Preparation method of Keemun camellia seed fermented oil

Abstract

The invention provides a preparation method of Keemun camellia seed fermented oil, and belongs to the technical field of vegetable oil production. The preparation method comprises the following steps: S1, preparing recombinant saccharomycetes; s2, jointly fermenting the antarctic torulopsis, the recombinant saccharomycetes and the Keemun camellia seed oil; s3, separating an oil phase from a water phase, and taking the oil phase; and S4, dehydrating the oil phase to obtain the Keemun camellia seed fermented oil. Preferably, according to the preparation method of the recombinant saccharomycetes in the step S1, phenylalanine deaminase, cinnamate hydroxylase, 4-coumaric acid CoA ligase, chalcone synthetase, chalcone reductase and peroxidase are expressed in saccharomyces cerevisiae. According to the Keemun camellia seed fermented oil prepared by the method disclosed by the invention, the characteristic components of the Keemun camellia seed oil can be remarkably improved, the antioxidant capacity of the Keemun camellia seed oil is enhanced while the skin feeling of the Keemun camellia seed oil is improved, and the application value of the Keemun camellia seed oil in cosmetics is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of vegetable oil production, and particularly to a preparation method of Qimen camellia seed fermented oil from tea leaves. Background Art

[0002] Qimen black tea is produced in Qimen County south of Huangshan Mountain in Anhui Province and is one of the top ten famous teas in China. The tea raw materials are selected from the local medium-leaf and mid-growing tea tree "Zhuyezhong" (also known as Qimenzhong) for production. The red tea has tight and thin strips, a beautiful appearance, rich and fresh aroma. After brewing, the soup color is orange-red and bright, the aroma is long, and the taste is relatively sweet and gentle. Qimen black tea belongs to Gongfu black tea, is a famous tea in Chinese history, and a famous black tea product. It was created by Anhui tea farmers during the Guangxu period, but the earliest historical record can be traced back to the Tea Classic by Lu Yu in the Tang Dynasty. It is produced in Qimen, Dongzhi, Chizhou, Shitai, Yixian in Anhui Province, and Fuliang in Jiangxi Province.

[0003] When Qimen black tea is used in food, because it is rich in vitamins, it can relieve fatigue and soothe the stomach, making people's body metabolism and nutritional structure tend to a normal state and better absorb the nutrients in food. In addition, Qimen black tea also has a wide range of uses in cosmetics. The components such as tea polyphenols, catechins, and theanine contained in Qimen black tea enable Qimen black tea to balance the free radicals in the skin, promote skin metabolism and growth, and thus maintain the good state of the skin. Qimen black tea also has significant antioxidant and anti-aging effects, as well as good skin repair effects.

[0004] In recent years, the skin care concept of "nourishing the skin with oil" has gained popularity. Camellia seed oil contains rich unsaturated fatty acids and components such as tea polyphenols, and has functions such as moisturizing, sun protection and freckle removal, anti-aging, and skin repair and regeneration, so it has been widely used in cosmetics. However, the content of functional active substances in the camellia seed oil prepared by the existing technology is not high, resulting in insufficiently significant skin care effects in aspects such as antioxidant. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the present invention provides a preparation method of Qimen camellia seed fermented oil. The Qimen camellia seed fermented oil prepared by this method can significantly improve the characteristic components of Qimen camellia seed oil, enhance the antioxidant ability of Qimen camellia seed oil while improving its skin feel, and enhance its application value in cosmetics.

[0006] The technical solution of the present invention is as follows: A preparation method of Qimen camellia seed fermented oil, the preparation method comprising the following steps: S1: Prepare a recombinant yeast; S2: Co-ferment Yarrowia antarctica, recombinant yeast and Qimen camellia seed oil; S3: Separate the oil phase from the water phase and take the oil phase; S4: Dehydrating the oil phase to obtain the Qimenshan camellia seed fermented oil.

[0007] Preferably, the method for preparing the recombinant yeast in step S1 is to express phenylalanine deaminase, cinnamate hydroxylase, 4-coumarate CoA ligase, chalcone synthase, chalcone reductase and peroxidase in Saccharomyces cerevisiae.

[0008] More preferably, the protein sequence of the phenylalanine deaminase is shown as SEQ ID No.1, the protein sequence of the cinnamate hydroxylase is shown as SEQ ID No.2, the protein sequence of the 4-coumarate CoA ligase is shown as SEQ ID No.3, the protein sequence of the chalcone synthase is shown as SEQ ID No.4, the protein sequence of the chalcone reductase is shown as SEQ ID No.5, and the protein sequence of the peroxidase is shown as SEQ ID No.6.

[0009] Preferably, the fermentation process in step S2 comprises the following steps: M1: The recombinant yeast and Antarctic Saccharomyces cerevisiae were inoculated into yeast seed culture medium to form seed liquid; M2: Inoculate the seed liquid of the recombinant yeast and Antarctic Saccharomyces cerevisiae into the fermentation medium for pre-fermentation; M3: After pre-fermentation, add Qimenshan camellia oil and phenylalanine to continue fermentation.

[0010] More preferably, the composition of the yeast seed culture medium in step M1 is: 18-22 g / L peptone, 9-12 g / L yeast extract, and 18-23 g / L glucose; the culture conditions are culturing at 28-32° C. for 20-26 h.

[0011] More preferably, the fermentation medium in step M2 comprises: 10-22 g / L rice, 18-23 g / L soybean meal, and 8-11 g / L glucose; and the pre-fermentation condition is culturing at 25-42° C. for 12-144 h.

[0012] Furthermore, the fermentation temperature of the pre-fermentation process in step M2 is preferably 28-32° C., and the fermentation time is preferably 24-48 h.

[0013] More preferably, the inoculation amounts of the seed solutions of the recombinant yeast and Antarctic Saccharomyces cerevisiae in step M2 are 4-6%, respectively.

[0014] More preferably, in step M3, the amount of Qimenshan camellia oil added is 10-900% of the weight of the initial fermentation medium; the amount of phenylalanine added is 10-40 g / L; and the continued fermentation time is 40-60 h.

[0015] Furthermore, in step M3, the amount of Qimenshan camellia oil added is preferably 50-200%, and the amount of phenylalanine added is preferably 15-25 g / L.

[0016] Preferably, the method for separating the oil phase and the water phase in step S3 comprises at least one of static separation, centrifugal separation and membrane separation.

[0017] Preferably, the method for dehydrating the oil phase in step S4 comprises at least one of vacuum dehydration, anhydrous sodium sulfate dehydration, and anhydrous calcium chloride dehydration.

[0018] The beneficial technical effects of the present invention are: 1. The present invention conducts metabolomics analysis on Keemun black tea and several other common black teas to determine the characteristic components of Keemun black tea, and then recombinant yeast is used to produce the characteristic components of Keemun black tea through the recombinant yeast metabolic network. Finally, Antarctic pseudosaccharomyces, recombinant yeast and Keemun mountain camellia oil are co-fermented to significantly increase the content of the characteristic components of Keemun black tea in the obtained Keemun mountain camellia oil.

[0019] 2. The present invention characterizes the differences in characteristic components between Keemun black tea and other common black teas, determines the characteristic components of Keemun black tea, and thus obtains the reason why Keemun black tea has excellent efficacy. The characteristic components of Keemun black tea in the Keemun Mountain Camellia Seed Fermented Oil prepared by the present invention are significantly increased, so that the Keemun Mountain Camellia Seed Oil has excellent efficacy, especially the antioxidant property is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is an overlay of the base peak ion current (BPI) of the QC sample in Example 2; Figure 2 The base peak ion current (BPI) diagram (left) and ion spectrum (Ionmap) diagram (right) of the Dianhong Jinzhen DHJZ sample in Example 2; Figure 3 is the principal component analysis (PCA) graph of different samples in Example 2 (Level 2); Figure 4 Cluster dendrogram (A) and correlation analysis (B) of different samples in Example 2 (Level 2); Figure 5 This is the abundance analysis of different black tea samples at the superclass level in Example 2; Figure 6The classification of all metabolites of Qihong Alpine Golden Needle QHGSJZ and Dianhong Golden Needle DHJZ in Example 2; Figure 7 The classification of all metabolites of Qihong Alpine Golden Needle QHGSJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY in Example 2; Figure 8 The classification of all metabolites of Qihong Alpine Golden Needle Tea QHGSJZ and No. 2 Black Tea 2HHC in Example 2; Fig. 9 The classification of all metabolites of Qihong Alpine Golden Needle Tea QHGSJZ and Fumingyuan Jinjunmei Black Tea FMYJJMHC in Example 2; Fig.10 The classification of all metabolites of Qihong Alpine Golden Needle Tea QHGSJZ and Yuyuehong Black Tea YYHHC in Example 2; Fig.11 The classification of all metabolites of Dianhong Jinzhen DHJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY in Example 2; Fig.12 This is a schematic diagram of the arrangement of each gene in Example 3 in the yeast genome, wherein: PAL: phenylalanine deaminase; C4H: 4-cinnamate hydroxylase; 4CL: 4-coumarate CoA ligase; CHS: chalcone synthetase; CHR: chalcone reductase; PRX: peroxidase; Pgk1, PTEF1, PTDH3, PITR1, PTDH1, PINO1 are gk1, TEF1, TDH3, ITR1, TDH1, INO1 promoters, respectively; TCYC1 is CYC1 terminator; Fig.13 The content change of dihydroxyaurone during the fermentation process in Example 4; Fig.14 This is a comparison chart of dihydroxyaurone content in Qimenshan tea seed fermented oil and Qimenshan tea seed crude oil in Test Example 1; Fig.15 This is a comparison chart of total flavonoids content between Qimenshan tea seed fermented oil and Qimenshan tea seed crude oil in Test Example 2; Fig.16 This is a comparison chart of the antioxidant capacity of Qimen Mountain Camellia Seed Fermented Oil and Qimen Mountain Camellia Seed Crude Oil in Test Example 2. DETAILED DESCRIPTION

[0021] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Analysis method of characteristic components of Keemun black tea In order to obtain the characteristic components of Keemun black tea, ultra performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS) was used to analyze the components of Keemun black tea, including Qihong High Mountain Golden Needle (QHGSJZ), Dianhong Golden Needle (DHJZ), Ninghong Gongfu Black Tea Chuyun (NHGFHCCY), No. 2 Black Tea (2HHC), Fumingyuan Jinjunmei Black Tea (FMYJJMHC) and Yuyuehong Black Tea (YYHHC).

[0023] The specific method is: Waters Vion-IMS system is used for non-targeted metabolomics acquisition, including ultra-high performance liquid chromatography Acquity UPLC I-Class Plus and high-resolution ion mobility-time of flight Vion-IMS QTof mass spectrometry. UNIFI (v 1.9) is used for data acquisition.

[0024] Chromatographic conditions were: Waters ACQUITY UPLC BEH C18 1.7 µm, 2.1 mm*100 mm; mobile phase A: 98% H2O, 2% ACN, 0.1% FA; mobile phase B: 98% ACN, 2% H2O, 0.1% FA. Flow rate 0.4 mL / min, column temperature 40°C. A 1.5 min equilibrium time was inserted between each two gradient injections. The gradient elution settings are shown in Table 1 below, and the reagents used are shown in Table 2.

[0025] Table 1. Gradient elution table

[0026] Table 2. Test reagents

[0027] The mass spectrometry conditions were as follows: acquisition and analysis mode: Sensitivity; capillary voltage: ESI+ 2.5 kV, ESI- 2 kV; ion source temperature 120 °C; desolvation temperature 500 °C; desolvation gas flow 800 L / h; reverse cone gas flow 50 L / h.

[0028] Data acquisition was performed in data-independent MSE mode with a mass range of 50-1000 m / z, a collection time of 0.3 s per cycle, a fragmentation energy of 10 eV-45 eV, and acquisition in positive and negative ion modes. During the acquisition process, leucine-enkephalin (556.2766 m / z in positive ion mode and 554.2620 m / z in negative ion mode) was injected as an online mass calibrant to maintain mass accuracy.

[0029] The data collection method was as follows: Waters Progenesis QI (v 3.0.3.0) was used to deconvolute the LC-MS raw data. The QC file was selected as a reference for peak extraction, alignment, and area normalization. Metlin2019; NIST; plant self-built databases were used for compound qualitative comparison, with isotope similarity >80%, parent ion mass deviation set to 10 ppm, and daughter ion mass deviation set to 20 ppm. In positive ion mode, the summed ions were set to [M + H]+, [M + Na ]+, and in negative ion mode, the summed ions were set to [MH ]-, [M + FA−H]-.

[0030] For the search results, compounds with a total score (Score) greater than 40 and a product ion matching value (FragmentationScore) greater than 20 are considered Level 2 reliable qualitative compounds that match both the primary parent ion spectrum and the secondary fragmentation spectrum. Compounds with a product ion matching score less than 20 but a total score greater than 36 are considered Level 3 qualitative compounds that can be matched by the primary parent ion spectrum.

[0031] Data analysis methods: MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca / ) was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to depict the overall differences between samples and explore differential metabolites (metabolites with VIP value ≥ 1.0 and P value ≤ 0.05 were considered differential metabolites). In addition, WekemoBioincloud (https: / / www.bioincloud. tech) was used to complete hierarchical cluster analysis (HCA). Enriched pathways were analyzed using MetPA (www. metaboanalyst.ca). The results are expressed as the standard ± deviation of the mean. Analysis of variance (ANOVA) was used to study the differences between treatments and samples. Statistical analysis was performed using the R software package (http: / / www.R-project.org).

[0032] Example 2: Detection of characteristic components of Keemun black tea and result processing (1) QC repeatability and sample spectra: Taking the positive ion mode as an example, 4 QC samples were collected and the base peak ion current (BPI) overlay was visualized. The QC repeatability was good, indicating that the results were reliable (e.g. Figure 1 as shown).

[0033] Taking the positive ion mode and the Dianhong Jinzhen DHJZ sample as an example, the BPI graph shows that the sample has a good detection peak shape and a large peak capacity. The 3D mass spectrometry ion map (Ion map) shows that the sample's ion distribution and retention time distribution are uniform (such as Figure 2 as shown).

[0034] (2) Database search result statistics: In this analysis, 21,725 ​​ions were collected in the positive ion mode and 17,047 ions were collected in the negative ion mode. After comparison and manual screening, at Level 2, the number of reliably identified compounds was 1,319 in the positive ion mode and 625 in the negative ion mode. At Level 3, the number of reliably identified compounds was 1,819 in the positive ion mode and 1,181 in the negative ion mode.

[0035] After deleting ions with missing values ​​> 80%, normalizing the total area, transforming the data to log2 (x+1), filling missing values ​​with a minimum value of 50%, and deleting ions with QC CV > 30%, a total of 1241 Level 2 compounds and 1663 Level 3 compounds had reliable quantitative information in the positive ion mode; a total of 581 Level 2 compounds and 1027 Level 3 compounds had reliable quantitative information in the negative ion mode. Therefore, the positive ion mode was selected for subsequent analysis.

[0036] (3) PCA analysis: PCA is an unsupervised analysis, mainly used to distinguish key differences between samples and observe the degree of aggregation and dispersion of samples. The closer the distance, the more similar the metabolite composition, and the farther the distance, the greater the difference in the metabolome. Figure 3 As shown in the figure, the horizontal and vertical coordinates PC1 and PC2 represent the degree of model fitting respectively. It can be seen that the distances between different black tea samples are relatively large, PC1 accounts for 34.9% of the total variation, and PC2 accounts for 27.8% of the total variation, indicating that the metabolite composition of different black teas is quite different.

[0037] (4) Cluster analysis and correlation analysis of different black tea samples: Cluster dendrogram and correlation analysis of different samples are shown in Figure 2. Figure 4 Cluster analysis and correlation analysis both reflect the similarity of metabolomes in different samples. The closer the metabolomes of different black tea samples are, the closer the clusters are, and the higher the correlation coefficient is.

[0038] The results showed that the metabolic compositions of Qihong high mountain golden needle QHGSJZ were similar to those of Yuyue black tea YYHHC (correlation coefficient 0.92), No. 2 black tea 2HHC (correlation coefficient 0.84), Ninghong Gongfu black tea Chuyun NHGFHCCY (correlation coefficient 0.82), Dianhong golden needle DHJZ (correlation coefficient 0.75), and Fumingyuan Jinjunmei black tea FMYJJMHC (correlation coefficient 0.70).

[0039] (5) Abundance analysis of different black tea samples at superclass level: Abundance analysis of different black tea samples at superclass level Figure 5 As shown in the figure, the two largest categories of all black tea samples are Lipids and lipid-like molecules and Phenylpropanoids and polyketides. Since most metabolites do not have accurate Chinese names, in order to avoid ambiguity, the official English names are used below.

[0040] For the classification of lipids and lipid-like molecules, the content is from high to low: No. 2 black tea 2HHC> Hong Gongfu black tea Chuyun NHGFHCCY> Yuyue black tea YYHHC> Qihong high mountain golden needle QHGSJZ> Fumingyuan Jinjunmei black tea FMYJJMHC> Dianhong golden needle DHJZ; For the classification of Phenylpropanoids and polyketides, the content from high to low is: Dianhong Jinzhen DHJZ> Qihong Gaoshan Jinzhen QHGSJZ> Yuyuehong black tea YYHHC> Fumingyuan Jinjunmei black tea FMYJJMHC> Hong Gongfu black tea Chuyun NHGFHCCY> No. 2 black tea 2HHC.

[0041] Lipids and lipid-like molecules are primary metabolites, which are mainly related to the metabolism of cells to maintain life activities; Phenylpropanoids and polyketides are secondary metabolites, and the literature reports that these secondary metabolites are often closely related to their efficacy. Therefore, for different black teas, the differences in Phenylpropanoids and polyketides are analyzed.

[0042] (6) Metabolome differences analysis of different black tea samples: ① Qihong Alpine Golden Needle QHGSJZ and Dianhong Golden Needle DHJZ As shown in Figure 6 and Table 3, under the category of Phenylpropanoids and polyketides, compared with Dianhong Jinzhen DHJZ, Qihong Gaoshan Jinzhen QHGSJZ had 10 compounds that increased significantly, namely biochanin a 7-(6-methylmalonylglucoside), axillarin, petunidin, cyanidin-rhamnoside, 1,3-diferuloylglycerol, isorhamnetin 3-(2-apiosylrutinoside), and 1,3-diferuloylglycerol. The main compounds with significantly decreased levels in Qihong Alpine Golden Needle QHGSJZ were 11 compounds, and the 10 compounds with the largest increase were adenocarpine, penduletin, kaempferol-coumaroylrutinoside, afzelin, tricetin, isoscoparin-glucoside, mauritianin, formononetin 7-(6''-malonylglucoside), and 10-hydroxy-1,1-dihydroxy-1-dihydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2-hydroxy-1-dihydroxy-2-hydroxy-2- Malonyl glucoside), 3'-prenylnaringenin (3'-isopentenylnaringenin), hydroxydaidzein-diglucoside (hydroxydaidzein-diglucoside).

[0043] Table 3. Differential metabolites of Qihong alpine golden needle QHGSJZ and Dianhong golden needle DHJZ under phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0044] ② Qihong Alpine Golden Needle Tea QHGSJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY As shown in Figure 7 and Table 4, under the category of Phenylpropanoids and polyketides, compared with Ninghong Gongfu Black Tea Chuyun NHGFHCCY, Qihong High Mountain Golden Needle QHGSJZ has 21 compounds that have increased significantly, and the 10 compounds with the largest increase are tri-o-galloyl-beta-glucose, cetraric acid, hellicoside, petunidin, herniarin, dihydroxyaurone, apiforol, dihydroxy-dimethoxycoumarin, epiafzelechin-(4beta->6)-epicatechin 3,3'-digallate, and 1,3-tetrachlorobenzene. The contents of 13 compounds decreased significantly in Qihong Alpine Golden Needle QHGSJZ, and the 10 compounds with the largest decrease were penduletin, santin, pseudobaptigenin, 6,8-diprenylnaringenin, tricetin, isoscoparin-glucoside, pelargonidin-glucoside, kaempferol-coumaroylrutinoside, dihydroxy-6,7-methylenedioxyflavanone, and limocitrin 3-rhamnoside.

[0045] Table 4. Differential metabolites of Qihong Alpine Golden Needle QHGSJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY under Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0046] ③ Qihong Alpine Golden Needle Tea QHGSJZ and No. 2 Black Tea 2HHC As shown in Figure 8 and Table 5, under the category of Phenylpropanoids and polyketides, compared with No. 2 black tea 2HHC, there are 26 compounds that have increased significantly in Qihong High Mountain Golden Needle QHGSJZ, and the 10 compounds with the largest increase are isorhamnetin 3-(6''-malonylglucoside), leucodelphinidin 3-[galactosyl-(1->4)-glucoside], kaempferide-rhamnoside, demethylsuberosin, quercetin 3-o-xylosyl-glucuronide, The main compounds that decreased significantly in Qihong Alpine Golden Needle QHGSJZ were penduletin, santin, pseudobaptigenin, adenocarpine, 6,8-diprenylnaringenin, delphinidin 3-sambubioside, isoscoparin-glucoside, and 1,2-dihydroxy-6-methoxyflavone 5-rhamnoside. Glucoside), pelargonidin-glucoside (pelargonidin-glucoside).

[0047] Table 5. Differential metabolites of Qihong Alpine Golden Needle QHGSJZ and No. 2 black tea 2HHC under Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0048] ④ Qihong Alpine Golden Needle Tea QHGSJZ and Fumingyuan Jinjunmei Black Tea FMYJJMHC As shown in Figure 9 and Table 6, under the category of Phenylpropanoids and polyketides, compared with Fumingyuan Jinjunmei Black Tea FMYJJMHC, Qihong Alpine Golden Needle QHGSJZ had 28 compounds that increased significantly, and the 10 compounds with the largest increase were leucodelphinidin 3-[galactosyl-(1->4)-glucoside], kaempferide-rhamnoside, apiforol, cetraric acid, biochanin a 7-(6-methylmalonylglucoside), trihydroxy-dimethoxyisoflavone, and 1,2-dimethoxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1,3-dihydroxy-1 The main compounds that decreased significantly in Qihong Alpine Golden Needle QHGSJZ were adenocarpine, 3-demethylsimmondsin 2'-ferulate, penduletin, santin, tri-o-galloyl-beta-glucose, petunidin, epiafzelechin-(4beta->6)-epicatechin 3,3'-digallate; the main compounds that decreased significantly in Qihong Alpine Golden Needle QHGSJZ were adenocarpine, 3-demethylsimmondsin 2'-ferulate, penduletin, santin, tricetin, isoscoparin-glucoside, and cyanidin-rhamnoside.

[0049] Table 6. Differential metabolites of Qihong Alpine Golden Needle Tea QHGSJZ and Fumingyuan Jinjunmei Black Tea FMYJJMHC under Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0050] ⑤ Qihong Alpine Golden Needle Tea QHGSJZ and Yuyuehong Black Tea YYHHC As shown in Figure 10 and Table 7, under the category of Phenylpropanoids and polyketides, compared with Yuyuehong black tea YYHHC, Qihong high mountain golden needle QHGSJZ had 25 compounds that increased significantly. The 10 compounds with the largest increase were demethylsuberosin, kaempferide-rhamnoside, leucodelphinidin 3-[galactosyl-(1->4)-glucoside], cyanidin 3-xyloside, genistin, epigallocatechin 3-cinnamic acid, and epigallocatechin 3-glucoside. The contents of 14 compounds in Qihong Alpine Golden Needle QHGSJZ decreased significantly, and the 10 compounds with the largest decrease were penduletin, santin, tricetin, cyclokievitonehydrate, isoscoparin-glucoside, pelargonidin-glucoside, isovitexin-glucoside, 6-hydroxyluteolin, formononetin, and safflower oil. 7-(6''-malonylglucoside) (7-(6''-malonylglucoside)), dihydroxy-6,7-methylenedioxyflavanone (dihydroxy-6,7-methylenedioxyflavanone).

[0051] Table 7. Differential metabolites of Qihong Alpine Golden Needle Tea QHGSJZ and Yuyuehong Black Tea YYHHC under Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0052] ⑥ Dianhong Golden Needle DHJZ and Ninghong Gongfu Black Tea NHGFHCCY As shown in Figure 11 and Table 8, under the category of Phenylpropanoids and polyketides, compared with Ninghong Gongfu Black Tea Chuyun NHGFHCCY, Dianhong Jinzhen DHJZ had 10 compounds with significantly increased contents, namely afzelin, adenocarpine, mauritianin, tri-o-galloyl-beta-glucose, herniarin, formononetin 7-(6''-malonylglucoside), cetraric acid, kaempferol-coumaroylrutinoside, kaempferide-rhamnoside and kaempferol methyl ether- rhamnoside), hellicoside; the main 9 compounds that decreased significantly in Dianhong Jinzhen DHJZ were biochanin a 7-(6-methylmalonylglucoside), 6,8-diprenylnaringenin, pseudobaptigenin, petunidin, isorhamnetin 3-(2g-apiosylrutinoside), 1,3-diferuloylglycerol, hydroxy-dimethoxyflavan, fisetin, and hydroxycoumarin.

[0053] Table 8. Differential metabolites of Dianhong Jinzhen DHJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY under Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0054] A comprehensive comparison of the above-mentioned metabolome differences showed that in the Phenylpropanoids and polyketides superclass, dihydroxyaurone was the characteristic metabolite of Qihonghong High Mountain Golden Needle QHGSJZ, and its content was significantly higher than that of the other five types of black tea.

[0055] Example 3: Construction of recombinant yeast Based on the analysis results of Example 2, the present invention constructs a recombinant yeast that produces dihydroxyaurone and is used for the production of Qimenshan camellia seed fermented oil. The construction steps of the recombinant yeast are as follows: Phenylalanine deaminase, cinnamate hydroxylase, 4-coumarate CoA ligase, chalcone synthase, chalcone reductase and peroxidase were codon optimized and gene synthesis was performed by Shanghai Bioengineering. In order to integrate the above genes into the Saccharomyces cerevisiae genome, rDNA was selected as the multi-copy integration site. The target gene was inserted between the intact non-transcribed spacer regions NTS1 and NTS2. The arrangement of the genes finally integrated into the genome is shown in the figure. Fig.12 The protein sequences of the above six enzymes are shown in SEQ ID No.1-SEQ ID No.6 respectively.

[0056] The CRISPR-Cas9 gene editing method was used to integrate these genes into the yeast genome. The specific steps are as follows: (1) First, the Cas9 expression cassette was integrated into Saccharomyces cerevisiae, and the Cas9 protein was expressed through the constitutive promoter TEF1. Saccharomyces cerevisiae was purchased from Shanghai Weidi Biotechnology Co., Ltd.; (2) Construction of fragments for homologous recombination: Since the fragment is too long and difficult to construct in one go, the above fragment is divided into three segments, and the adjacent fragments contain about 1000 bp homologous arms for homologous recombination and insertion into the genome. Fragment I: NTS2-Pgk1-PAL-PTEF1; Fragment II: PTEF1-C4H-PTDH3-4CL-PITR1; Fragment III: PITR1-CHS-PTDH1-CHR-PINO1-PRX-TCYC1-NTS1. The construction of the three fragments was completed by Overlap PCR.

[0057] (3) Construction of gRNA plasmid for gene editing. The gRNA plasmid was constructed according 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 Saccharomycescerevisiae[J]. FEMS yeast research, 2015, 15(2): fov004.), and the URA marker was selected as the screening marker.

[0058] (4) Yeast transformation: The three homologous recombination fragments constructed in step (2) and the gRNA plasmid constructed in step (3) were transferred into yeast competent cells, and positive clones were screened using SC-URA plates. SC-URA plate culture medium was purchased from Shanghai Weidi Biotechnology. The obtained positive clones were verified by colony PCR.

[0059] gRNA plasmid deletion: The correct cloned strain obtained in step (4) was cultured overnight in YPD liquid culture to lose the gRNA plasmid, and screened using SC+FOA plates to obtain recombinant yeast. The YPD liquid culture medium consists of 20 g / L peptone, 10 g / L yeast powder, and 20 g glucose. The SC+FOA plate culture medium consists of 6.7 g / L YNB, 0.77 g / L CSM culture medium, and 0.8 g / L FOA. YNB culture medium was purchased from Solebao Biotechnology, and CSM culture medium was purchased from Hepeng Biotechnology.

[0060] Example 4: Production of dihydroxyaurone by recombinant yeast fermentation (1) Seed liquid culture: The recombinant yeast constructed in Example 3 was inoculated into a yeast seed culture medium containing 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, and cultured at 30°C for 24 h.

[0061] (2) Fermentation: The recombinant yeast was cultured in a 1 L bioreactor to produce dihydro-xyaurone. The initial medium volume was 0.25 L. The initial medium composition was 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / LMgSO4·7H2O, 20 g / L glucose, 2 mL / L trace element solution, and 2 mL / L vitamin mixture.

[0062] The composition of the trace element solution is: 3.0 g / L FeSO4·7H2O, 4.5 g / L ZnSO4·7H2O, 4.5 g / L CaCl2·2H2O, 0.84 g / L MnCl2·2H2O, 0.3 g / L CoCl2·6H2O, 0.3 g / L CuSO4·5H2O, 0.4 g / L Na2MoO4·2H2O, 1.0 g / L H3BO3, 0.1 g / L KI, 19.0 g / L Na2EDTA·2H2O.

[0063] The composition of the vitamin mixture is: 0.05 g / L D-biotin, 1.0 g / L D-pantothenic acid hemi-calcium salt, 1.0 g / L thiamine-hydrochloride, 1.0 g / L pyridoxine-hydrochloride, 1.0 g / L niacin, 0.2 g / L 4-aminobenzoic acid and 25.0 g / L inositol.

[0064] The fermentation temperature was controlled at 30°C, the initial stirring was set at 800 rpm / min, and the stirring rate was gradually increased to a maximum of 1200 rpm / min according to the dissolved oxygen situation. The dissolved oxygen level was maintained above 40% by controlling the stirring rate and ventilation flow rate. The pH was controlled at 5.6 using KOH and hydrochloric acid. 800 g / L glucose was used as feed.

[0065] In order to increase the rate of dihydroxyaurone fermentation by recombinant yeast, phenylalanine was added during the reaction to encourage yeast to convert phenylalanine into dihydroxyaurone. After the reaction, the dihydroxyaurone content was detected according to the method described in the literature (Farag MA, Deavours BE, de Fátima A, et al. Integratedmetabolite and transcript profiling identify a biosynthetic mechanism forhispidol in Medicago truncatula cell cultures[J]. Plant physiology, 2009, 151(3): 1096-1113.). Fig.13 It can be seen that after 96 h of fermentation, the content of dihydroxyaurone reached 2.35 g / L.

[0066] Example 5: Preparation method of Qimenshan camellia seed fermented oil (1) Antarctic Saccharomyces cerevisiae and the recombinant yeast prepared in Example 3 were inoculated into yeast seed culture medium, wherein the yeast seed culture medium contained 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, and cultured at 30°C for 24 h. Antarctic Saccharomyces cerevisiae was purchased from Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0067] (2) The prepared Antarctic pseudoyeast seed liquid and recombinant yeast were transferred to the fermentation medium at an inoculation rate of 5%, respectively, and cultured at 30°C for 12 h. Then, Qimenshan camellia oil and 15 g / L phenylalanine were added at 100% of the initial fermentation medium volume, and the culture was continued for 48 h. The fermentation medium is: 20 g / L rice, 20 g / L soybean powder, and 10 g / L glucose.

[0068] (3) After fermentation, centrifuge at 10,000 r / min for 10 min to separate the oil phase and the water phase, and take the upper oil phase.

[0069] (4) Add anhydrous sodium sulfate and centrifuge at 10,000 r / min for 10 min to dehydrate the oil phase to obtain the finished Qimenshan camellia seed fermented oil.

[0070] Test Example 1: Determination of dihydroxyaurone content in Qimenshan camellia seed fermented oil The content of dihydroxyaurone in the Qimen Mountain tea seed fermented oil and the Qimen Mountain tea seed crude oil prepared in Example 5 was measured respectively. Fig.14 It can be seen that the content of dihydroxyaurone in Qimen Mountain camellia seed fermented oil is significantly increased, which is 93.3 times that of Qimen Mountain camellia seed crude oil.

[0071] Test Example 2: Determination of flavonoid content and antioxidant capacity of Qimenshan camellia seed fermented oil The total flavonoids content of the Qimen Mountain tea seed crude oil and the Qimen Mountain tea seed fermented oil prepared in Example 5 were measured respectively. The results are as follows: Fig.15 As shown in the figure, it can be seen that the total flavonoids content of Qimenshan tea seed fermented oil is 426% higher than that of Qimenshan tea seed crude oil.

[0072] The antioxidant capacity of the Qimen Mountain tea seed crude oil and the Qimen Mountain tea seed fermented oil prepared in Example 5 was determined by the DPPH free radical scavenging ability experiment. The results are as follows: Fig.16 As shown in the figure, the DPPH free radical scavenging rate of Qimen Mountain tea seed fermented oil is 82.2%, while the DPPH free radical scavenging rate of Qimen Mountain tea seed crude oil is 21.6%. The antioxidant capacity of Qimen Mountain tea seed fermented oil is significantly improved.

[0073] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing Qimenshan camellia seed fermented oil, characterized in that: The preparation method comprises the following steps: S1: preparing a recombinant yeast; S2: co-fermenting Antarctic Saccharomyces cerevisiae, recombinant yeast and Qimenshan camellia oil; S3: Separate the oil phase from the water phase and take the oil phase; S4: dehydrating the oil phase to obtain the Qimenshan camellia seed fermented oil; The preparation method of the recombinant yeast in step S1 is to express phenylalanine deaminase, cinnamate hydroxylase, 4-coumarate CoA ligase, chalcone synthase, chalcone reductase and peroxidase in Saccharomyces cerevisiae; The protein sequence of the phenylalanine deaminase is shown in SEQ ID No.1, the protein sequence of the cinnamate hydroxylase is shown in SEQ ID No.2, the protein sequence of the 4-coumarate CoA ligase is shown in SEQ ID No.3, the protein sequence of the chalcone synthase is shown in SEQ ID No.4, the protein sequence of the chalcone reductase is shown in SEQ ID No.5, and the protein sequence of the peroxidase is shown in SEQ ID No.

6.

2. The preparation method according to claim 1, characterized in that: The fermentation process in step S2 comprises the following steps: M1: The recombinant yeast and Antarctic Saccharomyces cerevisiae were inoculated into yeast seed culture medium to form seed liquid; M2: Inoculate the seed liquid of the recombinant yeast and Antarctic Saccharomyces cerevisiae into the fermentation medium for pre-fermentation; M3: After pre-fermentation, add Qimenshan camellia oil and phenylalanine to continue fermentation.

3. The preparation method according to claim 2, characterized in that: The composition of the yeast seed culture medium in step M1 is: 18-22 g / L peptone, 9-12 g / L yeast extract, and 18-23 g / L glucose; the culture conditions are culturing at 28-32° C. for 20-26 h.

4. The preparation method according to claim 2, characterized in that: The fermentation medium composition of step M2 is: 10-22 g / L rice, 18-23 g / L soybean meal, and 8-11 g / L glucose; the pre-fermentation condition is culturing at 25-42° C. for 12-144 h.

5. The preparation method according to claim 2, characterized in that: In step M2, the inoculation amounts of the seed solutions of the recombinant yeast and Antarctic Saccharomyces cerevisiae are 4-6% respectively.

6. The preparation method according to claim 2, characterized in that: In step M3, the amount of Qimenshan camellia oil added is 10-900% of the weight of the initial fermentation medium; the amount of phenylalanine added is 10-40 g / L; and the continued fermentation time is 40-60 h.

7. The preparation method according to claim 1, characterized in that: The method for separating the oil phase and the water phase in step S3 includes at least one of static separation, centrifugal separation, and membrane separation.

8. The preparation method according to claim 1, characterized in that: The method for dehydrating the oil phase in step S4 includes at least one of vacuum dehydration, anhydrous sodium sulfate dehydration, and anhydrous calcium chloride dehydration.

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