A method for preparing fermented oil from Qimen tea seeds

Abstract

This invention provides a method for preparing fermented oil from Qimen Mountain camellia seeds, belonging to the field of vegetable oil production technology. The preparation method includes the following steps: S1: preparing a recombinant yeast; S2: co-fermenting *Saccharomyces antarctica*, the recombinant yeast, and Qimen Mountain camellia seed oil; S3: separating the oil phase and the aqueous phase, and collecting the oil phase; S4: dehydrating the oil phase to obtain the fermented oil from Qimen Mountain camellia seeds. Preferably, the recombinant yeast in step S1 is prepared by expressing phenylalanine deaminase, cinnamic acid hydroxylase, 4-coumarate-CoA ligase, chalcone synthase, chalcone reductase, and peroxidase in *Saccharomyces cerevisiae*. The fermented oil from Qimen Mountain camellia seeds prepared by the method of this invention can significantly improve the characteristic components of Qimen Mountain camellia seed oil, improve the skin feel of Qimen Mountain camellia seed oil, enhance its antioxidant capacity, and increase its application value in cosmetics.

Description

Technical Field

[0001] This invention relates to the field of vegetable oil production technology, and in particular to a method for preparing fermented oil from Qimen Mountain tea seeds. Background Technology

[0002] Qimen black tea, produced in Qimen County south of Huangshan Mountain in Anhui Province, is one of China's top ten famous teas. The tea leaves are made from the local medium-leaf, medium-growing tea variety "Zhuye" (also known as Qimen variety). It features tightly rolled, beautiful red leaves, a rich and refreshing aroma, and a bright orange-red liquor with a lingering fragrance and a sweet and mellow taste. Qimen black tea belongs to the Gongfu black tea category and is a famous historical and premium black tea in China. While it was created by Anhui tea farmers during the Guangxu era, historical records can be traced back to Lu Yu's *The Classic of Tea* in the Tang Dynasty. It is produced in Qimen, Dongzhi, Chizhou, Shitai, and Yixian counties in Anhui Province, as well as Fuliang in Jiangxi Province.

[0003] When used in food, Keemun black tea, rich in vitamins, can relieve fatigue, soothe the stomach and intestines, and help the body's metabolism and nutritional structure return to a normal state, thus improving the absorption of nutrients from food. In addition, Keemun black tea also has wide applications in cosmetics. The tea polyphenols, catechins, and theanine it contains help balance free radicals in the skin, promote skin metabolism and growth, and maintain healthy skin. Keemun black tea also has significant antioxidant and anti-aging effects, as well as good skin repair properties.

[0004] In recent years, the skincare concept of "nourishing the skin with oil" has gained widespread popularity. Camellia seed oil is rich in unsaturated fatty acids and tea polyphenols, which have moisturizing, sun protection, anti-aging, and skin regeneration effects, thus finding wide application in cosmetics. However, the content of functional active substances in camellia seed oil prepared using current technology is not high, resulting in less significant skincare effects such as anti-oxidation. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a method for preparing fermented camellia seed oil from Qimen Mountain. The fermented camellia seed oil prepared by this method significantly enhances the characteristic components of Qimen Mountain camellia seed oil, improving its skin feel and antioxidant capacity, thus increasing its application value in cosmetics.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing fermented oil from Qimen Mountain tea seeds, the method comprising the following steps:

[0008] S1: Prepare a recombinant yeast strain;

[0009] S2: Ferment Antarctic yeast strains, recombinant yeast strains and Qimen tea seed oil together;

[0010] S3: Separate the oil phase from the aqueous phase and collect the oil phase;

[0011] S4: Dehydrate the oil phase to obtain the fermented oil of Qimenshan tea seeds.

[0012] Preferably, the recombinant yeast in step S1 is prepared by expressing phenylalanine deaminase, cinnamic acid hydroxylase, 4-coumaric acid CoA ligase, chalcone synthase, chalcone reductase and peroxidase in Saccharomyces cerevisiae.

[0013] More preferably, the protein sequence of the phenylalanine deaminase is shown in SEQ ID No. 1, the protein sequence of the cinnamic acid hydroxylase is shown in SEQ ID No. 2, the protein sequence of the 4-coumaric acid-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.

[0014] Preferably, the fermentation process in step S2 includes the following steps:

[0015] M1: Recombinant yeast and Antarctic yeast strain were inoculated into yeast seed culture medium and cultured to form seed liquid;

[0016] M2: The seed culture of recombinant yeast and Antarctic yeast was inoculated into the fermentation medium for pre-fermentation;

[0017] M3: After pre-fermentation, Qimenshan camellia seed oil and phenylalanine are added for further fermentation.

[0018] More preferably, the yeast seed culture medium in step M1 consists of: 18-22 g / L peptone, 9-12 g / L yeast extract, and 18-23 g / L glucose; and is cultured at 28-32°C for 20-26 h.

[0019] More preferably, the fermentation medium in step M2 consists of: 10-22 g / L rice, 18-23 g / L soybean flour, and 8-11 g / L glucose; the pre-fermentation conditions are cultured at 25-42℃ for 12-144 h.

[0020] Furthermore, the fermentation temperature of the pre-fermentation process described in step M2 is preferably 28-32℃, and the fermentation time is preferably 24-48 h.

[0021] More preferably, the inoculation amount of the recombinant yeast and Antarctic yeast seed liquid in step M2 is 4-6%, respectively.

[0022] More preferably, in step M3, the amount of Qimenshan tea seed 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 fermentation time is 40-60 h.

[0023] Furthermore, the preferred amount of Qimenshan camellia seed oil added in step M3 is 50-200%, and the preferred amount of phenylalanine added is 15-25 g / L.

[0024] Preferably, the method for separating the oil phase and the aqueous phase in step S3 includes at least one of static separation, centrifugal separation, and membrane separation.

[0025] Preferably, 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.

[0026] The beneficial technical effects of this invention are as follows:

[0027] 1. This invention identifies the characteristic components of Qimen black tea by conducting metabolomics analysis on Qimen black tea and several other common black teas. Then, through the metabolic network of recombinant yeast, the recombinant yeast obtained can produce the characteristic components of Qimen black tea. Finally, by co-fermenting Antarctic yeast, recombinant yeast and Qimen tea seed oil, the content of the characteristic components of Qimen black tea in the obtained Qimen tea seed oil is significantly increased.

[0028] 2. This invention characterizes the differences in characteristic components between Qimen black tea and other common black teas, identifies the characteristic components of Qimen black tea, and thus explains the reasons for its superior efficacy. The fermented oil of Qimen tea seeds prepared by this invention shows a significant increase in the characteristic components of Qimen black tea, thereby giving the Qimen tea seed oil its superior efficacy, especially its significantly enhanced antioxidant properties. Attached Figure Description

[0029] Figure 1 This is an overlay of the base peak ion chromatogram (BPI) of the QC sample in Example 2;

[0030] Figure 2 The image shows the base peak ion current map (BPI) (left) and ion spectrum (Ionmap) (right) of the Dianhong Jinzhen DHJZ sample in Example 2.

[0031] Figure 3 Principal component analysis (PCA) plots (Level 2) of different samples in Example 2;

[0032] Figure 4Clustering dendrogram (A) and correlation analysis (B) of different samples in Example 2 (Level 2);

[0033] Figure 5 Abundance analysis of different black tea samples at the superclass level in Example 2;

[0034] Figure 6 The classification of all metabolites of Qihong Alpine Golden Needle QHGSJZ and Dianhong Golden Needle DHJZ in Example 2;

[0035] Figure 7 The classification of NHGFHCCY total metabolites of Qihong Gaoshan Jinzhen QHGSJZ and Ninghong Gongfu Black Tea in Example 2;

[0036] Figure 8 The classification of 2HHC metabolites of Qihong Gaoshan Jinzhen QHGSJZ and No. 2 black tea in Example 2;

[0037] Figure 9 The classification of all metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and Fumingyuan Jinjunmei (FMYJJMHC) black tea in Example 2 is shown below.

[0038] Figure 10 The classification of all metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and Yuyuehong Black Tea (YYHHC) in Example 2;

[0039] Figure 11 The classification of NHGFHCCY total metabolites in Dianhong Jinzhen DHJZ and Ninghong Gongfu Black Tea in Example 2;

[0040] Figure 12 This is a schematic diagram of the arrangement of each gene in the yeast genome in Example 3, where,

[0041] PAL: Phenylalanine deaminase; C4H: 4-cinnamic acid hydroxylase; 4CL: 4-coumarate-CoA ligase; CHS: Chalcone synthase; CHR: Chalcone reductase; PRX: Peroxidase; Pgk1, PTEF1, PTDH3, PITR1, PTDH1, and PINO1 are the promoters of gk1, TEF1, TDH3, ITR1, TDH1, and INO1, respectively; TCYC1 is the terminator of CYC1.

[0042] Figure 13 The change in dihydroxyaurone content during fermentation in Example 4;

[0043] Figure 14 A comparison chart of dihydroxyaurone content in fermented oil and crude oil of Qimenshan tea seeds in Test Example 1;

[0044] Figure 15 A comparison chart of the total flavonoid content of fermented Qimenshan tea seed oil and crude Qimenshan tea seed oil in Test Example 2;

[0045] Figure 16 This is a comparison chart of the antioxidant capacity of fermented Qimenshan camellia seed oil and crude Qimenshan camellia seed oil in Test Example 2. Detailed Implementation

[0046] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0047] Example 1: Method for analyzing characteristic components of Qimen black tea

[0048] To obtain the characteristic components of Qimen black tea, ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS) was used to analyze the components of Qimen black tea, including Qimen High Mountain Golden Needle (QHGSJZ), Dianhong Golden Needle (DHJZ), Ninghong Gongfu Black Tea (NHGFHCCY), No. 2 Black Tea (2HHC), Fumingyuan Jinjunmei Black Tea (FMYJJMHC), and Yuyuehong Black Tea (YYHHC).

[0049] The specific method was as follows: Non-target metabolomics data were acquired using the Waters Vion-IMS system, including ultra-high performance liquid chromatography (Acquity UPLC I-Class Plus) and high-resolution ion mobility-time-of-flight (Vion-IMS) QTof mass spectrometry. Data acquisition was performed using UNIFI (v 1.9).

[0050] The chromatographic conditions were as follows: 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. The flow rate was 0.4 mL / min, and the column temperature was 40℃. A 1.5 min equilibration time was inserted between every two gradient elutions. The gradient elution settings are shown in Table 1 below, and the reagents used are shown in Table 2.

[0051] Table 1. Gradient elution table

[0052]

[0053] Table 2. Test Reagents

[0054]

[0055] The mass spectrometry conditions were as follows: acquisition and analysis mode: Sensitivity; capillary voltage: ESI+ 2.5kV, ESI- 2kV; ion source temperature: 120℃; desolvation gas temperature: 500℃; desolvation gas flow rate: 800 L / h; reverse cone gas flow rate: 50 L / h.

[0056] Data acquisition employed a data-independent acquisition mode (MSE), with a mass range of 50-1000 m / z and a cycle acquisition time of 0.3 s. The fragmentation energy ranged from 10 eV to 45 eV, and acquisition was conducted in both positive and negative ion modes. During the acquisition process, leucine-enkephalin (positive ion mode: 556.2766 m / z; negative ion mode: 554.2620 m / z) was simultaneously injected as an online mass calibrator to maintain mass accuracy.

[0057] Data collection methods included: Waters Progenesis QI (v 3.0.3.0) was used to deconvolve the raw LC-MS data. QC files were selected as references for peak extraction, alignment, and area normalization. Qualitative compound identification was performed using Metall2019, NIST, and a self-built plant database, with isotope similarity >80%. The parent ion mass deviation was set to 10 ppm, and the daughter ion mass deviation was set to 20 ppm. In positive ion mode, the added ions were set as [M + H]+ and [M + Na]+, while in negative ion mode, the added ions were set as [MH]- and [M + FA−H]-.

[0058] For the search results, compounds with a total score matching value greater than 40 and a fragmentation score greater than 20 are considered Level 2 reliable qualitative compounds where both the primary parent ion spectrum and the secondary fragmentation spectrum match. Compounds with a fragmentation score less than 20 but a total score greater than 36 are considered Level 3 qualitative compounds where the primary parent ion spectrum can match.

[0059] Data analysis methods included: Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca / ) to characterize overall differences between samples and explore differentially expressed metabolites (metabolites with a VIP value ≥ 1.0 and a p-value ≤ 0.05 were considered differentially expressed metabolites). Hierarchical cluster analysis (HCA) was performed using WekemoBioincloud (https: / / www.bioincloud.tech). Enriched pathways were analyzed using MetPA (www.metaboanalyst.ca). Results are expressed as mean ± standard deviation. Analysis of variance (ANOVA) was used to investigate differences between treatments and samples. Statistical analysis was performed using the R software package (http: / / www.R-project.org).

[0060] Example 2: Detection and Result Processing of Characteristic Components in Qimen Black Tea

[0061] (1) QC repeatability and sample spectra:

[0062] Taking positive ion mode as an example, four QC samples were collected. Under the visualization of the base peak ion chromatogram (BPI) overlay, the QC repeatability was good, indicating that the results are reliable (e.g., Figure 1 (As shown).

[0063] Taking the Dianhong Jinzhen DHJZ sample in positive ion mode as an example, the BPI chromatogram 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 ion distribution and retention time distribution of the sample are uniform (e.g., Figure 2 (As shown).

[0064] (2) Statistics of search results:

[0065] In this analysis, 21,725 ​​ions were collected in positive ion mode and 17,047 ions were collected in negative ion mode. After comparison and manual screening, at Level 2, 1,319 compounds were reliably identified in positive ion mode and 625 compounds were reliably identified in negative ion mode. At Level 3, 1,819 compounds were reliably identified in positive ion mode and 1,181 compounds were reliably identified in negative ion mode.

[0066] After sequentially deleting ions with missing values ​​>80%, normalizing the total area, performing log2(x+1) transformation, filling missing values ​​with the 50% minimum, and deleting ions with QC CV >30%, reliable quantitative information was found for 1241 Level 2 compounds and 1663 Level 3 compounds in the positive ion mode; in the negative ion mode, reliable quantitative information was found for 581 Level 2 compounds and 1027 Level 3 compounds. Therefore, the positive ion mode was selected for subsequent analysis.

[0067] (3) PCA analysis:

[0068] PCA is an unsupervised analysis primarily used to distinguish key differences between samples and observe the degree of sample aggregation and dispersion. The closer the samples are, the more similar their metabolite composition; the farther apart they are, the greater the difference in their metabolome. PCA score plots (such as...) Figure 3 As shown in the figure, the horizontal and vertical axes PC1 and PC2 represent the model fit. It can be seen that the distances between different black tea samples are relatively large. PC1 accounts for 34.9% of the total variance, and PC2 accounts for 27.8% of the total variance, indicating that the metabolite composition of different black teas varies greatly.

[0069] (4) Cluster analysis and correlation analysis of different black tea samples:

[0070] Clustering dendrograms and correlation analysis of different samples, as follows Figure 4 As shown, both cluster analysis and correlation analysis reflect the similarity of metabolomes among different samples. The more similar the metabolomes of different black tea samples, the closer the clusters are, and the higher the correlation coefficient.

[0071] The results showed that the metabolic composition similarity of Qihong Gaoshan Jinzhen (QHGSJZ) was as follows: Yuyuehong 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 Jinzhen (DHJZ) (correlation coefficient 0.75), and Fumingyuan Jinjunmei black tea FMYJJMHC (correlation coefficient 0.70).

[0072] (5) Abundance analysis of different black tea samples at the superclass level:

[0073] Abundance analysis of different black tea samples at the superclass level, as follows: Figure 5As shown, the two largest categories among all black tea samples are Lipids and lipid-like molecules and Phenylpropanoids and polyketides. Since most metabolites do not have accurate Chinese names, the official English names will be used below to avoid ambiguity.

[0074] For the classification of lipids and lipid-like molecules, their contents from largest to smallest are as follows: No. 2 black tea 2HHC > Honggongfu black tea Chuyun NHGFHCCY > Yuyue black tea YYHHC > Qihong Gaoshan Jinzhen QHGSJZ > Fumingyuan Jinjunmei black tea FMYJJMHC > Dianhong Jinzhen DHJZ.

[0075] The classification of Phenylpropanoids and polyketides, from highest to lowest content, is as follows: Dianhong Jinzhen (DHJZ) > Qihong Gaoshan Jinzhen (QHGSJZ) > Yuyuehong (YYHHC) > Fumingyuan Jinjunmei (FMYJJMHC) > Honggongfu (NHGFHCCY) > No. 2 (2HHC)

[0076] Lipids and lipid-like molecules are primary metabolites, mainly involved in cellular metabolism for maintaining life. Phenylpropanoids and polyketides are secondary metabolites, and literature reports that these secondary metabolites are often closely related to their efficacy. Therefore, this study analyzes the differences in phenylpropanoids and polyketides among different types of black tea.

[0077] (6) Analysis of differences in metabolomics among different black tea samples:

[0078] ① Qihong Alpine Golden Needle Tea QHGSJZ and Dianhong Golden Needle Tea DHJZ

[0079] As shown in Figure 6 and Table 3, under the category of Phenylpropanoids and polyketides, compared with Yunnan Golden Needle (DHJZ), Qihong Alpine Golden Needle (QHGSJZ) showed a significant increase in 10 compounds, in descending order of importance: biochanin a 7-(6-methylmalonylglucoside), axillarin, petunidin, cyanidin-rhamnoside, 1,3-diferuloylglycerol, and isorhamnetin 3-(2g-apiosylrutinoside). The compounds that showed a significant decrease in *Apigenin* saccharide rutin, hydroxycoumarin, fisetin, dihydroxyaurone, and 2''-op-coumaroylastragalin were: adenocarpine, penduletin, kaempferol-coumaroylrutinoside, afzelin, tritricetin, isoscoparin-glucoside, mauritianin, and formononetin 7-(6''-malonylglucoside). Malonyl glucoside, 3'-prenylnaringenin, and hydroxydaidzein-diglucoside.

[0080] Table 3. Differential metabolites of Qihong Alpine Golden Needle (QHGSJZ) and Yunnan Golden Needle (DHJZ) under the influence of phenylpropanoids and polyketides (p < 0.05, VIP > 1, FC ≥ 2 or FC ≤ 0.5).

[0081]

[0082] ② Qihong High Mountain Golden Needle (QHGSJZ) and Ninghong Gongfu Black Tea (NHGFHCCY)

[0083] As shown in Figure 7 and Table 4, under the category of Phenylpropanoids and polyketides, compared with Ninghong Gongfu Black Tea (NHGFHCCY), Qihong Gaoshan Jinzhen (QHGSJZ) showed a significant increase in 21 compounds. The 10 compounds with the largest increase were, in descending order: tri-o-galloyl-beta-glucose, cetraric acid, helicoside, petunidin, herniarin, dihydroxyaurone, apiforol, dihydroxy-dimethoxycoumarin, and epiafzelechin-(4β→6)-epicatechin 3,3'-digallate. The main compounds that showed a significant decrease in the levels of digalactoside, afzelin, and Qihong Alpine Golden Needle (QHGSJZ) were 13 compounds. The 10 compounds with the largest decrease were penduletin, santin, pseudoobaptigenin, 6,8-diprenylnaringenin, tricetin, isoscoparin-glucoside, pelargonidin-glucoside, kaempferol-coumaroylrutinoside, dihydroxy-6,7-methylenedioxyflavanone, and limocitrin 3-rhamnoside.

[0084] Table 4. Differential metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and Ninghong Gongfu Black Tea Chuyun (Chuyun) under the influence of NHGFHCCY (p < 0.05, VIP > 1, FC ≥ 2 or FC ≤ 0.5).

[0085]

[0086] ③ Qimen High Mountain Golden Needle Tea QHGSJZ and No. 2 Black Tea 2HHC

[0087] As shown in Figure 8 and Table 5, under the category of Phenylpropanoids and polyketides, compared with No. 2 black tea 2HHC, Qihong Gaoshan Jinzhen QHGSJZ showed a significant increase in 26 compounds. The 10 compounds with the largest increase were, in descending order: isorhamnetin 3-(6''-malonylglucoside), leucodelphinidin 3-[galactosyl-(1→4)-glucoside], kaempferide-rhamnoside, demethylsuberosin, and quercetin 3-o-xylosyl-glucuronide. The compounds that significantly decreased in Qihong Alpine Golden Needle (QHGSJZ) were penduletin, santin, pseudoobaptigenin, adenocarpine, 6,8-diprenylnaringenin, delphinidin 3-sambubioside, and isoscoparin-glucoside. pelargonidin-glucoside.

[0088] Table 5. Differential metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and No. 2 Black Tea (2HHC) under the influence of Phenylpropanoids and polyketides (p < 0.05, VIP > 1, FC ≥ 2 or FC ≤ 0.5).

[0089]

[0090] ④ Qimen High Mountain Golden Needle Tea (QHGSJZ) and Fumingyuan Golden Jun Mei Black Tea (FMYJJMHC)

[0091] As shown in Figure 9 and Table 6, under the category of Phenylpropanoids and polyketides, compared with Fumingyuan Jinjunmei Black Tea FMYJJMHC, Qihong Gaoshan Jinzhen QHGSJZ showed a significant increase in 28 compounds. The 10 compounds with the largest increase were, in descending order: leucodelphinidin 3-[galactosyl-(1→4)-glucoside], kaempferide-rhamnoside, apiforol, cetraricacid, biochanin A 7-(6-methylmalonylglucoside), and trihydroxy-dimethoxyisoflavone. Dimethoxy isoflavones, hispiduloside, tri-o-galloyl-beta-glucose, petunidin, epiafzelechin-(4beta->6)-epicatechin 3,3'-digallate; The seven compounds that significantly decreased in Qihong Alpine Golden Needle (QHGSJZ) were adenocarpine, 3-demethylsimmondsin 2'-ferulate, penduletin, santin, tritricetin, isoscoparin-glucoside, and cyanidin-rhamnoside.

[0092] Table 6. Differential metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and Fumingyuan Jinjunmei Black Tea (FMYJJMHC) under the influence of phenylpropanoids and polyketides (p < 0.05, VIP > 1, FC ≥ 2 or FC ≤ 0.5).

[0093]

[0094] ⑤ Qimen Black Tea (QHGSJZ) and Yuyue Black Tea (YYHHC)

[0095] As shown in Figure 10 and Table 7, under the category of Phenylpropanoids and polyketides, compared with Yu Yue Hong black tea YYHHC, Qi Hong Gao Shan Jin Zhen QHGSJZ showed a significant increase in 25 compounds. The 10 compounds with the largest increase were, in descending order: demethylsuberosin, kaempferide-rhamnoside, leucodelphinidin 3-[galactosyl-(1→4)-glucoside], cyanidin 3-xyloside, genistin, epigallocatechin 3-cinnamic acid. Cinnamon acid), helicoside, hispiduloside, pinostrobin 5-glucoside, kaempferol 3-(di-p-coumarylglucoside); The main compounds that showed a significant decrease in Qihong Alpine Golden Needle (QHGSJZ) were 14 compounds, with the 10 compounds showing the greatest decrease being penduletin, santin, tritricetin, cyclokievitonehydrate, isoscoparin-glucoside, pelargonidin-glucoside, isovitexin-glucoside, 6-hydroxyluteolin, and formononetin. 7-(6''-malonylglucoside), dihydroxy-6,7-methylenedioxyflavanone.

[0096] Table 7. Differential metabolites of Qihong Gaoshan Jinzhen (QHGSJZ) and Yuyuehong Black Tea (YYHHC) under the influence of phenylpropanoids and polyketides (p < 0.05, VIP > 1, FC ≥ 2 or FC ≤ 0.5).

[0097]

[0098] ⑥ Yunnan Golden Needle Black Tea (DHJZ) and Ninghong Gongfu Black Tea (NHGFHCCY)

[0099] As shown in Figure 11 and Table 8, under the category of Phenylpropanoids and polyketides, compared with Ninghong Gongfu Black Tea's initial flavor (NHGFHCCY), Dianhong Jinzhen (DHJZ) showed a significant increase in 10 compounds: afzelin, adenocarpine, mauritianin, tri-o-galloyl-beta-glucose, herniarin, formononetin 7-(6''-malonylglucoside), cetraric acid, kaempferol-coumaroylrutinoside, and kaempferide-rhamnoside. Rhamnose glycoside), helicoside; the main nine compounds that significantly decreased in Yunnan black peony DHJZ were biochanin a 7-(6-methylmalonylglucoside), 6,8-diprenylnaringenin, pseudoobaptigenin, petunidin, isorhamnetin 3-(2g-apiosylrutinoside), 1,3-diferuloylglycerol, hydroxy-dimethoxyflavan, fisetin, and hydroxycoumarin.

[0100] Table 8. Differential metabolites of Yunnan Golden Needle DHJZ and Ninghong Gongfu Black Tea Chuyun NHGFHCCY under the influence of Phenylpropanoids and polyketides (p<0.05, VIP>1, FC≥2 or FC≤0.5)

[0101]

[0102] Based on a comprehensive comparison of the differences in the above metabolomes, dihydroxyaurone is a characteristic metabolite of Qihonghong Gaoshan Jinzhen (QHGSJZ) tea in the superclass of Phenylpropanoids and polyketides, and its content is significantly higher than that of the other five types of black tea leaves.

[0103] Example 3: Construction of recombinant yeast

[0104] Based on the analysis results of Example 2, this invention constructed a recombinant yeast strain that produces dihydroxyaurone and used it for the production of fermented oil from Qimen tea seeds. The construction steps of the recombinant yeast strain are as follows:

[0105] Codon optimization was performed on phenylalanine deaminase, cinnamic acid hydroxylase, 4-coumarate-CoA ligase, chalcone synthase, chalcone reductase, and peroxidase, and the genes were synthesized by Shanghai Sangon Biotech. To integrate these genes into the *Saccharomyces cerevisiae* genome, rDNA was selected as the multicopy integration site. The target gene was inserted between the intact non-transcribed spacers NTS1 and NTS2. The final arrangement of the genes integrated into the genome is shown below. Figure 12 As shown. The protein sequences of the above six enzymes are shown in SEQ ID No. 1-SEQ ID No. 6, respectively.

[0106] These genes were integrated into the yeast genome using CRISPR-Cas9 gene editing. The specific steps are as follows:

[0107] (1) First, the Cas9 expression cassette was integrated into the Saccharomyces cerevisiae. The Cas9 protein was expressed through the constitutive promoter TEF1. The Saccharomyces cerevisiae was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0108] (2) Construction of fragments for homologous recombination: Due to the large length of the fragment, it is difficult to construct it in one go. Therefore, the above fragment was divided into 3 segments, with adjacent fragments containing approximately 1000 bp homologous arms for insertion into the genome via homologous recombination. 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 all three fragments was performed using overlap PCR.

[0109] (3) Construction of gRNA plasmids for gene editing. The gRNA plasmids were 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.

[0110] (4) Yeast transformation: The three homologous recombination fragments constructed in step (2) and the gRNA plasmid constructed in step (3) were transformed into competent yeast cells, and positive clones were screened using SC-URA plates. SC-URA plates were purchased from Shanghai Weidi Biotechnology. The obtained positive clones were verified by colony PCR.

[0111] gRNA plasmid deletion: The correct cloned strain obtained in step (4) was cultured overnight in YPD liquid culture to cause gRNA plasmid loss, and then screened using SC+FOA plates to obtain recombinant yeast. The YPD liquid culture medium consisted of 20 g / L peptone, 10 g / L yeast extract, and 20 g glucose. The SC+FOA plate culture medium consisted of 6.7 g / L YNB, 0.77 g / L CSM medium, and 0.8 g / L FOA. The YNB medium was purchased from Solarbio, and the CSM medium was purchased from Herpon Biotechnology.

[0112] Example 4: Production of dihydroxyaurone by recombinant yeast fermentation

[0113] (1) Seed culture: The recombinant yeast strain 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.

[0114] (2) Fermentation culture: Recombinant yeast was cultured in a 1 L bioreactor to produce dihydro-xyaurone. The initial culture medium volume was 0.25 L. The initial culture medium composition was 7.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 20 g / L glucose, 2 mL / L trace element solution and 2 mL / L vitamin mixture.

[0115] The composition of the trace element solution is as follows: 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, and 19.0 g / L Na2EDTA·2H2O.

[0116] The vitamin mixture consists of: 0.05 g / L D-biotin, 1.0 g / L D-pantothenic acid hemicalcium salt, 1.0 g / L thiamine hydrochloride, 1.0 g / L pyridoxine hydrochloride, 1.0 g / L nicotinic acid, 0.2 g / L 4-aminobenzoic acid, and 25.0 g / L inositol.

[0117] Fermentation temperature was controlled at 30℃, with initial stirring at 800 rpm / min, gradually increasing to a maximum of 1200 rpm / min based on dissolved oxygen levels. Dissolved oxygen levels were maintained above 40% by controlling the stirring rate and aeration flow rate. pH was controlled at 5.6 using KOH and hydrochloric acid. The feed consisted of 800 g / L glucose.

[0118] To increase the rate of dihydroxyaurone production by recombinant yeast fermentation, phenylalanine was added during the reaction to encourage the 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 for hispidol in Medicago truncatula cell cultures[J]. Plant physiology, 2009, 151(3): 1096-1113.). Figure 13 It can be seen that after 96 hours of fermentation, the dihydroxyaurone content reached 2.35 g / L.

[0119] Example 5: Preparation method of fermented oil from Qimen tea seeds

[0120] (1) Antarctic yeast and the recombinant yeast prepared in Example 3 were inoculated into yeast seed culture medium, which consisted of 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose, and cultured at 30°C for 24 h. Antarctic yeast was purchased from Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0121] (2) The prepared Antarctic yeast seed culture and recombinant yeast were transferred to the fermentation medium at an inoculation rate of 5%, and cultured at 30°C for 12 h. Then, Qimenshan tea seed 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 consisted of 20 g / L rice, 20 g / L soybean flour, and 10 g / L glucose.

[0122] (3) After fermentation, centrifuge at 10000 r / min for 10 min to separate the oil phase and the water phase, and take the upper oil phase.

[0123] (4) Add anhydrous sodium sulfate, centrifuge at 10000 r / min for 10 min to dehydrate the oil phase and obtain the finished Qimenshan tea seed fermented oil.

[0124] Test Example 1: Determination of dihydroxyaurone content in fermented oil from Qimen Mountain tea seeds

[0125] The content of dihydroxyaurone in the fermented oil and crude oil of Qimenshan tea seeds prepared in Example 5 was determined respectively. Figure 14 It can be seen that the content of dihydroxyaurone in fermented Qimenshan tea seed oil is significantly increased, reaching 93.3 times that of crude Qimenshan tea seed oil.

[0126] Test Example 2: Determination of Flavonoid Content and Antioxidant Capacity in Fermented Oil from Qimen Mountain Camellia Seeds

[0127] The total flavonoid content of crude Qimenshan tea seed oil and fermented Qimenshan tea seed oil prepared in Example 5 were determined separately, and the results are as follows: Figure 15 As shown in the figure, the total flavonoid content of fermented Qimenshan tea seed oil is 426% higher than that of crude Qimenshan tea seed oil.

[0128] The antioxidant capacity of Qimenshan camellia seed crude oil and the fermented Qimenshan camellia seed oil prepared in Example 5 was determined by the DPPH free radical scavenging capacity experiment. The results are as follows: Figure 16 As shown in the figure, the DPPH free radical scavenging rate of Qimenshan tea seed fermentation oil is 82.2%, while that of Qimenshan tea seed crude oil is 21.6%. The antioxidant capacity of Qimenshan tea seed fermentation oil is significantly improved.

[0129] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, and for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. A method for preparing fermented oil from Qimen tea seeds, characterized in that, The preparation method includes the following steps: S1: Prepare a recombinant yeast strain; S2: Ferment Antarctic yeast strains, recombinant yeast strains and Qimen tea seed oil together; S3: Separate the oil phase from the aqueous phase and collect the oil phase; S4: Dehydrate the oil phase to obtain the fermented oil from Qimenshan tea seeds; The method for preparing the recombinant yeast in step S1 involves expressing phenylalanine deaminase, cinnamic acid hydroxylase, 4-coumaric acid 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 cinnamic acid hydroxylase is shown in SEQ ID No. 2, the protein sequence of the 4-coumaric acid-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. The fermentation process described in step S2 includes the following steps: M1: Recombinant yeast and Antarctic yeast strain were inoculated into yeast seed culture medium and cultured to form seed liquid; M2: The seed culture of recombinant yeast and Antarctic yeast was inoculated into the fermentation medium for pre-fermentation; M3: After pre-fermentation, Qimenshan camellia seed oil and phenylalanine are added for further fermentation.

2. The preparation method according to claim 1, characterized in that, The yeast seed culture medium in step M1 consists of: 18-22 g / L peptone, 9-12 g / L yeast extract, and 18-23 g / L glucose; the culture conditions are 20-26 h at 28-32 ℃.

3. The preparation method according to claim 1, characterized in that, The fermentation medium in step M2 consists of: 10-22 g / L rice, 18-23 g / L soybean flour, and 8-11 g / L glucose; the pre-fermentation conditions are 12-144 h at 25-42 ℃.

4. The preparation method according to claim 1, characterized in that, The inoculation amounts of the recombinant yeast and Antarctic yeast seed culture described in step M2 are 4-6%, respectively.

5. The preparation method according to claim 1, characterized in that, In step M3, the amount of Qimenshan tea seed oil added is 10-900% of the initial fermentation medium weight; the amount of phenylalanine added is 10-40 g / L; and the fermentation time is 40-60 h.

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

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

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