Preparation method and application of ganoderma spore fermentation oil

Abstract

The invention relates to a preparation method and application of ganoderma lucidum spore fermentation oil, and the preparation method comprises the following steps: fermenting lactobacillus, recombinant saccharomycetes and ganoderma lucidum spore oil together, and separating an oil phase to obtain the ganoderma lucidum spore fermentation oil, wherein squalene epoxidase, lanosterol synthetase and cytochrome P450 enzyme are expressed in the recombinant saccharomycetes. The ganoderma lucidum spore fermentation oil prepared by the preparation method can improve the skin feeling of ganoderma lucidum spore oil, increases the content of ganoderma lucidum triterpenes in the ganoderma lucidum spore oil, and can be better applied to cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a preparation method and application of Ganoderma lucidum spore fermentation oil. Background Art

[0002] With the enhancement of consumers' health and environmental protection awareness, the requirements for the naturalness and safety of cosmetic ingredients are increasing day by day. Consumers are more inclined to choose products containing natural plant extracts, believing that these ingredients are milder and less irritating to the skin. Ganoderma lucidum, as a traditional natural Chinese medicine, has a long history of use. Its spore oil is regarded as a natural and green cosmetic raw material and has been recognized by the majority of consumers. Ganoderma lucidum spore oil is rich in various active ingredients, such as triterpenoids, unsaturated fatty acids, etc., and has various physiological functions such as immune regulation, anti-tumor, and antioxidant. Among them, Ganoderma triterpenoids are the characteristic components of Ganoderma lucidum spore oil. Ganoderma triterpenoids have various effects such as whitening, anti-inflammatory, antioxidant, and repairing photo-damage, and can achieve functions such as repairing dry skin, enhancing skin elasticity, and reducing skin wrinkles.

[0003] In recent years, fermentation technology based on synthetic biology as the underlying technology has played an important role in the application of cosmetics. For example, CN115381744A reports the application of a Ganoderma lucidum fermentation broth in cosmetics. However, there is no report on the fermentation technology of Ganoderma lucidum spore oil. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a preparation method and application of Ganoderma lucidum spore fermentation oil. The Ganoderma lucidum spore fermentation oil prepared by this preparation method can improve the skin feel of Ganoderma lucidum spore oil, increase the content of Ganoderma triterpenoids in Ganoderma lucidum spore oil, and be better applied to cosmetics.

[0005] In the first aspect of the present invention, a preparation method of Ganoderma lucidum spore fermentation oil is proposed, which includes the following steps:

[0006] Co-ferment Lactobacillus and recombinant yeast with Ganoderma lucidum spore oil, and then separate the oil phase to obtain Ganoderma lucidum spore fermentation oil; wherein, squalene epoxidase, lanosterol synthase, and cytochrome P450 enzyme are expressed in the recombinant yeast.

[0007] In the present invention, through the co-fermentation of recombinant yeast, Lactobacillus and Ganoderma lucidum spore oil, while improving the skin feel of Ganoderma lucidum spore oil, the content of Ganoderma triterpenoids is increased.

[0008] In some embodiments of the present invention, the protein sequence of the squalene epoxidase is as shown in SEQ ID No.1.

[0009] In some embodiments of the present invention, the protein sequence of the lanosterol synthase is as shown in SEQ ID No. 2.

[0010] In some embodiments of the present invention, the cytochrome P450 enzyme includes cytochrome P450 enzyme 1 and cytochrome P450 enzyme 2, wherein the protein sequence of cytochrome P450 enzyme 1 is as shown in SEQ ID No. 3, and the protein sequence of cytochrome P450 enzyme 2 is as shown in SEQ ID No. 4.

[0011] In some embodiments of the present invention, cytochrome P450 reductase is also expressed in the recombinant yeast.

[0012] In some preferred embodiments of the present invention, the protein sequence of the cytochrome P450 reductase is as shown in SEQ ID No. 5.

[0013] In some embodiments of the present invention, the method for constructing the recombinant yeast is to integrate the squalene epoxidase gene, lanosterol synthase gene and cytochrome P450 enzyme gene into the yeast genome by using the CRISPR-Cas9 gene editing method. In the present invention, through the design and transformation of yeast, yeast can be made to produce ganoderic triterpenoids.

[0014] In some preferred embodiments of the present invention, the method for constructing the recombinant yeast specifically includes:

[0015] S1. Integrate the Cas9 expression cassette into the yeast;

[0016] S2. Construct a homologous recombination fragment:

[0017] NTS2-PSED1-ERG1-PTDH2-ERG7-TCYC1-NTS1;

[0018] S3. Construct a gRNA plasmid for gene editing;

[0019] S4. Transfer the constructed homologous recombination fragment and gRNA plasmid into the yeast, and screen for positive clones using an SC-URA plate;

[0020] S5. Culture the positive clones overnight in a YPD liquid medium to lose the gRNA plasmid, and screen using an SC+FOA plate medium to obtain the recombinant yeast;

[0021] Among them, steps S1-S3 can be carried out in any order.

[0022] In the present invention, by modifying the metabolic pathway of yeast, the production of ganoderic triterpenoids can be achieved. Further, the production capacity of ganoderic triterpenoids can be improved by means of strain mutagenesis. Finally, the ganoderma spore fermentation oil obtained by fermentation can improve the feel of the oil while increasing the content of triterpenoids in the ganoderma spore oil.

[0023] In some preferred embodiments of the present invention, the yeast is Saccharomyces cerevisiae.

[0024] In some preferred embodiments of the present invention, the composition of the YPD liquid medium includes 20 g / L peptone, 10 g / L yeast extract, and 20 g glucose.

[0025] In some preferred embodiments of the present invention, the composition of the SC + FOA plate medium includes 6.7 g / L YNB, 0.77 g / L CSM medium, and 0.8 g / L FOA.

[0026] In some embodiments of the present invention, the recombinant yeast further improves the production efficiency of ganoderic triterpenoids in the recombinant yeast by means of mutagenesis. In the present invention, the production capacity of ganoderic triterpenoids is further improved by means of strain mutagenesis.

[0027] In some preferred embodiments of the present invention, the mutagenesis method includes at least one of low-temperature plasma mutagenesis, chemical mutagenesis, and ultraviolet mutagenesis.

[0028] In some embodiments of the present invention, the co-fermentation of Lactobacillus, recombinant yeast, and ganoderma spore oil includes:

[0029] M1. Select the Lactobacillus and the recombinant yeast and culture them into seed liquid;

[0030] M2. Inoculate the seed liquid into the fermentation medium and perform pre-fermentation;

[0031] M3. Add the ganoderma spore oil and continue the fermentation.

[0032] In some preferred embodiments of the present invention, the temperature of the fermentation is 25 - 42 °C, preferably 28 - 32 °C; and / or the time of the fermentation is 12 - 144 h, preferably 24 - 48 h; and / or the addition amount of the ganoderma spore oil is 10% - 90% of the weight of the fermentation medium, preferably 20% - 50%. In the present invention, the fermentation refers to the entire process of co-fermentation, including the entire process of co-fermentation of M1 - M3.

[0033] In some preferred embodiments of the present invention, the composition of the fermentation medium is: 80 g / L glucose, 80 g / L corn steep powder, 1 g / L urea, KH 2 PO4 6 g / L, MgSO 4 1 g / L, NaNO 3 2 g / L, squalene 20 g / L.

[0034] In some embodiments of the present invention, the method for separating the oil phase includes separating the oil phase and the water phase by at least one method among static separation, centrifugal separation, and membrane separation.

[0035] In some embodiments of the present invention, it further includes dehydrating the separated oil phase.

[0036] In some preferred embodiments of the present invention, the dehydration method includes at least one of vacuum dehydration, anhydrous sodium sulfate dehydration, and anhydrous calcium chloride dehydration.

[0037] The second aspect of the present invention provides Ganoderma lucidum spore fermentation oil prepared by the above method.

[0038] The third aspect of the present invention provides the application of the above Ganoderma lucidum spore fermentation oil in cosmetics.

[0039] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0040] The preparation method of the Ganoderma lucidum spore fermentation oil of the present invention can, through the oil fermentation technology, while improving the skin feel of the oil, enhance specific active ingredients in the oil, thereby enhancing the efficacy of the oil; through the fermentation of Ganoderma lucidum spore oil, the enhancement of the content of Ganoderma triterpenoids in Ganoderma lucidum spore oil is beneficial to the application of Ganoderma lucidum spore oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention in combination with the drawings, wherein,

[0042] Figure 1 is a schematic diagram of the arrangement of the squalene epoxidase and lanosterol synthase genes in the yeast genome in Example 1 of the present invention; wherein, ERG1: squalene epoxidase; ERG7: lanosterol synthase.

[0043] Figure 2 is a plasmid map for expressing cytochrome P450 enzyme 1, cytochrome P450 enzyme 2, and cytochrome P450 reductase in Example 1 of the present invention.

[0044] Figure 3 is the determination result of the content of Ganoderma triterpenoids in Example 5 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0046] Example 1 Construction of recombinant yeast

[0047] The squalene epoxidase and lanosterol synthase genes were cloned from the Saccharomyces cerevisiae genome. The amino acid sequences of squalene epoxidase and lanosterol synthase are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. They were further integrated into the Saccharomyces cerevisiae genome, and rDNA was selected as the multi-copy integration site. The target genes were inserted between the complete non-transcribed spacer regions NTS1 and NTS2. The arrangement of each gene finally integrated into the genome is as follows Figure 1 shown. These genes were integrated into the yeast genome by the CRISPR-Cas9 gene editing method. The specific steps are as follows:

[0048] (1) First, the Cas9 expression cassette was integrated into the yeast. The Cas9 protein was expressed by the constitutive promoter TEF1;

[0049] (2) The fragment for homologous recombination was constructed by Overlap PCR. The final fragment composition was: NTS2-PSED1-ERG1-PTDH2-ERG7-TCYC1-NTS1. (The TCYC1 sequence refers to the literature: Rebecca E Zordan, Yuxia Ren, Shih-Jung Pan, Giuseppe Rotondo, Alejandro De Las Joseph Iluore, Brendan P Cormack, Expression Plasmids for Use in Candida glabrata, G3 Genes|Genomes|Genetics, Volume 3, Issue 10, 1 October 2013, Pages 1675–1686; The PSED1 and PTDH2 sequences refer to the literature: Promoter-Library-Based Pathway Optimization for Efficient(2S)-Naringenin Production from p-Coumaric Acid in Saccharomyces cerevisiae)

[0050] (3) Construct the gRNA plasmid for gene editing. Refer to the method described in the literature (Mans R, van Rossum HM, Wijsman M, et al. CRISPR / Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae [J]. FEMS yeast research, 2015, 15(2): fov004.) to construct the gRNA plasmid, and select the URA marker as the screening marker.

[0051] (4) Yeast transformation: Transfer the homologous recombination fragment constructed in step 2 and the gRNA plasmid constructed in step 3 into yeast competent cells, and screen for positive clones using SC-URA plates (without URA). The SC-URA plate medium is purchased from Shanghai Weidi Biotechnology. The obtained positive clones are verified by colony PCR.

[0052] (5) gRNA plasmid deletion: The correct cloned strain obtained in step 4 is cultured overnight in YPD (Yeast Extract Peptone Dextrose) liquid medium to cause the loss of the gRNA plasmid, and screened using SC+FOA plates to obtain recombinant yeast. The composition of the YPD liquid medium is 20 g / L peptone, 10 g / L yeast powder, and 20 g glucose. The composition of the SC+FOA plate medium is 6.7 g / L YNB, 0.77 g / L CSM medium, and 0.8 g / L FOA (5-fluoroorotic acid). The YNB medium is purchased from Solarbio Life Sciences, and the CSM medium is purchased from Hepan Biotechnology.

[0053] The recombinant yeast obtained through the above steps is named Sc01.

[0054] To achieve the production of ganoderic triterpenoids, cytochrome P450 enzyme 1 and cytochrome P450 enzyme 2 are overexpressed in Sc01 by plasmid expression. At the same time, to enhance the activities of cytochrome P450 enzyme 1 and cytochrome P450 enzyme 2, cytochrome P450 reductase shown in SEQ ID No. 5 is overexpressed in Sc01. The plasmid maps for the overexpression of cytochrome P450 enzyme 1, cytochrome P450 enzyme 2, and cytochrome P450 reductase are as Figure 2As shown. The plasmid transformation process uses the standard PEG / LiAc method (reference: Enquist-Newman, M., Faust, A., Bravo, D. et al. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505, 239–243 (2014). https: / / doi.org / 10.1038 / nature12771). The resulting recombinant yeast is named Sc02.

[0055] The sequence information is as follows:

[0056] SEQ ID No.1

[0057] MSAVNVAPELINADNTITYDAIVIGAGVIGPCVATGLARKGKKVLIVERDWAMPDRIVGELMQPGGVRALRSLGMIQSINNIEAYPVTGYTVFFNGEQVDIPYPYKADIPKVEKLKDLVKDGNDKVLEDSTIHIKDYEDDERERGVAFVHGRFLNNLRNITAQEPNVTRVQGNCIEILKDEKNEVVGAKVDIDGRGKVEFKAHLTFICDGIFSRFRKELHPDHVPTVGSSFVGMSLFNAKNPAPMHGHVIFGSDHMPILVYQISPEETRILCAYNSPKVPADIKSWMIKDVQPFIPKSLRPSFDEAVSQGKFRAMPNSYLPARQNDVTGMCVIGDALNMRHPLTGGGMTVGLHDVVLLIKKIGDLDFSDREKVLDELLDYHFERKSYDSVINVLSVALYSLFAADSDNLKALQKGCFKYFQRGGDCVNKPVEFLSGVLPKPLQLTRVFFAVAFYTIYLNMEERGFLGLPMALLEGIMILITAIRVFTPFLFGELIG

[0058] SEQ ID No.2

[0059] MTEFYSDTIGLPKTDPRLWRLRTDELGRESWEYLTPQQAANDPPSTFTQWLLQDPKFPQPHPERNKHSPDFSAFDACHNGASFFKLLQEPDSGIFPCQYKGPMFMTIGYVAVNYIAGIEIPEHERIELIRYIVNTAHPVDGGWGLHSVDKSTVFGTVLNYVILRLLGLPKDHPVCAKARSTLLRLGGAIGSPHWGKIWLSALNLYKWEGVNPAPPETWLLPYSLPMHPGRWWVHTRGVYIPVSYLSLVKFSCPMTPLLEELRNEIYTKPFDKINFSKNRNTVCGVDLYYPHSTTLNIANSLVVFYEKYLRNRFIYSLSKKKVYDLIKTELQNTDSLCIAPVNQAFCALVTLIEEGVDSEAFQRLQYRFKDALFHGPQGMTIMGTNGVQTWDCAFAIQYFFVAGLAERPEFYNTIVSAYKFLCHAQFDTECVPGSYRDKRKGAWGFSTKTQGYTVADCTAEAIKAIIMVKNSPVFSEVHHMISSERLFEGIDVLLNLQNIGSFEYGSFATYEKIKAPLAMETLNPAEVFGNIMVEYPYVECTDSSVLGLTYFHKYFDYRKEEIRTRIRIAIEFIKKSQLPDGSWYGSWGICFTYAGMFALEALHTVGETYENSSTVRKGCDFLVSKQMKDGGWGESMKSSELHSYVDSEKSLVVQTAWALIALLFAEYPNKEVIDRGIDLLKNRQEESGEWKFESVEGVFNHSCAIEYPSYRFLFPIKALGMYSRAYETHTL

[0060] SEQ ID No.3

[0061] MADPVLVLTAIAGAAYVFWLVFHHFIVRSPLDNLPSPPSSLFLGNLPDIIHRQSHIWWRHVSNTCGPATKVTAFFGIKMLYTFDPKAMYSILVKDTELYPKKTAADFTLFIGPGLLFAEGAQHRRQRKWLNPVFSVAQLRDISHVFYGVAYKLEEAIRHRVGTQSQDLDVNGWMARTTLEMLGQAGLGYSFDKFTEDSTDSYGEALKSFFPIINHVPLLNLFVVTLANHIPKWLMRRVLRLAVPFPHVLRLLTISETMQKRSTEIIQEKKTALQKGDKALMHQVGEGKDIMSVLLKSNMNAPTDAERLPDEELLAQMSTFILAGMDTTSNALSRILHLLALHPDVQEKLRHELAEAREVVGNGRDVPYDDLVKLPYLDAVCRETLRLHPPLNLIGRRAAKDTIVPLSAPVRGMDGTLVSEIMLPKDTFVLLGLQACNTNKKLWGEDAYEWKPERWLQPLPPMLEEARVPGVYSNLMSFSGGVRSCIGFKFSQLEMKVLLTTLLPAFSFELTDKPIIWNSSAVSYPTMDKDSTRPEMLLKVRALDC

[0062] SEQ ID No.4

[0063] MEDTQTLLYTLVAAFAVAYIIRWRTNPLNSIPTVGGPSAPILSYLSAINFLFHGRELLTEGYQKFHNSAFKVALIDQWLVVVCGPLLDEYRKRSDDELSFIEGVEDVIRTRYTLGPEWLNDPYHVDIIKEKLMRTLPVVLPDVIDELTYAVPDYIPATEDDWVSMPVMKTMLNVVARVSNRVFVGLPVCRNQKFLDIAIAFTIDVVKDRTIINVFPDSLKSYVAWLTTNVRASIRHAIPHIKPLLDERKAKILENGVGENWPGKPNDMLEWILEQAIPRNSSDESIASRILLVNFAAIHTSSNSITHALFDLAAAPQYLQPLREEIEPIIAAEGWTKAAMGKMWKVDSFLRESQRFNGIGLTSVTRKAMKEVTLSNGTVIPKGALVVAASYPTHHDDAIYENANTFDPFRFSRMREVEGEGMKHQFVNTSPEYISFGHGRHACPGRFFAANELKAILAYIVVNYDLKIAGDGERPPNMYIAANVVPSPKGEILFRKRKVTA

[0064] SEQ ID No.5:

[0065]

[0066] Example 2 Mutagenesis of Recombinant Yeast

[0067] The obtained recombinant bacteria were mutagenized by the method of Atmospheric and Room Temperature Plasma (ARTP). The recombinant yeast obtained in Example 1 was cultured for about 12 h, 5 mL of the bacterial solution was taken, the cells were collected by centrifugation, and washed twice with ice-cold water. Then, the ARTP system was used for mutagenesis. The parameter settings were as follows: (1) the radio frequency power input was set to 100 W, and the helium gas flow rate was set to 10 standard liters per minute (SLM); (2) the distance between the plasma torch nozzle outlet and the glass slide was set to 2 mm; (3) different treatment times were selected, namely 80 s, 100 s, 120 s, 140 s, 160 s, 180 s, and 200 s. After treatment, the glass slide was rinsed with ice-cold water to construct an ARTP mutant library. The mutant strains were transferred to YPD medium, resuscitated for 1 h, and the resuscitated bacterial solution was spread on YPD plate medium and cultured at 30 °C for 24 h. The obtained single colonies were inoculated into YPD medium and cultured at 30 °C for 12 h, and 5 g / L squalene was added to the YPD medium. After culturing for 12 h, the obtained culture solution was centrifuged, and the strain with the highest triterpene content was screened. The recombinant yeast with the highest triterpene content obtained was named Sc03.

[0068] The screening method for triterpene content was as follows: In a 96-well plate, 5 μL of the sample was added, followed by 5 μL of 5% vanillin-glacial acetic acid solution and 32 μL of concentrated sulfuric acid in sequence, gently mixed, and developed at 60 °C in a water bath for 20 min, then taken out. The reaction was terminated by quickly cooling in an ice-water bath for 5 min, 200 μL of glacial acetic acid was added, mixed well, and left standing at room temperature for 10 min. The absorbance was measured at a wavelength of 550 nm. The higher the absorbance value, the higher the triterpene content.

[0069] Example 3 Preparation Method of Ganoderma lucidum Spore Fermentation Oil

[0070] (1) Culture of recombinant yeast seed liquid: The recombinant yeast Sc03 was inoculated into yeast seed medium, and the yeast seed medium was: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L. It was cultured at 30 °C for 24 h.

[0071] (2) Culture of Lactobacillus seed liquid: The Lactobacillus was purchased from Chr. Hansen, and the medium was MRS medium, which was purchased from Jingzhou Yinjie Chemical Co., Ltd. The culture temperature was 37 °C, and the culture time was 12 h.

[0072] (3) Inoculate the recombinant yeast into the fermentation medium. The composition of the fermentation medium is as follows: glucose 80 g / L, corn steep powder 80 g / L, urea 1 g / L, KH 2 PO 4 6 g / L, MgSO 4 1 g / L, NaNO 3 2 g / L, squalene 20 g / L. The fermentation temperature is 30°C, the dissolved oxygen is controlled not lower than 15%, the air flux is 1.5 vvm, and the fermentation time is 72 h.

[0073] (4) After the fermentation is completed, use a ceramic membrane to remove the recombinant yeast cells and collect the membrane-filtered supernatant. Then inoculate the seed liquid of Lactobacillus (purchased from Chr. Hansen) into it. After culturing at 30°C for 4 h, add 30% of Ganoderma spore oil (purchased from Ganzhi Hall) based on the weight of the fermentation medium and continue the fermentation culture for 12 h.

[0074] (5) After the fermentation is completed, perform centrifugal separation and collect the upper oil phase.

[0075] (6) Add 3% anhydrous sodium sulfate to the oil phase, centrifuge to remove the anhydrous sodium sulfate, and obtain the final Ganoderma spore fermentation oil.

[0076] Example 4 Method for determining the content of ganoderic triterpenoids in Ganoderma spore oil

[0077] Refer to NY / T 3676-2020 "Spectrophotometric method for the determination of total triterpenoid content in Ganoderma lucidum" to determine the content of ganoderic triterpenoids in Ganoderma spore oil. The specific method is as follows:

[0078] 1. Reagent preparation:

[0079] 1) 5% vanillin-glacial acetic acid solution: Weigh 5 g of vanillin, add about 70 mL of glacial acetic acid to dissolve it, and then add glacial acetic acid to make the volume up to 100 mL after dissolution.

[0080] 2) Oleanolic acid standard stock solution: Accurately weigh 20 mg of oleanolic acid standard (accurate to 0.1 mg), dissolve it with anhydrous methanol and make the volume up to 100 mL. The mass concentration of oleanolic acid in this standard solution is 200 μg / mL. Store it sealed in a refrigerator at 4°C, and the validity period is 1 month.

[0081] 2. Sample extraction:

[0082] Weigh 0.5 g of the sample (accurate to 0.0001 g) into a 250 mL stoppered conical flask, accurately add 50 mL of anhydrous ethanol, tighten the stopper, shake well, place it in an ultrasonic extractor for ultrasonic extraction for 1 h, shake it frequently during this period, mix it evenly after extraction, take an appropriate volume and centrifuge it at 8000 r / min for 10 min, and take the supernatant as the sample extract for standby.

[0083] 3. Determination:

[0084] Using oleanolic acid as the standard. Take 0.2 mL, 0.4 mL, 0.5 mL, 0.6 mL, and 0.8 mL of the oleanolic acid standard solution at the bottom of test tubes respectively. The masses of the standard products are 40 μg, 80 μg, 100 μg, 120 μg, and 160 μg respectively. Place the test tubes in a water bath at 90°C - 100°C and evaporate to dryness. Take a blank test tube as the blank control, with the mass of oleanolic acid (μg) on the X-axis and the absorbance value on the Y-axis to make a standard curve.

[0085] Pretreatment of the test solution: Take 0.05 mL of the test solution at the bottom of a test tube and evaporate to dryness in a water bath at 90°C - 100°C.

[0086] Detection steps: After the test sample and the oleanolic acid standard solution are evaporated to dryness, add 0.1 mL of 5% vanillin - glacial acetic acid solution and 0.8 mL of perchloric acid in sequence, mix gently, develop color in a water bath at 60°C for 20 min, take out and quickly cool in an ice - water bath for 5 min to terminate the reaction, add 5 mL of glacial acetic acid, mix well, and let stand at room temperature for 10 min. Repeat the above steps for the blank control. Measure the absorbance at a wavelength of 550 nm.

[0087] 4. Data processing:

[0088] The content of total triterpenoids (calculated as oleanolic acid) in the sample is calculated according to formula (1);

[0089] X = (m1 × V1 × f) / (m2 × V2 × 10^6) × 100……………………(1)

[0090] In the formula:

[0091] X: The content of total triterpenoids in the sample, unit is grams per hundred grams (g / 100g);

[0092] m1: The amount of total triterpenoids (calculated as oleanolic acid) in the sample reaction solution obtained from the standard curve, unit is microgram (μg);

[0093] V1: The volume of anhydrous ethanol accurately added during extraction, unit is milliliter (mL);

[0094] f: The dilution factor of the sample solution;

[0095] m2: The mass of the sample, unit is gram (g);

[0096] V2: The volume of the sample extraction solution taken for colorimetric determination, unit is milliliter (mL);

[0097] 10^6: Conversion factor.

[0098] The determination results of total triterpenoids are expressed as the arithmetic mean of two determination results. The number of decimal places retained in the determination results is consistent with the method detection limit, and at most three significant figures are retained.

[0099] Example 5 Content of Ganoderma triterpenoids in Ganoderma spore oil and fermented Ganoderma spore oil

[0100] The contents of Ganoderma triterpenoids in Ganoderma spore oil and fermented Ganoderma spore oil were determined respectively, and the determination results are as Figure 3 shown. The content of Ganoderma triterpenoids in Ganoderma spore oil is 20%, and the content of Ganoderma triterpenoids in fermented Ganoderma spore oil is 26%. From this result, it can be seen that the content of Ganoderma triterpenoids in fermented Ganoderma spore oil has increased by nearly 30% compared with Ganoderma spore oil.

[0101] Example 6 Evaluation of the skin feel of Ganoderma spore oil and fermented Ganoderma spore oil

[0102] Eight trained sensory evaluators, four males and four females, a total of 16, were recruited to evaluate and score the fluidity, absorption ability, freshness, moisturizing property, etc. of the skin touch and smell of Ganoderma spore oil and fermented Ganoderma spore oil, and the average value was taken. Scoring criteria: 10 is the full score; 0 - 2 is poor; 3 - 5 is ordinary; 6 - 8 is good; 9 - 10 is very high. The evaluation results are shown in Table 1. It can be seen from Table 1 below that compared with Ganoderma spore oil, the fluidity and freshness of fermented Ganoderma spore oil have been significantly improved, and the absorption ability has also been slightly improved, and it does not affect its moisturizing property and smell.

[0103] Table 1 Comparison of the skin feel evaluation of Ganoderma spore oil and fermented Ganoderma spore oil

[0104] sample ganoderma spore oil fermented ganoderma spore oil fluidity 5.5 8.2 absorbability 7.9 8.3 refreshing degree 2.3 8.3 moisturizing property 7.8 7.9 odor 8.2 8.1

[0105] Based on the experimental processes and results of the above examples, the present invention has developed a fermented Ganoderma spore oil. Through the oil fermentation technology, while improving the skin feel of the oil, it can enhance specific active ingredients in the oil, thereby enhancing the efficacy of the oil; through the fermentation of Ganoderma spore oil, enhancing the content of Ganoderma triterpenoids in Ganoderma spore oil is beneficial to improving the application of Ganoderma spore oil.

[0106] Obviously, the above examples are only for clear illustration and are not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation modes here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for preparing Ganoderma lucidum spore fermented oil, characterized in that: The following steps are involved: After co-fermentation of lactobacillus, recombinant yeast and ganoderma spore oil, the oil phase is separated to obtain ganoderma spore fermented oil; wherein the recombinant yeast expresses squalene epoxidase, lanosterol synthase and cytochrome P450 enzyme.

2. The preparation method according to claim 1, characterized in that: The protein sequence of the squalene epoxidase is shown in SEQ ID No. 1; and / or, The protein sequence of the lanosterol synthase is shown in SEQ ID No. 2; and / or The cytochrome P450 enzyme includes cytochrome P450 enzyme 1 and cytochrome P450 enzyme 2, wherein the protein sequence of the cytochrome P450 enzyme 1 is shown as SEQ ID No.3, and the protein sequence of the cytochrome P450 enzyme 2 is shown as SEQ ID No.

4.

3. The preparation method according to claim 1, characterized in that: The recombinant yeast also expresses cytochrome P450 reductase; preferably, the protein sequence of the cytochrome P450 reductase is shown as SEQ ID No.

5.

4. The preparation method according to claim 1, characterized in that: The recombinant yeast is constructed by using a CRISPR-Cas9 gene editing method to integrate the squalene epoxidase gene, the lanosterol synthase gene and the cytochrome P450 enzyme gene into the yeast genome; or The construction method of the recombinant yeast specifically includes: S1, integrating the Cas9 expression cassette into yeast; S2, construction of homologous recombination fragment NTS2-PSED1-ERG1-PTDH2-ERG7-TCYC1-NTS1; S3, construction of gRNA plasmid for gene editing; S4, transferring the constructed homologous recombination fragment and gRNA plasmid into yeast, and screening positive clones using SC-URA plate culture medium; S5, culturing the positive clone in YPD liquid medium overnight to lose the gRNA plasmid, and screening using SC+FOA plates to obtain the recombinant yeast; Among them, steps S1-S3 can be performed in any order.

5. The preparation method according to claim 1, characterized in that: The recombinant yeast further improves the production efficiency of Ganoderma triterpenes by mutagenesis; preferably, the mutagenesis method includes at least one of low-temperature plasma mutagenesis, chemical mutagenesis and ultraviolet mutagenesis.

6. The preparation method according to claim 1, characterized in that: The method of fermenting the lactobacillus, the recombinant yeast and the ganoderma spore oil together comprises: M1, selecting the lactobacillus and the recombinant yeast, and culturing them into seed liquid; M2, inoculating the seed liquid into the fermentation medium for pre-fermentation; M3, adding the Ganoderma lucidum spore oil to continue fermentation.

7. The preparation method according to claim 6, characterized in that: The fermentation temperature is 25-42°C, preferably 28-32°C; and / or The fermentation time is 12-144h, preferably 24-48h; and / or The added amount of the Ganoderma lucidum spore oil is 10%-90% by weight of the fermentation medium, preferably 20-50%.

8. The preparation method according to claim 1, characterized in that: The method for separating the oil phase comprises: separating the oil phase and the water phase by at least one of static separation, centrifugal separation and membrane separation; preferably, it also comprises dehydrating the separated oil phase, and the dehydration method comprises at least one of vacuum dehydration, anhydrous sodium sulfate dehydration and anhydrous calcium chloride dehydration.

9. Ganoderma lucidum spore fermented oil obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the Ganoderma lucidum spore fermented oil as claimed in claim 9 in cosmetics.

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