Biosynthesis method for preparing royal jelly acid and use thereof in skin care

Abstract

A biosynthesis method for preparing royal jelly acid and the use thereof in skin care, which belong to the technical field of genetic engineering and biosynthesis. The royal jelly acid is produced by means of fermentation on the basis of yeast engineering bacteria using a culture medium containing trans-2-decenoic acid. An assessment at both skin cell level and skin model level shows that royal jelly acid can promote the synthesis of stratum corneum lipids and ceramide on aging skin and repair the skin barrier; and also can promote the synthesis of mitochondria ATP and NADPH in skin fibroblasts, reduce the active oxygen content of mitochondria, improve mitochondrial dysfunctions, and inhibit apoptosis. Further experiments show that royal jelly acid can repair DNA damage of fibroblasts caused by ultraviolet irradiation, reduce telomere shortening caused by cell division and replication, and strengthen the protection of chromosomal terminal regions. The prepared royal jelly acid has wide application prospects in the development of functional skin care products for skin repairing, aging resisting, etc.

Description

A biosynthetic method for preparing royal jelly acid and its application in skin care

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 202311048043.2 filed with the State Intellectual Property Office of China on August 21, 2023, entitled “A biosynthetic method for preparing royal jelly acid and its application in skin care”, the entire contents of which are incorporated by reference into the present invention and constitute a part of the present invention for all purposes. Technical Field

[0003] The invention belongs to the technical field of genetic engineering and biosynthesis, and particularly relates to a biosynthetic method for preparing royal jelly acid and its application in skin care. Background Art

[0004] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0005] Royal jelly acid, also known as 10-hydroxy-2-decenoic acid (10-HDA), is an unsaturated fatty acid found in royal jelly. It has a single source and is found naturally only in royal jelly, where it comprises only 1.4%-2.4%. Research on the effects of royal jelly has revealed that it possesses the same strong health and medical benefits as royal jelly, including antibacterial, anti-tumor, antioxidant, immune-enhancing, and blood sugar-regulating properties.

[0006] At present, the synthesis of royal jelly acid in China is mainly based on physical extraction and chemical synthesis. The extraction cost is high and cannot meet the large demand. At present, physical extraction is generally used to extract royal jelly from royal jelly. Due to the small amount of royal jelly, this extraction method is not suitable for large-scale production. A large amount of organic solvents is used, and the cost is high. In terms of chemical preparation, there are usually α-olefin and carbon tetrachloride addition hydrolysis method, carboxylic acid α-dehalogenation method and Knoevenagel condensation method, etc. However, most chemical synthesis methods have harsh reaction conditions, and the yield is low except for the condensation method. The double bond formed is a cis-trans mixture, which is difficult to extract. At present, the preparation of royal jelly acid by biosynthesis is still in the research stage, and the output is unstable. It is urgent to obtain functionally stable engineering strains in order to prepare royal jelly acid in batches.

[0007] The skin barrier forms a protective layer between the "inside" and the "outside," protecting against external damage. Epidermal lipids, such as ceramides, fatty acids, triglycerides, and cholesterol, are essential components for the formation and maintenance of the epidermal barrier. Fatty acids account for approximately 15-20% of the total weight of the stratum corneum lipids and play a crucial role in the structural stability and repair of epidermal barrier damage. A decrease in fatty acids can lead to impaired skin barrier function and contribute to various skin inflammations. Studies have shown that lipid content decreases significantly in aging skin, a key factor contributing to the weakening of skin barrier function. In the skin, ROS can react with the protective lipid bilayer in the stratum corneum to form lipid hydroperoxides, which can weaken the skin barrier. Therefore, maintaining the lipid and ceramide content of stratum corneum cells and reducing lipid peroxidation are effective means of repairing a damaged skin barrier. Under external environmental stimuli, premature cellular aging leads to reduced cell lifespan. The primary mechanism is damage to the nucleus and DNA. Abnormal nuclear shape disrupts nuclear function, including altered histone modification patterns, abnormal chromatin regeneration, impaired nuclear translocation, delayed DNA repair reactions, and shortened telomere length. Therefore, repairing damaged DNA and extending telomeres are key to combating cellular aging. Furthermore, mitochondria are the powerhouse of all normal cellular life, providing over 90% of the cell's energy and profoundly regulating energy metabolism, ROS and free radicals, oxidative stress, and inflammation. While mitochondria utilize oxygen molecules, they are also constantly damaged by free radicals, leading to mitochondrial dysfunction and skin aging. Therefore, to combat skin aging caused by external stimuli, we can strengthen the skin's lipid barrier, reduce mitochondrial damage, repair cellular DNA, and slow the shortening of telomeres.

[0008] Summary of the Invention

[0009] In response to the above-mentioned prior art, the present invention aims to provide a biosynthetic method for preparing royal jelly acid and its application in skin care. This invention utilizes engineered yeast to ferment royal jelly acid using a culture medium containing trans-2-decenoic acid. Evaluations conducted on skin cells and skin models have shown that royal jelly acid can promote the synthesis of lipids and ceramides in the stratum corneum of aged skin, repairing the skin barrier. It also promotes the synthesis of mitochondrial ATP and NADPH in skin fibroblasts, reduces mitochondrial reactive oxygen species, improves mitochondrial dysfunction, and inhibits apoptosis. Further experiments have revealed that royal jelly acid can repair DNA damage in fibroblasts exposed to ultraviolet light, slow telomere shortening caused by cell division and replication, and enhance protection of chromosome ends. Based on these research findings, the present invention was completed.

[0010] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0011] The first aspect of the present invention provides a method for preparing royal jelly acid based on a biosynthetic method, the method comprising:

[0012] The invention uses a yeast engineering bacterium containing at least a CYP153A33-CPRBM3 fusion gene to ferment and produce royal jelly acid using a culture medium containing trans-2-decenoic acid.

[0013] The nucleotide sequence of the CYP153A33-CPRBM3 fusion gene is shown in SEQ ID NO. 1. The fusion gene is codon-optimized to make it easier to express in Saccharomyces cerevisiae. BM3 The yeast engineering bacteria with the fusion gene can react with the substrate trans-2-decenoic acid to obtain the royal jelly acid, and the yield thereof can reach 190 mg / L.

[0014] Furthermore, the engineered yeast may also contain an auxiliary gene sil1p and / or an auxiliary gene crp5p.

[0015] The nucleotide sequence of the auxiliary gene sil1p is shown in SEQ ID NO. 2, and the nucleotide sequence of the auxiliary gene crp5p is shown in SEQ ID NO. 3. By introducing the auxiliary genes sil1p and crp5p, the ability of the yeast engineered bacteria to produce royal jelly acid can be further improved.

[0016] The method may further comprise any one or more steps of separating, purifying and drying the royal jelly acid. Therefore, the royal jelly acid may be a liquid preparation or a solid (such as granular) preparation.

[0017] The second aspect of the present invention provides the use of the above method and / or the royal jelly acid produced by the above method in any one or more of the following:

[0018] (a) increasing the content of ceramide and fatty acids in the epidermis to repair the skin barrier or preparing a product for increasing the content of ceramide and fatty acids in the epidermis to repair the skin barrier;

[0019] (b) repairing DNA damage in dermal fibroblasts or preparing a product for repairing DNA damage in dermal fibroblasts;

[0020] (c) Improving mitochondrial function, enhancing cell function, promoting metabolism or preparing products that improve mitochondrial function, enhance cell function, and promote metabolism;

[0021] (d) delaying telomere shortening caused by cell division and replication, protecting the integrity of chromosome terminal structure, or preparing a product that delays telomere shortening caused by cell division and replication, protecting the integrity of chromosome terminal structure.

[0022] Beneficial technical effects of one or more of the above technical solutions:

[0023] 1) Compared with physical extraction and chemical methods for preparing royal jelly acid, the above technical solution provides a method for preparing royal jelly acid by biosynthesis, which is natural, green and safe. At the same time, the above technical solution is the first to BM3 The fusion gene was codon-optimized and expressed in Saccharomyces cerevisiae to recombinantly express CYP153A33-CPR. BM3 After reacting with the substrate trans-2-decenoic acid, 10-HDA can be obtained with a yield of up to 190 mg / L, which is better than the 10-HDA yield obtained by the recombinant protein CYP539A7*-F0CPR* acting on decenoic acid reported in CN202211449654.3. In addition, the above technical scheme constructs CYP153A33-CPR BM3 The fusion genes were co-transformed and expressed with the auxiliary protein genes sil1p and crp5p in Saccharomyces cerevisiae, respectively, which can effectively increase the production of 10-HDA in yeast engineered bacteria. The auxiliary protein gene sil1p is connected to the protein 10-HDA production can reach 280 mg / L, and the auxiliary protein gene crp5p is connected to the fusion enzyme gene. The 10-HDA production can reach 246 mg / L, which are both better than the recombinant protein CYP539A7*-F0CPR*- reported in CN202211449654.3. After being connected to the auxiliary protein, the constructed recombinant bacteria acted on trans-2-decenoic acid to obtain 10-HDA production; in particular, the time for preparing 10-HDA by the Saccharomyces cerevisiae engineered bacteria constructed by the above technical solution was significantly shortened than the reaction time reported in CN202211449654.3. The reaction time of the engineering bacteria involved in CN202211449654.3 is 48 hours, and the CYP153A33-CPR in the above technical solution is 48 hours. BM3 By co-expressing the plasmid engineering bacteria with the auxiliary protein gene sil1p in Saccharomyces cerevisiae, 0.5g / L decenoic acid can reach the maximum conversion rate and product conversion amount after 36 hours of reaction, thus achieving excellent technical results.

[0024] 2) The royal jelly acid prepared by the above technical solution was confirmed on a 3D epidermal model to significantly increase the total amount of ceramide and the content of ceramide / protein, with an increase rate of 35.37% and 47.64%, respectively; it can also increase the total amount of fatty acids and the content of fatty acids / protein, with an increase rate of 27.51% and 39%, respectively. Therefore, it can increase the lipid and ceramide content in the epidermis, improve the barrier protection function of the skin, and improve the damaged barrier.

[0025] 3) The royal jelly acid prepared by the above technical solution reduces the content of 8-hydroxydeoxyguanosine and γ-H2AX protein in a fibroblast model, with inhibition rates of 45.09% and 47.25%, respectively, inhibiting DNA damage and preventing damage accumulation from leading to functional abnormalities; it can reduce the content of mitochondrial reactive oxygen species and the early apoptosis rate of cells in cells, with inhibition rates of 70.71% and 12.74%, respectively, and increase the content of mitochondrial adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH), with increase rates of 56.23% and 15.13%, respectively, protecting mitochondria from peroxidative damage and maintaining energy supply and material metabolism in cells; it can extend telomere length, with the relative T / S (TL) value increased by 31%, effectively delaying telomere shortening caused by cell division and replication in the aging process, strengthening the structural protection of chromosome ends, preventing chromosome ends from degradation or fusion, and cells losing division activity and aging, thereby preventing skin aging.

[0026] In summary, the royal jelly acid and its products obtained by the above technical solution have broad application prospects in the development of functional cosmetics such as skin repair and anti-aging, especially skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] FIG1 is an agarose gel electrophoresis diagram of the PCR amplification products in Example 1 of the present invention; wherein lane M is a marker; lanes 1-4 are CYP153A33-CPR BM3 Lanes 5-6 are samples of PCR amplification products of the auxiliary protein gene sil1p; lanes 7-8 are samples of PCR amplification products of the auxiliary protein gene crp5p.

[0029] Figure 2 is pESC-URA-CYP153A33-CPR in Example 1 of the present invention BM3 Plasmid construction diagram.

[0030] FIG3 is a diagram showing the construction of the pESC-URA-CYP153A33-CPRBM3-sil1p plasmid in Example 1 of the present invention.

[0031] FIG4 is a diagram showing the construction of the pESC-URA-CYP153A33-CPRBM3-crp5p plasmid in Example 1 of the present invention.

[0032] Figure 5 is an agarose gel electrophoresis diagram of the colony PCR products in Example 1 of the present invention; wherein lane M is a marker; lanes 1-4 are picked E. coli DH5α / pESC-URA-CYP153A33-CPR BM3 Different single colony PCR amplification product samples of strains; lanes 5-8 are picked E. coli DH5α / pESC-URA-CYP153A33-CPR BM3 Lanes 9-12 are samples of PCR amplification products from different single colonies of the Escherichia coli DH5α / pESC-URA-CYP153A33-CPRBM3-crp5p strain.

[0033] Figure 6 is a graph showing the 10-HDA yield after 48 hours of reaction under different engineered strains in Example 1 of the present invention; from left to right are the engineered strains of Saccharomyces cerevisiae BY4741 / pESC-URA-CYP153A33-CPR BM3 The content of trans-2-decenoic acid and 10-HDA in the strain; Saccharomyces cerevisiae engineered strain BY4741 / pESC-URA-CYP153A33-CPR BM3 -sil1p strain trans-2-decenoic acid and 10-HDA content; Saccharomyces cerevisiae engineered strain BY4741 / pESC-URA-CYP153A33-CPR BM3 -crp5p strain trans-2-decenoic acid and 10-HDA content.

[0034] FIG. 7 is an engineered yeast strain BY4741 / pESC-URA-CYP153A33-CPR in Example 1 of the present invention. BM3 -sil1p and Saccharomyces cerevisiae engineered strain BY4741 / pESC-URA-CYP539A7-F0CPR-sil1p production line graph at different times.

[0035] FIG8 is a diagram showing the regulation of ceramide and fatty acid expression in a 3D epidermal model by royal jelly acid in Example 2 of the present invention.

[0036] FIG9 is a graph showing the detection results of royal jelly acid on DNA damage products 8-hydroxydeoxyguanosine and γ-H2AX in fibroblasts in Example 3 of the present invention.

[0037] FIG10 is a graph showing the immunofluorescence staining results of royal jelly acid on the DNA damage product γ-H2AX in fibroblasts in Example 3 of the present invention.

[0038] Figure 11 is a graph showing the detection results of royal jelly acid on mitochondrial function-related indicators in fibroblasts in Example 4 of the present invention; wherein, the mitochondrial function-related indicators are the average value of the relative total optical density (IOD) of ROS in (a); the mitochondrial ATP content in (b); the early cell apoptosis rate in (c); and the NADPH content in (d).

[0039] FIG12 is a graph showing the staining results of royal jelly acid on active oxygen in fibroblasts in Example 4 of the present invention.

[0040] FIG13 is a diagram showing the effect of royal jelly acid on telomere length of fibroblasts in Example 5 of the present invention.

[0041] FIG14 is a diagram showing changes in the red zone of the subjects using the skin care lotion on the 14th and 28th days in Example 6 of the present invention.

[0042] FIG15 is a graph showing wrinkle changes of the subjects on days 14 and 28 after using the skin care lotion in Example 6 of the present invention. DETAILED DESCRIPTION

[0043] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0044] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0045] The present invention will now be further described with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. Experimental conditions not specified in the examples are generally based on conventional conditions or those recommended by the reagent company. Reagents and consumables used in the following examples are commercially available unless otherwise specified.

[0046] In a typical embodiment of the present invention, a method for preparing royal jelly acid based on a biosynthetic method is provided, the method comprising:

[0047] will contain at least CYP153A33-CPR BM3 The yeast engineered bacteria with fusion genes use a culture medium containing trans-2-decenoic acid to ferment and produce royal jelly acid (10-hydroxy-2-decenoic acid, 10-HDA).

[0048] The nucleotide sequence of the CYP153A33-CPRBM3 fusion gene is shown in SEQ ID NO. 1. The fusion gene is codon-optimized to make it easier to express in Saccharomyces cerevisiae. BM3 The yeast engineering bacteria with the fusion gene can react with the substrate trans-2-decenoic acid to obtain the royal jelly acid, and the yield thereof can reach 190 mg / L.

[0049] Furthermore, the engineered yeast may also contain an auxiliary gene sil1p and / or an auxiliary gene crp5p.

[0050] The nucleotide sequence of the auxiliary gene sil1p is shown in SEQ ID NO. 2, and the nucleotide sequence of the auxiliary gene crp5p is shown in SEQ ID NO. 3. By introducing the auxiliary genes sil1p and crp5p, the ability of the yeast engineered bacteria to produce royal jelly acid can be further improved.

[0051] In the present invention, the original starting strain of the above-mentioned engineered yeast bacteria is Saccharomyces cerevisiae. Furthermore, the original starting strain can be Saccharomyces cerevisiae BY4741. Saccharomyces cerevisiae BY4741 is a strain auxotrophic for methionine, leucine, histidine, and uracil. It is a commonly used laboratory strain and is widely used in studies such as sodium and potassium ion balance, cellular salt tolerance, absorption of various metal ions, heavy metal toxicity, the effects of various sugars and carbon sources on eukaryotic cell growth, and the absorption and transport of peroxides and superoxides. It is commercially available.

[0052] Specifically, the yeast engineering bacteria are constructed by the following method: BM3 The recombinant expression vector of the fusion gene is introduced into the original starting strain.

[0053] Wherein, the recombinant expression vector is expressed by the above CYP153A33-CPR BM3 The fusion gene is effectively linked to an expression vector, which is any one or more of a viral vector, a plasmid, a phage, a cosmid or an artificial chromosome; in another specific embodiment of the present invention, the expression vector is a plasmid, more specifically, the expression vector can be a pESC-URA plasmid.

[0054] Furthermore, the recombinant expression vector may also be connected to the auxiliary gene sil1p and / or the auxiliary gene crp5p.

[0055] Furthermore, the method for preparing royal jelly acid based on biosynthesis specifically includes:

[0056] S1. The yeast engineering bacteria were inoculated into a uracil-deficient seed culture medium with a pH of 5.0-6.0, and cultured overnight at 25-35°C with shaking. The seed solution was heated to an initial OD 600 The OD value was 0.3-0.5, and the uracil-deficient fermentation medium was added and cultured at 25-35℃ with shaking until the OD value was 600 After the bacterial solution is 1.0-1.2, collect the bacteria;

[0057] S2. Resuspending the bacterial cells collected in step S1 in the uracil-deficient fermentation medium in step S1, then adding trans-2-decenoic acid to the medium, and producing royal jelly acid after fermentation and cultivation.

[0058] Wherein, in said step S1, the uracil-deficient seed culture medium is composed of a basal medium YNB, 1.5%-2.5% glucose by mass concentration, and 1.0-1.5 g / L of a uracil-deficient amino acid mixture;

[0059] The uracil-deficient fermentation medium comprises the following components: a basic medium YNB, 3.5%-4.5% galactose, 2mM 5-aminolevulinic acid (5-ALA), and 1.0-1.5g / L of a uracil-deficient amino acid mixture.

[0060] In step S2, the bacterial cells are collected by centrifuging the bacterial solution at 3500-4000 rpm for 10-15 minutes and collecting the precipitate;

[0061] The fermentation culture conditions are 25-35° C. for 24-72 hours, such as 24, 36, 48, 60, and 72 hours. Studies have shown that the engineered yeast of the present invention can achieve maximum conversion rate and product conversion after reacting with 0.5 g / L trans-2-decenoic acid for 36 hours, thus being more conducive to practical industrial production.

[0062] The concentration of trans-2-decenoic acid added to the culture medium is 0.1-2.0 g / L, further 0.5-1.0 g / L.

[0063] Furthermore, the method may further comprise any one or more steps of separating, purifying and drying the royal jelly acid. Therefore, the royal jelly acid may be a liquid preparation or a solid (such as granular) preparation, which is not specifically limited herein.

[0064] In another embodiment of the present invention, the use of the above method and / or the royal jelly acid prepared by the above method in any one or more of the following is provided:

[0065] (a) increasing the content of ceramide and fatty acids in the epidermis to repair the skin barrier or preparing a product for increasing the content of ceramide and fatty acids in the epidermis to repair the skin barrier;

[0066] (b) repairing DNA damage in dermal fibroblasts or preparing a product for repairing DNA damage in dermal fibroblasts;

[0067] (c) Improving mitochondrial function, enhancing cell function, promoting metabolism or preparing products that improve mitochondrial function, enhance cell function, and promote metabolism;

[0068] (d) delaying telomere shortening caused by cell division and replication, protecting the integrity of chromosome terminal structure, or preparing a product that delays telomere shortening caused by cell division and replication, protecting the integrity of chromosome terminal structure.

[0069] The product can be food, medicine or cosmetics. Further, the cosmetics are skin care products, which can effectively repair the skin and resist aging.

[0070] Of course, the cosmetics may also contain any other ingredients permitted in the cosmetics field, including but not limited to emulsifiers, emollients, moisturizers, and thickeners. Those skilled in the art may select and add these ingredients based on actual circumstances. Accordingly, the specific dosage forms of the cosmetics may include creams, lotions, lotions, gels, powders, aerosols, patches, films, etc., without specific limitation herein.

[0071] The present invention is further explained by the following examples, but is not intended to limit the scope of the present invention. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples are generally carried out under conventional conditions, with the specific conditions being noted. The reagents and drugs used in the examples are all common commercially available products.

[0072] Example 1 Preparation of Royal Jelly Acid by Yeast Engineering

[0073] The CYP153A33 / M228L-CPRBM3 fusion gene was codon-optimized according to the codon preference of Saccharomyces cerevisiae. The optimized nucleotide sequence is shown in SEQ ID NO.1. The nucleotide sequences of the auxiliary protein genes sil1P and cpr5P in Saccharomyces cerevisiae are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. The amplification product results are shown in Figure 1. The target band appeared and the band was single. After full gene synthesis, it was cloned into the Saccharomyces cerevisiae expression vector pESC-URA between the BamHI and NotⅠ restriction sites to obtain the recombinant expression vector pESC-URA-CYP153A33 / M228L-CPRBM3. BM3(Figure 2). They were then co-expressed with the auxiliary protein genes sil1P (SEQ ID NO. 2) and cpr5P (SEQ ID NO. 3) from Saccharomyces cerevisiae. The auxiliary protein genes were cloned into the Saccharomyces cerevisiae expression vector pESC-URA between the ClaⅠ and EcoRI restriction sites to obtain the recombinant expression vectors pESC-URA-CYP153A33 / M228L-CPR. BM3 -sil1P (Figure 3) and pESC-URA-CYP153A33 / M228L-CPR BM3 -cpr5P (Figure 4). The recombinant expression vector was transformed into Escherichia coli DH5α for culture, and colony PCR identification was performed. The identification results are shown in Figure 5. The target band appeared and the band was single, indicating that the colony was a positive clone. DNA sequencing and comparison showed that the recombinant sequence was correct. The recombinant expression plasmids were chemically transformed into Saccharomyces cerevisiae BY4741, and the recombinant transformants were screened in uracil-deficient medium to obtain high-copy recombinant Saccharomyces cerevisiae BY4741-pESC-URA-CYP153A33 / M228L-CPR BM3 , S.cerevisiae BY4741-pESC-URA-CYP153A33 / M228L-CPR BM3 -sil1P, S.cerevisiae BY4741-pESC-URA-CYP153A33 / M228L-CPR BM3 - cpr5P.

[0074] The above-mentioned engineering bacteria produced 10-hydroxy-2-decenoic acid as follows: a single colony of the yeast engineering bacteria was inoculated into a uracil-deficient seed culture medium (basic medium YNB 6.7g / L, uracil-deficient amino acid mixture 1.29g / L, glucose with a mass concentration of 2%), and cultured overnight at 30°C and 200rpm. The seed solution was heated to an initial OD 600 The culture medium was inoculated into a uracil-deficient fermentation medium (basic medium YNB 6.7 g / L, uracil-deficient amino acid mixture 1.29 g / L, 4% galactose, 2 mM 5-ALA) at pH 5.5 at 0.4, and the culture was induced by shaking at 30°C until the bacterial solution OD 600 After the pH value reaches 1.0, the cells are collected; the collected cells are resuspended in the uracil-deficient fermentation medium with the same components as above, and trans-2-decenoic acid is added to a final concentration of 0.5 g / L. After culturing at 30°C for 36 hours, the cells are extracted with ethyl acetate, dried, and tested to obtain 10-hydroxy-2-decenoic acid. The yield and conversion rate of 10-hydroxy-2-decenoic acid produced by the three engineered strains of Saccharomyces cerevisiae are shown in Figure 6. BM3After reacting with the substrate trans-2-decenoic acid, 10-HDA can be obtained with a yield of up to 190 mg / L, constructing CYP153A33-CPR BM3 The fusion gene was co-expressed with the auxiliary protein genes sil1p and crp5p in Saccharomyces cerevisiae, which can effectively increase the production of 10-HDA in yeast engineering bacteria. The auxiliary protein gene sil1p linked to the protein 10-HDA yield can reach 280 mg / L, and the auxiliary protein gene crp5p linked to the fusion enzyme gene can reach 246 mg / L. At the same time, as shown in Figure 7, CYP153A33-CPR BM3 The plasmid-engineered bacteria co-expressing the auxiliary protein gene sil1p in Saccharomyces cerevisiae can achieve the maximum conversion rate and product conversion amount after reacting with 0.5g / L decenoic acid for 36 hours, which is better than the yeast engineered bacteria BY4741 / pESC-URA-CYP539A7*-F0CPR*-sil1p involved in CN202211449654.3.

[0075] Example 2: Effect of Royal Jelly Acid on the Regulation of Ceramides and Fatty Acids in a 3D Epidermal Model

[0076] The models were transferred to a 6-well plate pre-filled with 3.7 mL / well model culture medium and incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. The PC group contained pironic acid WY14643 at a final concentration of 50 μM, and the sample group contained royal jelly acid at a final concentration of 0.0625 mg / ml. After incubation, each model was cut in half. One half was used for protein determination, and the other half was used for lipid extraction and placed in a 6-well plate (models were stored at -80°C). 1 mL of trypsin was added to each well of the 6-well plate, and the plates were incubated in an incubator for 30 minutes. Some water was added to the 6-well plate lid, and the model and nylon membrane were separated with tweezers. The sticky material on the stratum corneum was also peeled off with tweezers. The stratum corneum was dried with paper, and then the stratum corneum was placed in a glass tube. 1 mL of chloroform-methanol mixture (chloroform: methanol = 1:1) was added to each glass tube, and the mixture was ultrasonicated on ice for 30 min. The supernatant was transferred to a sample bottle and blown dry. 160 μL of acetonitrile-isopropanol mixture (acetonitrile: isopropanol = 1:1) was added to the dried sample bottle, and then 20 μL of ceramide C12 internal standard solution (ceramide internal standard solution concentration 100 μg / mL, internal standard solvent is acetonitrile and isopropanol solution in a ratio of 1:1) was added. Ultrasonication for 10 min, shaking to dissolve for 30 min, transferred to a sample centrifuge tube, centrifuged at 12000 rpm for 10 min, and 100 μL of the upper layer was placed in a 250 μL liner tube. The sample was collected using a ThermoFisher Orbitrap QExactive TM LC-MS detection was performed using a quadrupole-orbital ion trap high-resolution mass spectrometer.

[0077] Mass spectrometry conditions for lipid analysis were as follows: Mass spectrometry analysis was performed using a quadrupole-orbitrap mass spectrometer equipped with a thermoelectrospray ion source. The positive and negative ion source voltages were 3.7 kV and 3.5 kV, respectively. The capillary heating temperature was 320°C, the sheath gas pressure was 30 psi, and the auxiliary gas pressure was 10 psi. The volumetric heating evaporation temperature was 300°C. Both the sheath and auxiliary gases were nitrogen, and the collision gas was nitrogen at a pressure of 1.5 mTorr. The primary full scan parameters were: resolution 70,000, and an automatic gain control target of 1 × 10 6 , maximum isolation time 50 ms, mass-to-charge ratio scanning range 50-1500; the LC / MS system was controlled by Xcalibur 2.2SP1.48 software, and data acquisition was also controlled by the software.

[0078] The LC-MS detection parameters are shown in Table 1 below.

[0079] Table 1 LC-MS specific detection parameters

[0080] The elution program of LC-MS is shown in Table 2 below.

[0081] Table 2 LC-MS elution program

[0082] The structural basis for the skin's barrier protection is the "brick-gray" structure of the stratum corneum. The "brick" structure is mainly composed of transmembrane proteins and intramembrane proteins of corneocytes, which are cross-linked with each other under the action of transglutaminase to form a protein membrane sheath. The "gray" is composed of lipid capsules formed by cross-linking extracellular lipids through esterification. The two are further cross-linked through esterification reactions to form a highly sealed brick wall structure, which together resist the external environment and act as a permeability barrier. Ceramides and fatty acids are the main lipids that make up the lipid capsule. Through LC-MS experimental detection, it was found that the results shown in Figure 8 showed that royal jelly acid treatment of the 3D epidermal model of the skin can significantly promote the expression of ceramides and fatty acids, with an increase rate of 47.64% and 39.0% respectively, which can effectively restore the sebum barrier.

[0083] Example 3 Effect of royal jelly acid on damage and repair of fibroblasts

[0084] Fibroblasts were cultured at a rate of 2.2 × 10 5Cells were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate reached 40-60%, 2 mL of culture medium containing the test substance at the corresponding concentration was added to each well according to different groups. BC was the blank control group, which did not receive any drug intervention and was cultured with only 2 ml of normal culture medium; NC was the negative control group, which did not receive any drug intervention and was cultured with only 2 ml of normal culture medium; PC was the positive control group, which contained VE at a final concentration of 7 μg / ml; and the sample group contained royal jelly acid at a final concentration of 0.25 mg / ml. After 24 hours of incubation, the NC, PC, and sample groups were irradiated with UVA at a dose of 30 J / cm 2 After irradiation, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours. After incubation, the cell culture supernatant was collected and tested according to the operating instructions of the 8-hydroxydeoxyguanosine (8-OHDG) kit. At the same time, the cells in the culture plate were fixed with 4% paraformaldehyde for 24 hours, and then immunofluorescence detection was performed using a DNA damage detection kit (γ-H2AX immunofluorescence method). Fluorescence microscopy was used to take pictures and the cells were detected. Image processing software was used for analysis. As shown in Figure 9, experimental testing revealed that ultraviolet irradiation attacks the 8th carbon atom of the guanine base in DNA molecules, leading to an increase in the content of the damage product 8-hydroxydeoxyguanosine (8-OHDG). It also phosphorylates H2AX Ser139, generating the damage product γ-H2AX. Treatment with royal jelly acid inhibited the expression of both DNA damage products, with inhibition rates for 8-OHDG and γ-H2AX of 45.09% and 47.25%, respectively. The immunofluorescence staining results in Figure 10 further confirm the above conclusion, indicating that royal jelly acid treatment can effectively inhibit the expression of the DNA damage product γ-H2AX in fibroblasts.

[0085] Example 4 Effect of Royal Jelly Acid on Mitochondrial Function of Fibroblasts

[0086] Fibroblasts were cultured at a rate of 2.2 × 10 5 Cells were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate reached 40-60%, 2 mL of culture medium containing the test substance at the corresponding concentration was added to each well according to different groups. BC was the blank control group, which did not receive any drug intervention and was cultured with only 2 ml of normal culture medium; NC was the negative control group, which did not receive any drug intervention and was cultured with only 2 ml of normal culture medium; PC was the positive control group, which contained VE at a final concentration of 7 μg / ml; and the sample group contained royal jelly acid at a final concentration of 0.25 mg / ml. After 24 hours of incubation, the NC, PC, and sample groups were irradiated with UVA at a dose of 30 J / cm2 After irradiation, the cells were placed in an incubator (37°C, 5% CO2) and cultured for 24 hours. + Mitochondrial function was detected by using the / NADPH detection kit (WST-8 method) and the JC-1 mitochondrial membrane potential detection kit instructions. As shown in Figure 11, experimental detection found that ultraviolet irradiation significantly increased the ROS content and cell apoptosis rate in fibroblasts, and significantly reduced the ATP and NADPH content in mitochondria. After intervention with 0.25 mg / ml royal jelly acid, the generation of ROS and cell apoptosis caused by ultraviolet irradiation were inhibited, with inhibition rates of 70.71% and 12.74%, respectively. At the same time, the expression of ATP and NADPH was also upregulated, with upregulation rates of 56.23% and 15.13%, respectively. Furthermore, as shown in Figure 12, intervention with 0.25 mg / ml royal jelly acid was able to effectively inhibit the production of reactive oxygen species in fibroblast mitochondria, thereby improving the body's antioxidant capacity.

[0087] Example 5 Effect of Royal Jelly Acid on Telomere Length of Fibroblasts

[0088] Fibroblasts were cultured at a rate of 2.2 × 10 5 Cells were seeded into 6-well plates at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate reached 40-60%, 2 mL of culture medium containing the test substance at the corresponding concentration was added to each well according to different groups. The BC group was a blank control, and the cells were derived from the back skin of 7-8-year-old children. No drug intervention was performed during the culture process. The NC group was a negative control, and the cells were derived from the back skin of 48-year-old adults. When the cell plating rate reached 40-60%, H2O2 was used for stimulation for 3 consecutive days, each stimulation for 2 hours, and then samples were collected for detection after 5 consecutive passages. The PC group was a positive control, and the cells were derived from 4 Cells from the back skin of an 8-year-old adult were stimulated with H2O2 for 2 hours each time for 3 consecutive days when the cell plating rate reached 40-60%. During this period, they were cultured in a medium containing cycloastragenol at a final concentration of 1 mg / ml. After 5 consecutive passages, samples were collected for testing. Cells in the sample group were derived from the back skin of a 48-year-old adult. When the cell plating rate reached 40-60%, they were stimulated with H2O2 for 2 hours each time for 3 consecutive days. During this period, they were cultured in a medium containing royal jelly acid at a final concentration of 0.25 mg / ml. After 5 consecutive passages, samples were collected for testing. Telomere length was detected using TAKARA's RNA extraction kit, RNA reverse transcriptase kit, and TB Green fluorescence quantification kit, respectively. The primer sequences and single-copy gene sequences used are shown in Table 3 below.

[0089] Table 3 Primer sequence information and product length for amplifying telomeres and single copy gene 36B4

[0090] The results are shown in Figure 13. Experimental tests found that the telomere length of aging cells in the NC group was significantly reduced, only 72% of the telomere length of the young BC group. After using cycloastragenol, the telomere length was significantly extended, which was 43.1% higher than that of the NC group. After intervention with royal jelly acid, the telomere length also showed a significant increase, which was 34.6% higher than that of the NC group. This shows that royal jelly acid can delay the shortening of telomeres caused by cell division and replication in the aging process, strengthen the structural protection of chromosome ends, and effectively prevent cells from losing their division activity and aging, thereby preventing the occurrence of skin aging.

[0091] Example 6 Application of Royal Jelly Acid in Efficacy-Based Skin Care Products

[0092] Table 4 Raw material formula of skin care lotion

[0093] Royal jelly acid is added to the skin care matrix emulsion according to the examples and content of Table 4, stirred and mixed at room temperature for 5 minutes, and a skin care emulsion is obtained. The above-mentioned skin care emulsion is preferably for cosmetics with repair and anti-aging effects, with sensitive muscle groups with damaged barriers over 35 years old as the object, and the testers are allowed to use the skin care emulsion continuously for 14 and 28 days, and VISIA is used to take photos of the subjects' faces. The beneficial effects of the skin care emulsion containing royal jelly acid on the skin barrier are evaluated by using the changes in the red zone before and after use, and the beneficial effects of the skin care emulsion containing royal jelly acid on skin aging are evaluated by using the changes in the number of wrinkles before and after use. The results show that after 28 days of using the skin care emulsion containing royal jelly acid, the red zone is significantly improved (Figure 14), and the number and depth of wrinkles are reduced (Figure 15), indicating that royal jelly acid has good repair and anti-aging effects.

[0094] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing royal jelly acid based on biosynthesis, characterized in that: The method comprises: Using a yeast engineered bacterium containing at least a CYP153A33-CPRBM3 fusion gene to ferment and produce royal jelly acid using a medium containing trans-2-decenoic acid; Wherein, the nucleotide sequence of the CYP153A33-CPRBM3 fusion gene is shown as SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The yeast engineering bacteria further contains an auxiliary gene sil1p and / or an auxiliary gene crp5p; Among them, the nucleotide sequence of the auxiliary gene sil1p is shown in SEQ ID NO.2, and the nucleotide sequence of the auxiliary gene crp5p is shown in SEQ ID NO.

3.

3. The method according to claim 1, characterized in that The original starting strain of the yeast engineering bacteria is Saccharomyces cerevisiae BY4741.

4. The method according to claim 1, characterized in that The yeast engineering bacteria is obtained by constructing the yeast engineering bacteria by the following method: BM3 The recombinant expression vector of the fusion gene is introduced into the original starting strain to obtain; The recombinant expression vector is obtained by effectively connecting the above-mentioned CYP153A33-CPRBM3 fusion gene to an expression vector, and the expression vector is a plasmid.

5. The method according to claim 4, characterized in that The recombinant expression vector is also connected to the auxiliary gene sil1p and / or the auxiliary gene crp5p.

6. The method according to any one of claims 1 to 5, characterized in that: The method for preparing royal jelly acid based on biosynthesis specifically comprises: S1. The yeast engineering bacteria were inoculated into a uracil-deficient seed culture medium with a pH of 5.0-6.0, and cultured at 25-35° C. overnight with shaking. The seed solution was grown at an initial OD 600 The uracil-deficient fermentation medium was inoculated and cultured at 25-35°C with shaking until the OD 600 After the bacterial solution reaches 1.0-1.2, collect the bacteria; S2. Resuspending the bacterial cells collected in step S1 in the uracil-deficient fermentation medium in step S1, then adding trans-2-decenoic acid to the medium, and obtaining royal jelly acid after fermentation and cultivation.

7. The method according to claim 6, characterized in that In the step S1, the uracil-deficient seed culture medium is composed of a basic culture medium YNB, a mass concentration of 1.5%-2.5% glucose, and a uracil-deficient amino acid mixture of 1.0-1.5 g / L; The uracil-deficient fermentation medium comprises the following components: a basic medium YNB, galactose with a mass concentration of 3.5%-4.5%, 2mM 5-aminolevulinic acid and 1.0-1.5g / L of a uracil-deficient amino acid mixture.

8. The method according to claim 6, characterized in that In step S2, the bacterial cells are collected by centrifuging the bacterial solution at 3500-4000 rpm for 10-15 min and collecting the precipitate; The fermentation culture conditions are culturing at 25-35°C for 24-72 hours; The concentration of trans-2-decenoic acid added to the culture medium is 0.1-2.0 g / L.

9. Use of the method according to any one of claims 1 to 8 and / or the royal jelly acid obtained according to any one of claims 1 to 8 in any one or more of the following: (a) increasing the content of ceramide and fatty acids in the epidermis, repairing the skin barrier or preparing a product for increasing the content of ceramide and fatty acids in the epidermis, and repairing the skin barrier; (b) repairing DNA damage in dermal fibroblasts or preparing a product for repairing DNA damage in dermal fibroblasts; (c) Improving mitochondrial function, enhancing cell function, promoting metabolism or preparing products that improve mitochondrial function, enhancing cell function, and promoting metabolism; (d) delaying the shortening of telomeres caused by cell division and replication, protecting the integrity of the chromosome terminal structure, or preparing a product that delays the shortening of telomeres caused by cell division and replication, protecting the integrity of the chromosome terminal structure.

10. The use according to claim 9, characterized in that: The product is food, medicine or cosmetics; the cosmetics are skin care products.

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