Quality evaluation method of Liupao tea

Abstract

The invention discloses a Liupao tea quality evaluation method, which comprises: carrying out solvent extraction on a Liupao tea sample, and carrying out centrifugal filtration to prepare a test sample solution; carrying out gradient elution separation on components in the test solution, and carrying out positive ion mode detection by adopting mass spectrometry to obtain original data containing primary mass spectrometry and secondary mass spectrometry; and deconvolving the original data into an abf format file, uploading the abf format file to MS-DIAL software, performing normalization processing to form an mgf format file and a peak area table, and representing the generated chemical nodes through a GNPS online analysis platform. 249 flavonoid compounds, 16 polypeptide compounds and 5 cyclic dipeptide derivatives are rapidly found and identified, and flavor and active substances in Liupao tea are rapidly and comprehensively analyzed.

Description

Technical Field

[0001] The invention belongs to the technical field of food quality analysis and detection, and particularly relates to a quality assessment method for Liubao tea. Background Art

[0002] Liubao tea is a unique microbial fermented tea produced in Liubao Township, Cangwu County, Wuzhou City, Guangxi Province. Its production process includes two stages: primary fermentation and secondary fermentation. Liubao tea processing methods are generally categorized as traditional or modern, depending on whether or not secondary microbial fermentation is involved. Currently, the Liubao tea processing industry faces significant challenges. Traditional fermentation methods are significantly influenced by uncontrollable factors, such as microbial community structure, resulting in poor product quality consistency and low standardization. Modern fermentation methods attempt to overcome these issues. However, Liubao tea products developed using existing fermentation technologies suffer from poor consistency, low standardization, and poor sensory properties, making it difficult to meet market demand for functional products with targeted flavors.

[0003] Due to the complex compositional changes in Liubao tea during fermentation, the chemical characteristics of the unique flavor of Liubao tea produced by microbial fermentation remain unclear, making it difficult to accurately separate and identify these complex compounds using traditional techniques. Therefore, developing a rapid and comprehensive systematic analysis method for the flavor and active substances in Liubao tea can better assess the quality of Liubao tea under different fermentation conditions. Furthermore, developing fermentation strains that can improve the quality and flavor of Liubao tea based on these analytical methods is also a future trend in Liubao tea processing. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a method for evaluating the quality of Liubao tea. Liubao tea samples under different fermentation conditions are subjected to solvent extraction and centrifugal filtration to prepare a test solution; components in the test solution are separated by gradient elution, and positive ion mode detection is performed using a mass spectrometer to obtain raw data including a primary mass spectrum and a secondary mass spectrum; the raw data is deconvoluted into an abf format file and uploaded to MS-DIAL software, normalized to form an mgf format file and a peak area table, and characterized using the Global Natural Products Social Molecular Networking system (GNPS) online analysis platform.

[0005] Preferably, the following steps are also included: inferring the molecular formula based on the precursor ion mass, and then further analyzing the chemical nodes not annotated by GNPS based on the characteristic ion information and known node information of the secondary mass spectrum fragments, combined with literature or online databases.

[0006] Preferably, the online database is one or more of MassBank, MassBank of North America (MoNA) and METLIN.

[0007] Preferably, the solvent extraction of the Liubao tea sample comprises: extracting in an ultrasonic water bath using 80% methanol aqueous solution, 40% methanol aqueous solution and water in sequence.

[0008] Preferably, the components in the test solution are separated by gradient elution, comprising: taking the test solution, injecting it into an LC-MS liquid spectrometer, using octadecylsilane bonded silica gel as a filler, an aqueous solution containing 0.1% volume concentration of formic acid as mobile phase A, and an acetonitrile solution containing 0.1% volume concentration of formic acid as mobile phase B for gradient elution, the flow rate is 0.3 mL / min, and the injection volume is 1 μL.

[0009] Preferably, the gradient elution is performed according to the following program: 0-0.5 min, mobile phase A from 95% to 95%, mobile phase B from 5% to 5%; 0.5-4.5 min, mobile phase A from 95% to 93%, mobile phase B from 5% to 7%; 4.5-5.5 min, mobile phase A from 93% to 92.7%, mobile phase B from 7% to 7.3%; 5.5-7.5 min, mobile phase A from 92.7% to 92.2%, mobile phase B from 7.3% to 7.8%; 7.5-11.0 min, mobile phase A from 92.2% to 91%, mobile phase B from 7.8% to 9.0%; 11.0-13.0 min, mobile phase A from 91% to 86%, mobile phase B from 9 .0%→14.0%; 13.0~18.0 min, mobile phase A from 86%→77%, mobile phase B from 14%→23%; 18.0~19.0 min, mobile phase A from 77%→65%, mobile phase B from 23%→25%; 19.0~23.0 min, mobile phase A from 65%→53%, mobile phase B from 25%→47%; 23.0~28.0 min, mobile phase A from 53%→40%, mobile phase B from 47%→60%; 28.0~30.0 min, mobile phase A from 40%→5%, mobile phase B from 60%→95%; 30.0~33.0 min, mobile phase A from 5%→5%, mobile phase B from 95%→95%.

[0010] Preferably, the chromatographic column of the liquid chromatography-mass spectrometer and its specifications are Waters ACQUITY UPLC HSS T3, 2.1×100 mm, 1.8 μm.

[0011] Preferably, mass spectrometry is used for positive ion mode detection, including: the ion source is ESI; the backflush nitrogen temperature is 325°C, the backflush nitrogen flow rate is 11.0 L / min, the nebulizer gas flow rate is 45 psig, the auxiliary nitrogen temperature is 350°C, the auxiliary nitrogen flow rate is 11.0 L / min, the capillary voltage is 3500 V, the source collision voltage is 175 V, and the cone voltage is 65.0 V; the scan mode is full scan; the scan range is m / z 60 to 1700.

[0012] Preferably, the parameters for normalization processing using MS-DIAL software are: the MS1 tolerance for data acquisition is 0.01Da, and the MS2 tolerance is 0.025Da; the minimum peak height for peak detection is 1000 amplitudes, and the mass tolerance is 0.1Da; the deconvolution parameters are set as follows: the sigma window value is 0.1, the MS / MS abundance cutoff value is 5 amplitudes; the retention time tolerance is set to 0.1 minutes; in the characterization step of the generated chemical nodes through the GNPS online analysis platform, the cosine score threshold is set to 0.7, the minimum matching fragment ion is 6, the topK is set to 10, and the precursor ion mass tolerance is 0.02; the Cys format data is downloaded and imported into Cytoscape 3.7.0 software for visualization.

[0013] According to another aspect of the present invention, there is provided a use of Eurotium cristatum in reducing the content of ester catechins and increasing the content of non-ester catechins in the fermentation of Liubao tea.

[0014] According to another aspect of the present invention, there is provided a use of Bacillus coagulans in promoting the production of polypeptide compounds during Liubao tea fermentation.

[0015] According to another aspect of the present invention, there is provided a use of Liubao tea microbial metabolites in the preparation of Keap1-Nrf2 PPI inhibitors or ACE inhibitors.

[0016] This invention provides a systematic analysis method for Liubao tea that combines artificial inoculation, feature-based molecular networking, and liquid chromatography-mass spectrometry. This method addresses the poor quality consistency and low standardization of traditional fermented products, as well as the poor sensory properties of existing microbial secondary fermentation methods. Artificial inoculation with probiotics, combined with feature-based molecular networking and liquid chromatography-mass spectrometry, effectively separates the complex chemical components of Liubao tea during fermentation. The method rapidly discovered and identified 249 flavonoids, 16 polypeptides, and five cyclic dipeptide derivatives, providing a rapid and comprehensive analysis of the flavor and active substances in Liubao tea, enabling better quality assessment of novel Liubao tea flavor products. In addition, according to the research of the present invention, Eurotium cristatum can be used to reduce the content of ester catechins and increase the content of non-ester catechins in the fermentation of Liubao tea. Eurotium cristatum can also promote the biosynthesis of flavan-3-ol B-ring fission analogues and ethylpyrrolidone-substituted flavan-3-ol B-ring fission analogues; Bacillus coagulans can be used to promote the production of polypeptide compounds in the fermentation of Liubao tea; Liubao tea microbial metabolites can also be used as Keap1-Nrf2 PPI inhibitors or ACE inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A flow chart of the method provided according to the present invention is shown;

[0019] Figure 2 Shown is a sensory radar chart of a novel Liubao tea flavor product prepared according to the present invention;

[0020] Figure 3 The contents of the main components determined according to the present invention are shown;

[0021] Figure 4 A schematic diagram of a feature-based molecular network constructed according to the present invention is shown;

[0022] Figure 5 The relative abundance of flavan-3-ol B-ring fission analogs and ethylpyrrolidone-substituted flavan-3-ol B-ring fission analogs identified according to the present invention in Liubao tea samples is shown;

[0023] Figure 6 The relative abundance of polypeptide compounds and cyclic dipeptide derivatives identified according to the present invention in Liubao tea samples is shown;

[0024] Figure 7PCA and OPLS-DA analysis of compounds identified according to the present invention are shown;

[0025] Figure 8 A schematic diagram of molecular docking of flavan-3-ol B-ring cleavage analogues identified according to the present invention with Keap1 is shown;

[0026] Figure 9 A schematic diagram of molecular docking between the polypeptide compounds identified according to the present invention and ACE is shown. DETAILED DESCRIPTION

[0027] The examples listed below are intended to allow those skilled in the art to more clearly understand the present invention. It should be noted that the following examples do not limit the scope of protection claimed in the present invention and are only illustrative examples. Unless otherwise specified, the raw materials, reagents or devices mentioned in the following examples can be obtained from commercial channels or by known existing methods. The information of Liubao tea used in the present invention is shown in Table 1. Liubao tea samples and strains were provided by China Tea Technology (Beijing) Co., Ltd. and can also be purchased through commercial channels.

[0028] Table 1: Information of 4 Liubao tea samples

[0029]

[0030]

[0031] Taste Evaluation of Liubao Tea

[0032] The taste characteristics of tea were evaluated according to the standardized method in GB / T 23776-2018. Briefly, 3 g of each tea sample was steeped in 150 ml of boiling water for 8 minutes. The taste characteristics were evaluated based on six attributes: sweetness, umami, bitterness, sourness, astringency, and staleness. Each sample was evaluated in triplicate on different days. The intensity of each taste attribute in the Liubao tea samples was scored on a 10-point scale, with 0 indicating no perception, 5 indicating moderate intensity, and 10 indicating strong intensity. The final score was calculated based on the mean and processed using a one-way analysis of variance (ANOVA).

[0033] The endpoint of secondary microbial fermentation is determined based on sensory evaluation by experts and quantitatively evaluated. Figure 2As shown, the sensory characteristics of the new Liubao teas have undergone significant changes. While traditionally fermented Liubao teas are characterized by a woody aroma, the tea brewed with Bacillus coagulans (No. 10059) exhibits a stale aroma. In contrast, the tea brewed with Bacillus coagulans (JZ-1) and Eurotium cristatum (8730) exhibited cheesy and fungal floral aromas, respectively. Compared to traditional Liubao teas, the new Liubao teas exhibited significantly increased sweetness and umami, and significantly decreased bitterness (p < 0.001).

[0034] The present invention relates to the technical field of tea component determination and provides a method for detecting flavonoids and peptides in Liubao tea using a feature-based molecular network. The method comprises the following steps: ① Sample solution preparation: a Liubao tea sample is subjected to sample pretreatment to obtain a mixture solution; ② LC-MS analysis: the sample solution obtained by S1 is analyzed in a tandem liquid chromatography-mass spectrometry (LC-MS) instrument to effectively separate the flavonoids and peptide components in the Liubao tea; ③ Feature-based molecular network construction: the data is preprocessed using MS-DIAL and uploaded to GNPS to construct a feature-based molecular network. The molecular network is downloaded and imported into Cytoscape software for visualization and analysis. ④ Identification of flavonoid and peptide components: the compounds are preliminarily characterized using the GNPS database. Based on the characteristic ion information and known node information of the secondary mass spectrometry fragments, the compounds are further identified in combination with literature or online databases. The above method can rapidly detect and discover active peptides and flavonoid compounds in Liubao tea, thereby better evaluating the quality of microbially fermented Liubao tea. Specific embodiments

[0036] 1. LC-MS analysis of Liubao tea samples

[0037] 1.1 Preparation of test solution

[0038] A 100 mg tea sample was accurately weighed and extracted in an ultrasonic bath (40 kHz) for 30 minutes using 1 mL of 80% methanol in water, 1 mL of 40% methanol in water, and 1 mL of distilled water. After centrifugation at 15,890 g for 5 minutes, the supernatants were combined and mixed to prepare a Liubao tea sample solution.

[0039] 1.2 Selection of liquid chromatography conditions

[0040] A Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column was used as the elution column. Mobile phase A was 0.1% by volume formic acid in water, and mobile phase B was 0.1% by volume formic acid in acetonitrile. The flow rate was 0.3 mL / min, the column temperature was 35°C, and the injection volume was 1 μL. Gradient elution was performed according to the following procedure:

[0041] 0-0.5 min, mobile phase A from 95% to 95%, mobile phase B from 5% to 5%;

[0042] 0.5-4.5 min, mobile phase A from 95% to 93%, mobile phase B from 5% to 7%;

[0043] 4.5-5.5 min, mobile phase A from 93% to 92.7%, mobile phase B from 7% to 7.3%;

[0044] 5.5-7.5 min, mobile phase A from 92.7% to 92.2%, mobile phase B from 7.3% to 7.8%;

[0045] 7.5-11.0 min, mobile phase A from 92.2% to 91%, mobile phase B from 7.8% to 9.0%;

[0046] 11.0-13.0 min, mobile phase A from 91% to 86%, mobile phase B from 9.0% to 14.0%;

[0047] 13.0-18.0 min, mobile phase A from 86% to 77%, mobile phase B from 14% to 23%;

[0048] 18.0-19.0 min, mobile phase A from 77% to 65%, mobile phase B from 23% to 25%;

[0049] 19.0-23.0 min, mobile phase A from 65% to 53%, mobile phase B from 25% to 47%;

[0050] 23.0-28.0 min, mobile phase A from 53% to 40%, mobile phase B from 47% to 60%;

[0051] 28.0-30.0 min, mobile phase A from 40% to 5%, mobile phase B from 60% to 95%;

[0052] 30.0~33.0min, mobile phase A changes from 5%→5%, mobile phase B changes from 95%→95%.

[0053] High-resolution mass spectrometry detection conditions were as follows: detection in positive ion mode; ESI ion source; backflush nitrogen temperature 325°C, backflush nitrogen flow 11.0 L / min, nebulizer gas flow 45 psig, auxiliary nitrogen temperature 350°C, auxiliary nitrogen flow 11.0 L / min, capillary voltage 3500 V, source collision voltage 175 V, cone voltage 65.0 V; scan mode: full scan; scan range: m / z 60-1700.

[0054] 2. Construction of feature-based molecular networks

[0055] The raw data of LC-MS were converted into abf file format for analysis using MS-DIAL. Subsequently, the processed data were uploaded to the GNPS online workflow to create a molecular network. The molecular network data were then submitted to the MolNetEnhancer workflow to run the annotation of the chemical categories of the mass spectrometry nodes. A total of 18,342 mass spectrometry nodes were found in the feature-based molecular network (FBMN), which were divided into 22 clusters (number of nodes>3). The compounds annotated by MolNetEnhancer include a total of five chemical categories, including flavonoids, "amino acids, peptides and analogs", "alkaloids and nucleosides" and lipids, and are marked with different colors, such as Figure 4 shown.

[0056] During the above process, the LC-MS data raw files were converted to Abf format using the Reifycs Abf converter. Preprocessing was performed using MS-DIAL 4.8.0 software with the following parameters: data acquisition with an MS1 ​​tolerance of 0.01 Da and an MS2 tolerance of 0.025 Da; peak detection with a minimum peak height of 1000 amplitudes and a mass tolerance of 0.1 Da. Deconvolution parameters were set as follows: a sigma window of 0.1, an MS / MS abundance cutoff of 5 amplitudes, and a retention time tolerance of 0.1 min.

[0057] After preprocessing the mass spectrometry data using MS-DIAL, two files were exported for the Global Natural Products Network (GNPS) system: an mgf file for molecular networking and a peak area table. These files were then uploaded to GNPS to generate a molecular network. The cosine score threshold was set to 0.7, the minimum matching fragment ion was 6, the topK was set to 10, and the precursor ion mass tolerance was 0.02. The data were downloaded in Cys format and imported into Cytoscape 3.7.0 for visualization.

[0058] 3. Identification of flavonoids and peptides

[0059] The generated chemical nodes were initially characterized using the GNPS database. For the remaining nodes not annotated by the GNPS library, their molecular formulas were preliminarily inferred based on the precursor ion masses. Further analysis was performed based on the characteristic ion information of the secondary mass spectrometry fragments and known node information, combined with literature or online databases such as MassBank, MassBank of North America (MoNA), and METLIN.

[0060] First, the chemical nodes of Liubao tea samples were preliminarily characterized using the GNPS database. For example, the spectra of epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), tryptophan, valine, phenylalanine, etc. were successfully matched with the spectra in the GNPS library. For the remaining nodes that were not annotated by the GNPS library, their molecular formulas were preliminarily inferred based on the mass of the precursor ions, and then further analyzed based on the characteristic ion information of the secondary mass spectrometry fragments and the known node information, combined with literature or online databases (such as MassBank, MassBank of North America (MoNA), NIST and METLIN, etc.). In addition, based on the respective extracted ion chromatogram (EIC) peak areas, pie charts were used to clearly illustrate the abundance and proportion of each node in different sources, such as Figure 4 The identification results of 249 flavonoids are shown in Table 2. Among them, the relative abundance of flavan-3-ol B-ring fission analogues (such as Teadenol, Fuzhuanin B, Xanthocerin, etc.) and ethylpyrrolidone-substituted flavan-3-ol B-ring fission analogues (such as Ethylpyrrolidinonyl teadenol, Ethylpyrrolidinonyl fuzhuanin B, Ethylpyrrolidinonyl xanthocerin, etc.) increased significantly after fermentation with Eurotium cristatum. The relative abundances of these compounds in the four samples are shown in Table 2. Figure 5 shown.

[0061] Subsequently, 31 unique nodes connected to annotated amino acids (such as valine, phenylalanine, and leucine / isoleucine) were discovered in the third cluster. Based on spectral similarity between connected nodes, 16 were successfully annotated as peptides, including one cyclic dipeptide, one dipeptide, two tripeptides, three tetrapeptides, three pentapeptides, three hexapeptides, one octapeptide, one nonapeptide, and one tridecapeptide. The identification results are shown in Table 3. The relative abundance of peptide compounds in Liubao tea significantly increased after fermentation with Bacillus coagulans. Furthermore, a class of cyclic dipeptide derivatives with potential antibacterial and anticancer activities, including neoechinulin and echinulin, was discovered. The identification results are shown in Table 4. Variecolorin G, cristatumin B, and tardioxopiperazine A were reported in Liubao tea for the first time. These compounds are products of secondary metabolism of Eurotium cristatum, and their relative abundance significantly increased after fermentation with Eurotium cristatum. The relative abundances of representative peptide compounds and cyclic dipeptide derivatives in the four samples are shown in Figure 2. Figure 6 shown.

[0062] Table 2: Identification of 249 flavonoids in Liubao tea

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Table 3: Identification of 16 peptides in Liubao tea

[0075]

[0076] Table 4: Identification of five cyclic dipeptide derivatives in Liubao tea

[0077]

[0078]

[0079] The significant differences in the components between traditional Liubao tea and microbial secondary fermentation Liubao tea were analyzed by principal component analysis ( Figure 7 ). As can be seen from the score chart, there are significant differences between traditional Liubao tea and Liubao tea fermented by Bacillus coagulans and Eurotium cristatum. Subsequently, OPLS-DA analysis was performed on the identified compounds, and compounds with differences were screened based on VIP ≥ 1 and t-test p value < 0.05. The up-regulated differential compounds screened in Liubao tea fermented by Eurotium cristatum mainly include non-ester catechins, flavan-3-ol B-ring fission analogs, ethylpyrrolidone-substituted flavan-3-ol B-ring fission analogs, and cyclic dipeptide derivatives. The significantly down-regulated differential compound is ester catechin. For Liubao tea fermented by Bacillus coagulans No. 10059 and JZ-1, the significantly up-regulated compounds screened are mainly amino acids and peptides.

[0080] After fermentation by Eurotium cristatum, the relative contents of ester catechins, including epigallocatechin gallate (EGCG), catechin gallate (CG), and epigallocatechin gallate (GCG), decreased. However, the contents of non-ester catechins, including epicatechin gallate (ECG), catechin (C), epicatechin (EC), and epigallocatechin (GC), significantly increased (p < 0.0001). Exoenzymes secreted by Eurotium cristatum during Liubao tea fermentation facilitate the hydrolysis of ester catechins into their corresponding non-ester catechins. Subsequently, the B ring of the non-ester catechins is further oxidized by these enzymes, producing flavan-3-ol B-ring cleavage analogs. Furthermore, Eurotium cristatum may facilitate the degradation of theanine and its substitution onto the A ring of the flavan-3-ols, resulting in ethylpyrrolidone-substituted flavan-3-ol B-ring cleavage analogs. These results indicate that Eurotium cristatum plays a crucial role in catechin transformation, driving the biotransformation of ester-type catechins into the corresponding non-ester-type catechins, flavan-3-ol B-ring cleavage analogs, and ethylpyrrolidone-substituted flavan-3-ol B-ring cleavage analogs. Bacillus coagulans promotes protein hydrolysis into peptides and amino acids. We then further investigated the bioactivities of these microbially derived secondary metabolites to gain a deeper understanding of their functional roles in Liubao tea.

[0081] 4. Activity of Differential Microbial Metabolites in Liubao Tea

[0082] Activation of the Keap1-Nrf2-ARE pathway plays a crucial role in the antioxidant stress and neuroprotective effects of flavonoids. Compounds that bind to Keap1 may disrupt the Keap1-Nrf2 interaction, inhibit Keap1-dependent Nrf2 ubiquitination, lead to an increase in Nrf2 protein and translocation to the nucleus, bind to the antioxidant response element (ARE), thereby significantly activating the Nrf2-ARE signaling and reducing oxidative stress. CPUY192018 is a Keap1-Nrf2 PPI inhibitor that mimics the binding mode of Nrf2 and has a strong binding affinity for Keap1. Eight representative FBRFAs and a standard drug CPUY192018 were docked with the Kelch-Neh2 complex protein to analyze their binding potential. The results showed that all candidate compounds can effectively interact with Keap1, and the optimal mode binding energy of most compounds is low, ranging from -9.7kcal / mol to -8.5kcal / mol ( Figure 8). The standard drug CPUY192018 had the lowest binding energy of -9.7 kcal / mol, forming four hydrogen bonds with Keap1. fuzhuanin A had a similarly low binding energy to CPUY192018, formed two hydrogen bonds with Keap1, and interacted with residues Leu 557 and Val 463. In addition, the binding energy of the Teadenol A-Keap1 molecular docking was -8.5 kcal / mol. In this binding mode, the ligand formed two hydrogen bonds with Val 512 and Ala 510. It has been reported that teadenolA has in vitro antioxidant activity. The binding energies of other flavan-3-ol B-ring cleavage analogs were all lower than those of teadenolA, suggesting that they may contribute to the antioxidant activity of Liubao tea.

[0083] Many food-derived peptides have potential antihypertensive effects. For example, Leu / Ile-Pro has been reported as a potential ACE inhibitor with an IC50 of 130 μM. In order to evaluate the antihypertensive potential of peptides from Liubao tea, seven representative peptides were screened and subjected to computer simulation molecular docking with the well-known ACE inhibitor lisinopril. Figure 9 As shown, all peptides can effectively interact with ACE, and their binding energies range from -7.5kcal / mol to -8.6kcal / mol in the optimal binding mode. The results showed that VPPFL had the lowest binding energy, followed by PVEP and GPFPL, with binding energies of -8.6, -8.3 and -7.9kcal / mol, respectively. In the optimal conformation of VPPFL-ACE, the ligand formed five hydrogen bonds with the ACE enzyme, interacting with residues Thr 282, Gln 281, Asn 277 and Glu 376. In addition, pentenoylation of aromatic compounds has also been reported to enhance biological activity. Pentenyl cyclic dipeptides have potential anti-inflammatory, antiviral and anticancer activities. These results suggest that microbial metabolites may contribute to the various effects of Liubao tea ( Figure 8-9 ).

[0084] The flavonoids include flavan-3-ols (catechin, epicatechin, gallocatechin, epigallocatechin, etc.), flavan-3-ol B-ring fission analogs (Teadenol, Fuzhuanin A, Fuzhuanin B, Teasperin, etc.), flavanones, flavanols, flavones, flavonols, ester-type catechins (catechin gallate, epicatechin gallate, gallocatechin gallate, epigallocatechin gallate, etc.), flavan alkaloids (ethylpyrrolidone-substituted flavan-3-ols and their B-ring fission analogs, etc.), flavonols, methylated flavonoids, biflavonoids, trimer catechins, and flavonoid glycosides.

[0085] The polypeptide compounds include cyclic (leucine / isoleucine-proline), leucine / isoleucine-proline, proline-glutamine-valine, proline-tyrosine-proline, leucine / isoleucine-glycine-proline-valine, proline-valine-glutamine-proline, cysteine-histidine-valine-proline, valine-proline-glutamine-leucine / isoleucine-glutamine-leucine / isoleucine-valine-proline-asparagine, glutamine-proline-phenylalanine-proline-leucine / isoleucine, cysteine-histidine-proline-valine-leucine / isoleucine Amino acid, valine-proline-proline-phenylalanine-leucine / isoleucine, valine-proline-leucine / isoleucine-threonine-proline-threonine, alanine-proline-phenylalanine-proline-glutamate-valine, threonine-aspartic acid-alanine-proline-serine-phenylalanine-serine-aspartic acid-leucine / isoleucine-proline-asparagine-proline-leucine / isoleucine, tyrosine-proline-valine-glutamate-proline-phenylalanine, glycine-serine-cysteine-cysteine-aspartic acid-serine-aspartic acid-proline.

[0086] In summary, the present invention provides a systematic analysis method for Liubao tea that combines artificial inoculation, feature-based molecular networking, and liquid chromatography-mass spectrometry. This method addresses the poor quality consistency and low standardization of traditional fermented products, as well as the poor sensory properties of existing microbial secondary fermentation methods. Artificial inoculation of probiotics, combined with feature-based molecular networking and liquid chromatography-mass spectrometry, effectively separates the complex chemical components of Liubao tea fermentation. 249 flavonoids, 16 polypeptides, and 5 cyclic dipeptide derivatives were quickly discovered and identified, providing a rapid and comprehensive analysis of representative active ingredients in Liubao tea, enabling better quality assessment of microbially fermented Liubao tea.

[0087] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for evaluating the quality of Liubao tea, characterized in that: Liubao tea samples under different fermentation conditions were extracted with solvents and centrifuged to prepare test solutions. The components in the test solution are separated by gradient elution, and detected by mass spectrometry in positive ion mode to obtain raw data including primary mass spectrometry and secondary mass spectrometry; The raw data were deconvoluted into an abf format file and uploaded to the MS-DIAL software, normalized to form an mgf format file and a peak area table, and the generated chemical nodes were characterized using the GNPS online analysis platform.

2. The method according to claim 1, characterized in that The following steps are also included: The molecular formula is inferred based on the precursor ion mass, and chemical nodes not annotated by GNPS are further analyzed based on the characteristic ion information and known node information of the secondary mass spectrometry fragments, combined with literature or online databases; the online databases are one or more of MassBank, MassBank of North America (MoNA), and METLIN.

3. The method according to claim 1, characterized in that Solvent extraction of Liubao tea samples includes: extracting in an ultrasonic water bath using 80% methanol aqueous solution, 40% methanol aqueous solution and water in sequence; performing gradient elution separation on the components in the test solution, including: taking the test solution, injecting it into an LC-MS liquid spectrometer, using octadecylsilane bonded silica gel as a filler, using 0.1% volume concentration of formic acid aqueous solution as mobile phase A, and 0.1% volume concentration of formic acid acetonitrile solution as mobile phase B for gradient elution, with a flow rate of 0.3 mL / min and an injection volume of 1 μL.

4. The method according to claim 3, characterized in that Gradient elution was performed according to the following procedure: 0-0.5 min, mobile phase A from 95% to 95%, mobile phase B from 5% to 5%; 0.5-4.5 min, mobile phase A from 95% to 93%, mobile phase B from 5% to 7%; 4.5-5.5 min, mobile phase A from 93% to 92.7%, mobile phase B from 7% to 7.3%; 5.5-7.5 min, mobile phase A from 92.7% to 92.2%, mobile phase B from 7.3% to 7.8%; 7.5-11.0 min, mobile phase A from 92.2% to 91%, mobile phase B from 7.8% to 9.0%; 11.0-13.0 min, mobile phase A from 91% to 86%, mobile phase B from 9.0% to 14.0%; 13.0-18.0 min, mobile phase A from 86% to 77%, mobile phase B from 14% to 23%; 18.0-19.0 min, mobile phase A from 77% to 65%, mobile phase B from 23% to 25%; 19.0-23.0 min, mobile phase A from 65% to 53%, mobile phase B from 25% to 47%; 23.0-28.0 min, mobile phase A from 53% to 40%, mobile phase B from 47% to 60%; 28.0-30.0 min, mobile phase A from 40% to 5%, mobile phase B from 60% to 95%; 30.0~33.0min, mobile phase A changes from 5%→5%, mobile phase B changes from 95%→95%.

5. The method according to claim 4, characterized in that The chromatographic column of the liquid chromatography-mass spectrometer and its specifications are Waters ACQUITY UPLC HSS T3, 2.1×100 mm, 1.8 μm.

6. The method according to claim 1, characterized in that Mass spectrometry was used for positive ion mode detection, including: ESI ion source; backflush nitrogen temperature of 325°C, backflush nitrogen flow of 11.0 L / min, nebulizer gas flow of 45 psig, auxiliary nitrogen temperature of 350°C, auxiliary nitrogen flow of 11.0 L / min, capillary voltage of 3500 V, source collision voltage of 175 V, cone voltage of 65.0 V; scan mode: full scan; scan range: m / z 60-1700.

7. The method according to any one of claims 1 to 6, characterized in that The parameters for normalization processing using MS-DIAL software are as follows: the MS1 tolerance for data acquisition is 0.01Da, and the MS2 tolerance is 0.025Da; the minimum peak height for peak detection is 1000 amplitudes, and the mass tolerance is 0.1Da; the deconvolution parameters are set as follows: the sigma window value is 0.1, the MS / MS abundance cutoff value is 5 amplitudes, and the retention time tolerance is set to 0.1 minutes; in the characterization step of the generated chemical nodes using the GNPS online analysis platform, the cosine score threshold is set to 0.7, the minimum matching fragment ion is 6, the topK is set to 10, and the precursor ion mass tolerance is 0.02; the Cys format data are downloaded and imported into Cytoscape 3.7.0 software for visualization.

8. Use of Eurotium cristatum in reducing the content of ester catechins and increasing the content of non-ester catechins in Liubao tea fermentation.

9. Use of Bacillus coagulans in promoting the production of polypeptide compounds during Liubao tea fermentation.

10. Use of Liubao tea microbial metabolites in the preparation of Keap1-Nrf2 PPI inhibitor or ACE inhibitor food.

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