and high production costs due to long fermentation times. However, the strain provided by this invention has high glycolipid yield and oil utilization, short fermentation time, and the obtained glycolipids are a mixture of sophorolipids, glycerol glycolipids, and glucose glycolipids.

[0009] The present invention also provides a microbial agent comprising *Bombyx mori* yeast as described above.

[0010] The bacterial cells described in this invention can be bacterial liquid, bacterial sludge, or granules containing *Bombus strophanthus* as described above; this invention does not limit the specific type. The bacterial liquid is obtained by fermentation of *Bombus strophanthus* as described above; the bacterial sludge is obtained by centrifugation and sedimentation of the bacterial liquid; and the granules are obtained by mixing the bacterial liquid or sludge with excipients, followed by drying and granulation. For example, the excipients include, but are not limited to, carriers (such as vermiculite, perlite, diatomaceous earth, activated carbon), fillers (such as calcium carbonate, kaolin, bentonite), nutrients (such as glucose, sucrose, peptone, yeast powder), and protectants (such as sodium alginate, gelatin, polyvinyl alcohol).

[0011] Furthermore, the present invention also provides a method for preparing the bacterial agent as described above, which includes inoculating the aforementioned *Bombyx mori* yeast into a seed culture medium and obtaining a seed liquid after fermentation.

[0012] The seed culture medium described in this invention comprises 50-250 g / L glucose, 2-20 g / L yeast extract, and Mg. 2+ 0.1~1.5 g / L, PO4 3- 0.1~1.5 g / L, pH value 4~7; preferably, the seed culture medium includes 80~120 g / L glucose, 15~25 g / L yeast extract, and Mg 2+ 0.5~1.5 g / L, PO4 3- 0.5~1.5 g / L, pH value 5.4~5.8.

[0013] In this embodiment of the invention, the magnesium ions are derived from one or more of MgSO4, MgCl2, magnesium glycine, magnesium threonate, magnesium citrate, magnesium glycine, and magnesium orotate. Preferably, the magnesium ions in the seed culture medium are MgSO4.

[0014] In this embodiment of the invention, PO4 3- The PO4 is derived from one or more of KH2PO4, NH4H2PO4, NaH2PO4, NaH2PO4·2H2O, K2HPO4, (NH4)2HPO4, Na2HPO4, Na2HPO4·12H2O, and H3PO4; preferably, the PO4 in the seed culture medium is... 3- It is KH2PO4.

[0015] In a specific embodiment, the seed culture medium includes 100 g / L glucose, 20 g / L yeast extract, 1 g / L MgSO4, 1 g / L KH2PO4, and pH 5.4~5.8.

[0016] In this embodiment of the invention, the fermentation conditions in the preparation of the microbial agent include culturing at 200 rpm and 28°C for 36-50 h.

[0017] The preparation method of the microbial agent of the present invention further includes the step of adding excipients to the seed liquid and then drying it.

[0018] Furthermore, the present invention also provides the application of the aforementioned *Bombyx mori* yeast, the aforementioned inoculum, or the inoculum prepared by the aforementioned method in the preparation of glycolipids.

[0019] In this invention, the glycolipids include sophorolipids, glycerol glycolipids, and / or glucose lipolipids.

[0020] Furthermore, the present invention also provides a method for preparing glycolipids, comprising: fermenting a culture medium containing oil in a bumblebee yeast as described above, a bacterial agent as described above, or a bacterial agent prepared by the method described above, to obtain a fermentation product containing glycolipids.

[0021] In this invention, the culture medium contains 50-150 g / L carbon source, 0.1-1.0 g / L nitrogen source, 30-200 mL / L oil, 0.1-2.0 g / L phosphorus source, 0.02-0.2 g / L magnesium salt, 0.01-0.5 mg / kg copper salt, and 0.034-1.32 g / L complex vitamins.

[0022] In this embodiment of the invention, the carbon source has a concentration of 50-150 g / L, preferably 80-120 g / L, based on reducing sugar content. The carbon source is selected from one or more of glucose, starch hydrolysate, fructose, maltose, sucrose, and molasses; preferably, the carbon source is glucose.

[0023] In this embodiment of the invention, the nitrogen source has a concentration of 0.1~1.0 g / L based on nitrogen element weight. Specifically, the nitrogen source is selected from one or more of yeast extract, peptone, beef extract, soy protein, corn steep liquor, ammonium sulfate, ammonium phosphate, and ammonia water. Preferably, the nitrogen source is yeast extract. The nitrogen content in the yeast extract is 8%~12%, and the content of the yeast extract in the culture medium is 0.1%~1%.

[0024] In this embodiment of the invention, the oil content in the culture medium is 30-200 mL / L. Specifically, the oil is selected from one or more of the following: camellia oil, prickly ash fruit oil, wheat germ oil, Pu-erh tea seed oil, rapeseed oil, corn oil, peanut oil, olive oil, soybean oil, and glycerin.

[0025] In this embodiment of the invention, the concentration of the phosphorus source in the culture medium, based on the weight of phosphorus, is 0.1~2.0 g / L; in a specific embodiment, the phosphorus source is selected from one or more of KH2PO4, NH4H2PO4, NaH2PO4, NaH2PO4·2H2O, K2HPO4, (NH4)2HPO4, Na2HPO4, Na2HPO4·12H2O, and H3PO4; preferably KH2PO4 and Na2HPO4·12H2O, with a ratio of 1:3~3:1, and more preferably 1:1.5~1.5:1.

[0026] In this embodiment of the invention, the concentration of magnesium salt in the culture medium, based on the weight of Mg, is 0.02~0.2 g / L; in a specific embodiment, the magnesium salt is selected from one or more of MgSO4, MgCl2, magnesium glycinate, magnesium threonate, magnesium citrate, magnesium glycinate, magnesium orotate, etc. Preferably, the magnesium salt in this invention is magnesium sulfate and / or magnesium chloride.

[0027] In this embodiment of the invention, the concentration of the copper salt in the culture medium is 0.01~0.5 mg / kg based on the weight of Cu. Specifically, the copper salt is one or more of CuSO4·5H2O, CuCl2, copper acetate, and copper nitrate; preferably, the copper salt in this invention is copper sulfate.

[0028] In this embodiment of the invention, the complex vitamins include: biotin and / or its salts, VB1 and / or its salts, VB2 and / or its salts, VB6 and / or its salts, D-calcium pantothenate and / or its salts, and niacin and / or its salts. In a specific embodiment, the complex vitamins include: biotin, VB1, VB2, VB6, D-calcium pantothenate, and niacin. In the culture medium, the concentrations of the complex vitamins are: biotin 0.001~0.02 g / L, VB1 0.01~0.2 g / L, VB2 0.002~0.05 g / L, VB6 0.001~0.05 g / L, D-calcium pantothenate 0.01~0.5 g / L, and niacin 0.01~0.5 g / L.

[0029] In this embodiment of the invention, the culture medium comprises: glucose 80-120 g / L, yeast extract 1-10 g / L, oil 30-200 mL / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.5-0.7 g / L, CuSO4·5H2O 0.05-0.2 mg / kg, biotin 0.001-0.02 g / L, VB1 0.01-0.2 g / L, VB2 0.002-0.05 g / L, VB6 0.001-0.05 g / L, D-calcium pantothenate 0.01-0.5 g / L, and nicotinic acid 0.01-0.5 g / L.

[0030] In some embodiments, the culture medium comprises: 100 g / L glucose, 5 g / L yeast extract, 30-200 mL / L oil, 3.0 g / L K2HPO4, 4.0 g / L Na2HPO4·12H2O, 0.6 g / L MgSO4, 0.1 mg / kg CuSO4·5H2O, 0.005 g / L biotin, 0.06 g / L VB1, 0.01 g / L VB2, 0.01 g / L VB6, 0.05 g / L D-calcium pantothenate, and 0.05 g / L nicotinic acid.

[0031] In a specific embodiment, the culture medium includes: glucose 120 g / L, Pu-erh tea seed oil 200 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.005 g / L, VB1 0.06 g / L, VB2 0.01 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, and niacin 0.05 g / L;

[0032] In other specific embodiments, the culture medium includes: glucose 120 g / L, tea seed oil 30 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.005 g / L, VB1 0.06 g / L, VB2 0.01 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, and nicotinic acid 0.05 g / L;

[0033] In other specific embodiments, the culture medium includes: glucose 120 g / L, wheat germ oil 200 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.002 g / L, VB1 0.01 g / L, VB2 0.003 g / L, VB6 0.006 g / L, D-calcium pantothenate 0.02 g / L, and nicotinic acid 0.02 g / L;

[0034] In other specific embodiments, the culture medium includes: 120 g / L glucose, 200 mL / L rapeseed oil, 5 g / L yeast extract, 3.0 g / L K2HPO4, 4.0 g / L Na2HPO4·12H2O, 0.6 g / L MgSO4, 0.1 g / L CuSO4·5H2O, 0.002 g / L biotin, 0.01 g / L VB1, 0.003 g / L VB2, 0.006 g / L VB6, 0.02 g / L D-calcium pantothenate, and 0.02 g / L nicotinic acid.

[0035] In this invention, the fermentation conditions include: inoculum size of 2%–10%, fermentation temperature of 20–32°C, fermentation time of 30–120 h, pH control of 2.0–6.0, airflow of 0.5–6.0 L / min, rotation speed of 200–700 rpm, and dissolved oxygen maintained above 20% during fermentation. The inoculum size during fermentation is 5%–10%, and the pH value is 3.0–5.0.

[0036] In a specific embodiment, the inoculum size was 10%, the pH was adjusted to 3.0, the temperature was 28°C, the air volume was 0.5-6.0 L / min, the rotation speed was 200-700 rpm, the dissolved oxygen content during fermentation was >20%, and the fermentation time was 120 h.

[0037] In a specific embodiment, the inoculum amount was 10%, the pH was adjusted to 3.0, the temperature was 28°C, the air volume was 0.5-6.0L / min, the rotation speed was 200-700rpm, the dissolved oxygen content during fermentation was >20%, and the fermentation time was 30h.

[0038] In a specific embodiment, the inoculum amount was 10%, the pH was adjusted to 5.0, the temperature was 28°C, the air volume was 0.5-6.0L / min, the rotation speed was 200-700rpm, the dissolved oxygen content during fermentation was >20%, and the fermentation time was 120h.

[0039] In a specific embodiment, the inoculum amount was 5%, the pH was adjusted to 4.0, the temperature was 30°C, the air volume was 0.5-6.0L / min, the rotation speed was 200-700rpm, the dissolved oxygen content during fermentation was >20%, and the fermentation time was 120h.

[0040] The preparation method of this invention further includes sterilization at 121°C for 30 min after fermentation, collection of the bottom lactone-type glycolipid after standing, and drying to obtain lactone-type glycolipid. The upper layer contains no unused oil; the pH is adjusted to 5.5, followed by solid-liquid separation. The separated solution is dried to obtain acidic glycolipid.

[0041] This invention provides *Saccharomyces simulans* strain with accession number CCTCC NO: M 2025061, and a method for preparing glycolipids. Glycolipids prepared using this strain have the advantages of short fermentation time, high oil utilization rate, and high glycolipid yield. Furthermore, the glycolipids obtained by this method are a mixture of sophorolipids, glycerol glycolipids, and glucose glycolipids, which can be widely used in pharmaceuticals, food, agriculture, environmental protection, cosmetics, and other fields, and can significantly reduce the production cost of glycolipids.

[0042] Biological Preservation Instructions

[0043] Starmerella bombicola FM-1 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on January 8, 2025, with accession number CCTCC NO: M2025061. Attached Figure Description

[0044] Figure 1 Results of strain identification;

[0045] Figure 2 It has a glycerol glycolipid structure;

[0046] Figure 3 It has a sophorolipid structure;

[0047] Figure 4 It has a glucose ester structure;

[0048] Figure 5 Comparison of the antioxidant capacity of glycolipids prepared by different processes;

[0049] Figure 6 For the moisturizing ability of glycolipids;

[0050] Figure 7 The impact of usage time on TWEL;

[0051] Figure 8 The effect of usage time on skin moisture content;

[0052] Figure 9 This is a flowchart of the glycolipid preparation process. Detailed Implementation

[0053] This invention provides *Bombyx mori* yeast and its application in the preparation of sophorolipids with anti-aging and / or moisturizing functions. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0054] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0055] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0056] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0057] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0058] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0059] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0060] The structures of the glyceroglycolipid, sophoroglycolipid, and glucose lipid described in this invention are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0061] The technical solutions involved in this invention mainly include:

[0062] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with Starmerella bombicola FM-1, and culture at 200rpm and 28℃ for 36-50h to obtain seed culture.

[0063] Seed culture medium composition:

[0064] Add 100 g / L glucose, 20 g / L yeast extract, 1 g / L MgSO4, and 1 g / L KH2PO4 to adjust the pH to 5.4-5.8.

[0065] 2. Glycolipid fermentation: Glycolipid fermentation is carried out in a 5L fermenter.

[0066] The carbon source is one or more of glucose, starch hydrolysate, fructose, maltose, sucrose, and molasses, with a concentration of 50-150 g / L based on reducing sugar, preferably 80-120 g / L.

[0067] The nitrogen source is one or more of yeast extract, peptone, beef extract, soy protein, corn steep liquor, ammonium sulfate, ammonium phosphate, and ammonia water, with a concentration of 0.1-1.0 g / L based on the weight of nitrogen element.

[0068] The oil is composed of one or more of the following: camellia oil, prickly pear fruit oil, wheat germ oil, Pu-erh tea seed oil, rapeseed oil, corn oil, peanut oil, olive oil, soybean oil, and glycerin. The amount of oil added is 30-200 mL / L.

[0069] The phosphorus source is one or more combinations of KH₂PO₄, NH₄H₂PO₄, NaH₂PO₄, NaH₂PO₄*2H₂O, K₂HPO₄, (NH₄)₂HPO₄, Na₂HPO₄, Na₂HPO₄*12H₂O, and H₃PO₄, preferably KH₂PO₄ and Na₂HPO₄*12H₂O, in a ratio of 1:3 to 3:1, more preferably 1:1.5 to 1.5:1. The phosphorus concentration is 0.1-2.0 g / L by weight.

[0070] The magnesium salt is one or more of MgSO4, MgCl2, magnesium glycinate, magnesium threonate, magnesium citrate, magnesium glycinate, magnesium oroticate, etc., preferably MgSO4 or MgCl2, with a concentration of 0.02-0.2 g / L based on the weight of Mg;

[0071] The copper salt is composed of one or more of CuSO4*5H2O, CuCl2, copper acetate, and copper nitrate, with a concentration of 0.01-0.5 mg / kg based on the weight of Cu.

[0072] Biotin 0.001-0.02 g / L, VB1 0.01-0.2 g / L, VB2 0.002-0.05 g / L, VB6 0.001-0.05 g / L, D-calcium pantothenate 0.01-0.5 g / L, niacin 0.01-0.5 g / L.

[0073] The seed fermentation broth inoculum should be 2-10%, the fermentation temperature 20-32℃, the fermentation time 30-120h, the pH controlled at 2.0-6.0, the air volume 0.5-6.0L / min, the rotation speed 200-700rpm, and the dissolved oxygen maintained above 20% during the fermentation process.

[0074] 3. After fermentation, adjust the pH to 3.0-8.0, sterilize at 121℃ for 30 minutes, collect the oily solution at the bottom after standing, and dry it to obtain lactone-type glycolipids; remove unused oil from the remaining solution, separate the solid and liquid to remove the cells, and dry it to obtain acidic glycolipids.

[0075] 4. Glycolipid products are obtained by liquid phase preparation.

[0076] The conditions for liquid phase include:

[0077] Chromatographic column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); column temperature: 45℃; mobile phase: A-water, B-acetonitrile; flow rate: 1 mL / min. The mobile phases are shown in the table below:

[0078] Table 1

[0079]

[0080] Testing has shown that the strains and methods described in this invention can produce effects including, but not limited to:

[0081] 1. Short fermentation time, high oil utilization rate and high sugar and lipid yield: Within 30h-120h, the utilization rate of 3%-20% oil reaches more than 94%, and the sugar and lipid content is as high as 233g / L, which significantly reduces the production cost of sugar and lipid.

[0082] 2. The glycolipids obtained after fermentation are mixed glycolipids: glycerol glycolipids (22%-39%), glucose glycolipids (13%-20%), sophorolipids (13%-41%), and other glycolipids (1%-9%). Among them, lactone-type glycolipids account for 0-23%, and acid-type glycolipids account for 77%-100%. It has moisturizing and anti-inflammatory effects and can be widely used in medicine, food, agriculture, environmental protection, cosmetics, and other fields.

[0083] The present invention will be further illustrated below with reference to the embodiments:

[0084] Example 1 Screening of Bumblebee Saccharomyces simulans FM-1

[0085] 1. Strains Collection and Purification: Wild honey was collected from the Qinling Mountains in Shaanxi Province. Honey samples were diluted with sterile water and inoculated into PDB medium (containing 50 μg / mL chloramphenicol and 50 μg / mL gentamicin) and cultured for 2 days. The bacterial suspension was then serially diluted, and 200 μL was spread onto PDA medium and incubated at 28°C for 3 days. Single colonies with different morphologies and colors were picked and streaked onto PDA medium to isolate and purify the fungi. After the bacteria grew, streaking was repeated three times to ensure the purity of the obtained strain.

[0086] 2. The purified strains were subjected to glycolipid fermentation to screen for strains with excellent glycolipid production capabilities.

[0087] Seed fermentation: Add 100 mL of seed culture medium to a 500 mL shake flask, inoculate with *Candida baccata* and *Saccharomyces simulans* FM-1, and culture at 200 rpm and 28 °C for 48 h to obtain seed culture.

[0088] Glycolipid fermentation: 500 mL of fermentation broth was inoculated with 50 mL of seed fermentation broth, at a temperature of 28℃ and a rotation speed of 200 rpm for 120 h.

[0089] The composition of the fermentation broth is shown below:

[0090] Glucose 120g / L, Pu-erh tea seed oil 50mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, biotin 0.005g / L, VB1 0.06g / L, VB2 0.01g / L, VB6 0.01g / L, D-calcium pantothenate 0.05g / L, niacin 0.05g / L.

[0091] Determination of sugar and lipid content: After fermentation, the sugar and lipid content in the fermentation broth was determined according to the sulfuric acid phenol method.

[0092] Strain identification and preservation: The strain with the highest glycolipid production, *Starmerella bombicola* FM-1, was sent to the China Industrial Microbial Culture Collection Center for identification and preservation. The strain was identified as *Starmerella bombicola*. Figure 1 As shown. It was subsequently deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2025061.

[0093] Example 2 (20% Pu-erh tea seed oil fermentation time 120h)

[0094] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0095] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 120h.

[0096] The composition of the fermentation broth is shown below:

[0097] Glucose 120g / L, Pu-erh tea seed oil 200mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, biotin 0.005g / L, VB1 0.06g / L, VB2 0.01g / L, VB6 0.01g / L, D-calcium pantothenate 0.05g / L, niacin 0.05g / L.

[0098] 3. After fermentation, sterilize at 121℃ for 30 minutes. After standing, collect the bottom lactone-type glycolipid and dry it to obtain lactone-type glycolipid. Remove the unused oil in the upper layer, adjust the pH to 5.5, then perform solid-liquid separation, collect the separated solution and dry it to obtain acidic glycolipid.

[0099] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0100] The oil utilization rate was 94%, the glycolipid concentration in the fermentation broth was 233 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 39% glycerol glycolipids, 34% sophorolipids, 20% glucose lipolipids, and 7% other glycolipids.

[0101] Example 3 (3% tea seed oil fermentation time 30h)

[0102] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0103] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 30h.

[0104] The composition of the fermentation broth is shown below:

[0105] Glucose 120g / L, Camellia seed oil 30mL / L, Yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, Biotin 0.005g / L, VB1 0.06g / L, VB2 0.01g / L, VB6 0.01g / L, D-calcium pantothenate 0.05g / L, Niacin 0.05g / L.

[0106] 3. After fermentation, sterilize at 121℃ for 30 min. No lactone-type glycolipids precipitate. There is no unused oil in the upper layer. Adjust the pH to 5.5 and sterilize at 121℃ for 30 min. Then, separate the solid and liquid to remove the mycelium. Collect the separated solution and dry it to obtain acidic glycolipids with a glycolipid concentration of 50 g / L.

[0107] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0108] The oil utilization rate was 100%, the glycolipid concentration in the fermentation broth was 50 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 34% glycerol glycolipids, 41% sophorolipids, 20% glucose lipolipids, and 5% other glycolipids.

[0109] Example 4 (20% wheat germ oil fermentation time 120h)

[0110] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0111] 2. Fermentation volume 3L, inoculate with 300mL seed fermentation broth, adjust pH to 5.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 120h.

[0112] The composition of the fermentation broth is shown below:

[0113] Glucose 120g / L, wheat germ oil 200mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, biotin 0.002g / L, VB1 0.01g / L, VB2 0.003g / L, VB6 0.006g / L, D-calcium pantothenate 0.02g / L, niacin 0.02g / L.

[0114] 3. After fermentation, sterilize at 121℃ for 30 minutes. After standing, collect the bottom lactone-type glycolipid and dry it to obtain the lactone-type glycolipid. Remove the unused oil from the upper layer; the oil utilization rate is 95%. Adjust the pH to 6.0, then perform solid-liquid separation. Dry the separated solution to obtain the acidic glycolipid. Weigh and calculate.

[0115] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0116] The oil utilization rate was 100%, the glycolipid concentration was 218 g / L, and the proportions of lactone-type glycolipids and acid-type glycolipids were 28% and 72%, respectively. The glycolipid mixture contained 28% glycerol glycolipids, 47% sophorolipids, 16% glucose lipolipids, and 9% other glycolipids.

[0117] Example 5 (20% rapeseed oil, fermentation time 120h)

[0118] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0119] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 4.0, temperature 30℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 120h.

[0120] The composition of the fermentation broth is shown below:

[0121] Glucose 120g / L, Pu'er rapeseed oil 200mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, biotin 0.002g / L, VB1 0.01g / L, VB2 0.003g / L, VB6 0.006g / L, D-calcium pantothenate 0.02g / L, niacin 0.02g / L.

[0122] 3. After fermentation, sterilize at 121℃ for 30 minutes. After standing, collect the bottom lactone-type glycolipid and dry it to obtain lactone-type glycolipid. There is no unused oil in the upper layer. Adjust the pH to 5.5, then perform solid-liquid separation. After separation, dry the solution to obtain acidic glycolipid.

[0123] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0124] The oil utilization rate was 100%, the glycolipid concentration was 227 g / L, and the proportions of lactone-type glycolipids and acid-type glycolipids were 77% and 23%, respectively. The glycolipid mixture contained 22% glycerol glycolipids, 57% sophorolipids, 13% glucose lipolipids, and 8% other glycolipids.

[0125] Comparative Example 1 (Starmerella bombicola ATCC 22214 strain 20% Pu-erh tea seed oil)

[0126] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate Starmerellabombicola ATCC 22214, and culture at 200rpm and 28℃ for 48h to obtain seed culture.

[0127] 2. Fermentation volume 3L, inoculate with 300mL seed fermentation broth, adjust pH to 5.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0128] The composition of the fermentation broth is shown below:

[0129] Glucose 120g / L, Pu-erh tea seed oil 200mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4*12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4*5H2O 0.1g / L, biotin 0.002g / L, VB1 0.01g / L, VB2 0.003g / L, VB6 0.006g / L, D-calcium pantothenate 0.02g / L, niacin 0.02g / L.

[0130] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer, adjust the pH to 6.0, and then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0131] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0132] The oil utilization rate was 63%, the glycolipid concentration was 177 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 36% glycerol glycolipids, 52% sophorolipids, 11% glucose lipolipids, and 1% other glycolipids.

[0133] Comparative Example 2 (Bumblebee Saccharomyces FM-1, 20% Pu-erh Tea Seed Oil, Biotin-free)

[0134] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0135] 2. Fermentation volume 3L, inoculate with 300mL seed fermentation broth, adjust pH to 5.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0136] The composition of the fermentation broth is shown below:

[0137] Glucose 120g / L, Pu-erh tea seed oil 200mL / L, yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4·12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4·5H2O 0.1g / L, VB1 0.01g / L, VB2 0.003g / L, VB6 0.006g / L, D-calcium pantothenate 0.02g / L, niacin 0.02g / L.

[0138] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer; the oil utilization rate is 66%. Adjust the pH to 6.0, then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0139] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0140] The oil utilization rate was 66%, the glycolipid concentration was 102 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 28% glycerol glycolipids, 47% sophorolipids, 21% glucose lipolipids, and 4% other glycolipids.

[0141] Control Example 3 (Bumblebee Saccharomyces simulans FM-1, without VB1)

[0142] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0143] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0144] The composition of the fermentation broth is shown below:

[0145] Glucose 120 g / L, Camellia seed oil 150 mL / L, Yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, Biotin 0.005 g / L, VB2 0.01 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, Niacin 0.05 g / L.

[0146] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer, adjust the pH to 6.0, and then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0147] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0148] The oil utilization rate was 59%, the glycolipid concentration was 83 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 9% glycerol glycolipids, 87% sophorolipids, and 4% other glycolipids.

[0149] Control Example 4 (Bumblebee Saccharomyces simulans FM-1, without VB2)

[0150] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with Bumblebee Saccharomyces simulans FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed culture.

[0151] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0152] The composition of the fermentation broth is shown below:

[0153] Glucose 120 g / L, Camellia seed oil 150 mL / L, Yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, Biotin 0.005 g / L, VB1 0.06 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, Niacin 0.05 g / L.

[0154] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer, adjust the pH to 6.0, and then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0155] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0156] The oil utilization rate was 57%, the glycolipid concentration was 99 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 15% glycerol glycolipids, 77% sophorolipids, 4% glucose lipolipids, and 4% other glycolipids.

[0157] Control Example 5 (Bumblebee Saccharomyces simulans FM-1, without VB6)

[0158] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with Bumblebee Saccharomyces simulans FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed culture.

[0159] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0160] The composition of the fermentation broth is shown below:

[0161] Glucose 120 g / L, Camellia seed oil 150 mL / L, Yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, Biotin 0.005 g / L, VB1 0.06 g / L, VB2 0.01 g / L, D-calcium pantothenate 0.05 g / L, Niacin 0.05 g / L.

[0162] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer; the oil utilization rate is 46%. Adjust the pH to 6.0, then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0163] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0164] The oil utilization rate was 46%, the glycolipid concentration was 76 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 3% glycerol glycolipids, 92% sophorolipids, 3% glucose lipolipids, and 2% other glycolipids.

[0165] Control Example 6 (Bumblebee Saccharomyces simulans FM-1, without D-calcium pantothenate)

[0166] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with Bumblebee Saccharomyces simulans FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed culture.

[0167] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0168] The composition of the fermentation broth is shown below:

[0169] Glucose 120g / L, Camellia seed oil 150mL / L, Yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4·12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4·5H2O 0.1g / L, Biotin 0.005g / L, VB1 0.06g / L, VB2 0.01g / L, VB6 0.01g / L, Niacin 0.05g / L.

[0170] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer; the oil utilization rate is 49%. Adjust the pH to 6.0, then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0171] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0172] The oil utilization rate was 49%, the glycolipid concentration was 88 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 11% glycerol glycolipids, 85% sophorolipids, 2% glucose lipolipids, and 2% other glycolipids.

[0173] Comparative Example 7 (Bumblebee Saccharomyces simulans FM-1, without niacin)

[0174] 1. Seed fermentation: Add 1000mL of seed culture medium to a 2000mL shake flask, inoculate with bumblebee yeast FM-1, and culture at 200rpm and 28℃ for 48h to obtain seed liquid.

[0175] 2. Fermentation volume 3L, inoculate with 300mL of seed fermentation broth, adjust pH to 3.0, temperature 28℃, air flow 0.5-6.0L / min, rotation speed 200-700rpm, dissolved oxygen content >20% during fermentation, fermentation time 144h.

[0176] The composition of the fermentation broth is shown below:

[0177] Glucose 120g / L, Camellia seed oil 150mL / L, Yeast extract 5g / L, K2HPO4 3.0g / L, Na2HPO4·12H2O 4.0g / L, MgSO4 0.6g / L, CuSO4·5H2O 0.1g / L, Biotin 0.005g / L, VB1 0.06g / L, VB2 0.01g / L, VB6 0.01g / L, D-calcium pantothenate 0.05g / L.

[0178] 3. After fermentation, sterilize at 121℃ for 30 minutes. No lactone-type glycolipids precipitate at the bottom layer. Remove the unused oil from the upper layer; the oil utilization rate is 42%. Adjust the pH to 6.0, then perform solid-liquid separation. After drying the separated solution, acidic glycolipids are obtained.

[0179] 4. Weigh 100 mg of glycolipid, prepare and characterize it by HPLC-MS, and freeze-dry the prepared solution to obtain a single glycolipid product.

[0180] The oil utilization rate was 42%, the glycolipid concentration was 83 g / L, and the proportion of acidic glycolipids was 100%. The glycolipid mixture contained 26% glycerol glycolipids, 65% sophorolipids, 5% glucose lipolipids, and 4% other glycolipids.

[0181] Example 6: In vitro antioxidant capacity test

[0182] Prepare DPPH standard solution: Weigh 4 mg of DPPH and dilute to 100 mL with anhydrous ethanol to prepare a 0.04 mg / mL DPPH solution.

[0183] 10% sample solution: The acidic glycolipids of Examples 2-5, the acidic glycolipids of Control Examples 1-7, and sophorolipids (commercially available) were prepared into solutions with a dry matter content of 10%.

[0184] Test group settings:

[0185] Table 2

[0186]

[0187] After mixing thoroughly, let stand at room temperature for 30 minutes. After that, centrifuge at 5000 rpm for 10 minutes and measure the absorbance of the supernatant at 517 nm. Two replicates were set up for each experimental group.

[0188] DPPH removal rate is calculated according to the following formula

[0189] DPPH removal rate % = %

[0190] A0—Absorbance of blank control

[0191] A1—Absorbance of the sample solution

[0192] A2—Absorbance of the sample control

[0193] Experimental results are as follows Figure 5 The results showed that, compared with the comparative example, the glycolipids prepared in each example had better antioxidant capacity.

[0194] Example 7 Hyaluronidase Inhibition Experiment

[0195] Experimental content

[0196] Blank group: Add 0.1 mL of acetate buffer to the test tube and incubate at 37℃ for 20 min; add 0.5 mL of acetate buffer and incubate at 37℃ for 20 min; add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution and incubate at 37℃ for 30 min; let stand at room temperature for 5 min, add 0.5 mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na₂CO₃ solution, freshly prepared) and 0.1 mL of 0.4 mol / L NaOH solution, boil in a water bath for 15 min, then immediately place in an ice bath for 5 min; add 1 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, freshly prepared), develop color at room temperature for 20 min, and measure the absorbance at 530 nm. Set up 4 replicates.

[0197] Positive control group: Add 0.1 mL of 2.5 mmol / L CaCl2 and 0.5 mL of 600 U / mL hyaluronidase to the test tube, and treat at 37℃ for 20 min; add 0.5 mL of 0.5 mg / L dexamethasone sodium phosphate solution, and treat at 37℃ for 20 min; add 0.5 mL of 0.5 mg / mL hyaluronic acid sodium solution, and treat at 37℃ for 30 min; let stand at room temperature for 5 min, add 0.5 mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na2CO3 solution, freshly prepared) and 0.1 mL of 0.4 mol / L NaOH solution, boil in a water bath for 15 min, and immediately in an ice bath for 5 min; add 1 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, freshly prepared), develop color at room temperature for 20 min, and then incubate at 530℃. The absorbance was measured at a wavelength of nm. Four parallel measurements were performed.

[0198] Example 3 group: Add 0.1 mL of 2.5 mmol / L CaCl2 and 0.5 mL of 600 U / mL hyaluronidase to the test tube and treat at 37℃ for 20 min; add 0.5 mL of 10% Example 3 glycolipid solution and treat at 37℃ for 20 min; add 0.5 mL of 0.5 mg / mL sodium hyaluronate solution and treat at 37℃ for 30 min; let stand at room temperature for 5 min, add 0.5 mL of acetylacetone solution (1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na2CO3 solution, freshly prepared) and 0.1 mL of 0.4 mol / L NaOH solution, boil in a water bath for 15 min, and immediately place on an ice bath for 5 min; add 1 mL of Ehrlich reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, freshly prepared), develop color at room temperature for 20 min, and measure its absorbance at a wavelength of 530 nm. Set 4 parallel lines.

[0199] The inhibition rate is calculated as follows:

[0200] Inhibition rate / % = %

[0201] In the formula:

[0202] The absorbance of enzyme A reaction group, that is, the absorbance of the reaction between hyaluronidase and hyaluronic acid without the addition of sample;

[0203] B represents the blank absorbance (acetic acid buffer replacing enzyme solution and experimental sample).

[0204] C represents the absorbance of the experimental group;

[0205] D represents the absorbance of the experimental group and the blank group (acetic acid buffer replaced enzyme solution).

[0206] 2. Experimental results are as follows Figure 6 The results showed that the glycolipid prepared in Example 3 had better moisturizing ability than sophorolipid alone.

[0207] Example 8: Human Efficacy Testing

[0208] Experimenter selection

[0209] Nine individuals aged 25-40 were selected, based on the following criteria:

[0210] (1) No acute inflammation or other skin diseases (such as rosacea, eczema, lupus erythematosus, seborrheic dermatitis, psoriasis, severe epidermal exfoliation, etc.);

[0211] (2) No highly sensitive constitution or cosmetic allergy;

[0212] 2. Experimental Environment

[0213] Temperature 20±1℃, humidity 45%-65%.

[0214] 3. Experimental Group

[0215] Control sample group: water

[0216] Test sample group: glycolipid solution obtained in Example 3 with a dry matter content of 10%.

[0217] 4. Experiment Content

[0218] (1) Mark the left arm of the subject: the test area sample group and the blank control area. The area of ​​each area is 9-25 cm². 2 The sample application rate was 0.02 g / cm³. 2 .

[0219] (2) Measurement data using Corneometer® CM825 for skin moisture content test and Tewameter®™300 for skin moisture loss test.

[0220] (3) Apply the control sample and the test sample to the experimental site of the subject in sequence.

[0221] (4) The subjects used a Corneometer to measure skin moisture content 1h, 2h, 3h and 4h after using the sample. ® CM825, Tewameter for testing skin moisture loss ® The TM300 was measured 5 times, and the average value was taken.

[0222] (5) Statistically analyze the measured values ​​and the changes in skin moisture and water loss.

[0223] Skin TEWL = (TEWL value after sample application / TEWL value before sample application) * 100%

[0224] 4. Experimental results are as follows Figures 7-8 The results showed that the glycolipid prepared in Example 3 had good hydrating and / or moisturizing effects.

[0225] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bumblebee Saccharomyces cerevisiae with preservation number CCTCC NO: M 2025061 ( Starmerella bombicola ).

2. A microbial agent comprising the bumblebee yeast of claim 1.

3. The method for preparing the microbial agent according to claim 2, comprising inoculating the bumblebee yeast of claim 1 into a seed culture medium and obtaining a seed liquid after fermentation.

4. The preparation method according to claim 3, characterized in that, The seed culture medium includes 50-250 g / L glucose, 2-20 g / L yeast extract, and Mg. 2+ 0.1~1.5 g / L, PO4 3- 0.1~1.5 g / L, pH value 4~7.

5. The preparation method according to claim 3 or 4, characterized in that, It also includes the steps of adding excipients to the seed liquid and then drying it.

6. The use of the *Bombyx mori* yeast of claim 1, the inoculum of claim 2, or the inoculum prepared by any one of claims 3 to 5 in the preparation of glycolipids.

7. The application according to claim 6, characterized in that, The glycolipids include sophorolipids, glycerol glycolipids, and / or glucose lipolipids.

8. A method for preparing glycolipids, comprising: Using the *Bombyx mori* yeast of claim 1, the inoculum of claim 2, or the inoculum prepared by any one of claims 3 to 5, fermentation is carried out in a culture medium containing oil to obtain a fermentation product containing glycolipids; wherein the oil is selected from 20% wheat germ oil, 20% rapeseed oil, 20% Pu-erh tea seed oil, or 3% tea seed oil.

9. The preparation method according to claim 8, characterized in that, The carbon source in the culture medium is selected from one or more of glucose, starch hydrolysate, fructose, maltose, sucrose, and molasses; The nitrogen source in the culture medium is selected from one or more of yeast extract, peptone, beef extract, soy protein, corn steep liquor, ammonium sulfate, ammonium phosphate, and ammonia water; The phosphorus source in the culture medium is selected from one or more of KH2PO4, NH4H2PO4, NaH2PO4, NaH2PO4·2H2O, K2HPO4, (NH4)2HPO4, Na2HPO4, Na2HPO4·12H2O, and H3PO4. The magnesium salt in the culture medium is selected from one or more of MgSO4, MgCl2, magnesium threonate, magnesium citrate, magnesium glycine, and magnesium oroticate. The copper salt in the culture medium is one or more of CuSO4·5H2O, CuCl2, copper acetate, and copper nitrate. The culture medium contains a complex of vitamins including: biotin and / or its salts, VB1 and / or its salts, VB2 and / or its salts, VB6 and / or its salts, D-pantothenic acid and / or its salts, and niacin and / or its salts.

10. The preparation method according to claim 8, characterized in that, The culture medium comprises: glucose 120 g / L, Pu-erh tea seed oil 200 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.005 g / L, VB1 0.06 g / L, VB2 0.01 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, and niacin 0.05 g / L. Alternatively, the culture medium may include: glucose 120 g / L, tea seed oil 30 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.005 g / L, VB1 0.06 g / L, VB2 0.01 g / L, VB6 0.01 g / L, D-calcium pantothenate 0.05 g / L, and niacin 0.05 g / L; Alternatively, the culture medium may include: glucose 120 g / L, wheat germ oil 200 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.002 g / L, VB1 0.01 g / L, VB2 0.003 g / L, VB6 0.006 g / L, D-calcium pantothenate 0.02 g / L, and nicotinic acid 0.02 g / L; Alternatively, the culture medium may include: glucose 120 g / L, rapeseed oil 200 mL / L, yeast extract 5 g / L, K2HPO4 3.0 g / L, Na2HPO4·12H2O 4.0 g / L, MgSO4 0.6 g / L, CuSO4·5H2O 0.1 g / L, biotin 0.002 g / L, VB1 0.01 g / L, VB2 0.003 g / L, VB6 0.006 g / L, D-calcium pantothenate 0.02 g / L, and nicotinic acid 0.02 g / L.

11. The preparation method according to any one of claims 8 to 10, characterized in that, The fermentation conditions include: inoculum size of 2% to 10%, fermentation temperature of 20 to 32°C, fermentation time of 30 to 120 hours, pH control of 2.0 to 6.0, air volume of 0.5 to 6.0 L / min, rotation speed of 200 to 700 rpm, and dissolved oxygen maintained above 20% during the fermentation process.

Images