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Tannase (E.C. 3.1.1.20) is hypothesized to be involved in the metabolism of gallates and gallic acid (GA) in pu-erh tea fermentation. In this work, we measured tannase in Aspergillus niger fermented tea leaves and confirmed the production of fungal tannase during pu-erh tea fermentation. A decrease in catechin and theaflavin gallates and a significant increase in GA content and the relative peak areas of ethyl gallate, procyanidin A2, procyanidin B2, procyanidin B3, catechin-catechin-catechin, epiafzelechin, and epicatechin-epiafzelechin [variable importance in the projection (VIP) > 1.0, p 1.5] were observed using high performance liquid chromatography (HPLC) and metabolomics analysis of tea leaves fermented or hydrolyzed by tannase. In vitro assays showed that hydrolysis by tannase or polymerization of catechins increased the antioxidant activity of tea leaves. In summary, we identified a metabolic pathway for gallates and their derivatives in tea leaves hydrolyzed by tannase as well as associated changes in gallate and GA concentrations caused by fungal tannase during pu-erh tea fermentation.

Macroalgae Laminaria japonica (MLJ) has been reported to exhibit various biological activities including improving immunity, anti-aging, anti-tumor, anti-atherosclerosis and anti-diabetic, but the protective mechanisms of MLJ consumption against non-alcoholic fatty liver disease (NAFLD) associated with hyperlipidemia remain poorly understood. This study demonstrated that MLJ consumption prevented high-fat diet (HFD)-induced NAFLD associated with hyperlipidemia in a rat model, and improved hyperlipidemia-related parameters, e.g. serum and hepatic lipid profiles. Moreover, histological analysis showed that MLJ reduced lipid deposition in adipocytes and hepatocytes compared with the HFD group. Such beneficial effects may be associated with the modulation of the intestinal microbiota, especially some key microbial phylotypes involved in lipid metabolism homeostasis. The underlying protective mechanisms of MLJ consumption against HFD-induced NAFLD associated with hyperlipidemia were also studied by ultra-high performance liquid chromatography with quadruple-time of flight mass spectrometry (UPLC-QTOF/MS)-based liver metabolomics coupled with pathway analysis. The metabolic pathway enrichment analysis of the differentially abundant hepatic metabolites indicated that primary bile acid biosynthesis metabolism and cysteine and methionine metabolism were the two main metabolic pathways altered by MLJ consumption when compared with the model group. The analysis of the transcription levels of liver-related genes by RT-qPCR and the expressions of liver-related proteins by immunohistochemistry (IHC) showed that MLJ consumption could regulate the levels of mRNA transcription and protein expression related to hepatic lipid metabolism. In short, this study indicates that MLJ could be developed as functional food supplement for the prevention or treatment of NAFLD associated with hyperlipidemia.

Developing an effective method to improve the quality of Pu-erh tea is of great scientific and commercial interest. In this work, Penicillium chrysogenum P1 isolated from Pu-erh tea was inoculated in sterilized or unsterilized sun-dreid green tea leaves to develop pure-culture fermentation (PF) and enhanced fermentation (EF) of Pu-erh tea. Spectrophotometry and high performance liquid chromatography determined that contents of free amino acids (FAA), total tea polyphenols and eight polyphenolic compounds in PF were significantly lower than these in non-inoculation control test (CK) (P 0.05), whereas the contents of soluble sugars and theabrownins (TB) in PF were significantly higher (P 0.05) than in CK. A total of 416 volatile compounds were detected by headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry. Comparison to CK, 124 compounds in PF were degraded or decreased significantly [Variable importance in projection [(VIP) 1.0, P 0.05, fold change (FC) 0.5], whereas 110 compounds in PF were formed or increased significantly (VIP 1.0, P 0.05, FC 2). Compared with normal fermentation (NF), the levels of gallic acid, (+)-catechin, (-)-epicatechin and 64 volatile compounds in EF were significantly lower (VIP 1.0, P 0.05, FC 0.5), whereas the levels of FAA and 39 volatile compounds were significantly higher (VIP 1.0, P 0.05, FC 2). Amplicon sequencing of fungal internal transcribed spacer 1 (ITS1) revealed that P. chrysogenum P1 didn't become the dominant fungus in EF; while the fungal communities in EF were different from those in NF, in that the relative abundances of Blastobotrys bambusae and P. chrysogenum in EF were higher, and the relative abundances of Aspergillus niger and Kluyveromyces marxianus in EF were lower. Overall, inoculation of P. chrysogenum in unsterilized sun-dreid green tea leaves changed the the fungal communities in fermentation of Pu-erh tea, and chemical compounds in fermented tea leaves, i.e., the levels of TB and the compounds responsible for the stale flavor, e.g., 2-amino-4-methoxybenzothiazole were increased, resulting in improvement of the sensory quality, including mellower taste and stronger stale flavor.

Post-fermented Pu-erh tea (PFPT) is a microbially-fermented tea with distinct sensory qualities and multiple health benefits. Aspergillus are the dominant fungi in the fermentation and the main contributors to the characteristics of PFPT, so their underlying functions warrant detailed study. Here, tea leaves were fermented by Aspergillus niger, Aspergillus tamarii and Aspergillus fumigatus, and resulting samples (designated as Asn, Ast and Asf, respectively) were analyzed by proteomic and metabolomic methods. Changes to the composition of flavonoids, glycerophospholipids, organo-oxygen compounds and fatty acids resulting from Aspergillus fermentation were observed. Carbohydrate-active enzymes, e.g., endoglucanases and cellulases, for degradation of cellulose, starch, lignin, pectin, xylan and xyloglucan were identified. Glycoside hydrolase, glycosyltransferases, tannase, laccases, vanillyl-alcohol oxidases and benzoquinone reductase were identified and hypothesized to catalyze hydrolysis, oxidation, polymerization and degradation of phenolic compounds. Together, functions of Aspergillius were demonstrated as production of enzymes to change concentrations and compositions of metabolites in tea leaves.

The aim of this study is to investigate the composition of the fatty acids and phospholipids derived from large yellow croaker (Pseudosciaena crocea) roe. The composition of the total lipids and the molecular species of phospholipids were determined by gas chromatography–mass spectroscopy (GC–MS) and high performance liquid chromatography–evaporative light scattering detector (HPLC–ELSD), respectively. The results showed that large yellow croaker roe had high levels of the total lipid (19.6% ± 1.32%, w/w) and phospholipid (61.2% ± 1.22% of the total lipid). The phospholipid was rich in docosahexaenoic acid (31.0% ± 0.19% of the total phospholipids), and the major phospholipid molecular species was phosphatidylcholine (PC, 61.06% ± 0.02% of the total phospholipids, w/w). It was concluded that large yellow croaker roe is expected to be a good resource of phospholipids with a high content of PC.

A simple and efficient RP high-performance liquid chromatography method was developed for simultaneous analysis of eight small antihypertensive peptides with tyrosine at the C-terminal in Laminaria japonica hydrolysates (LHs). The chromatography was performed on an Xterra MS-C18 column at 45C, with an aqueous mobile phase (0.1% trifluoroacetic acid, pH 2.8) acetonitrile in a linear gradient at the flow rate of 0.7 mL/min, and detected at 218 nm. The detection limits ranged from 0.0624 to 0.2365 μM. Correlation coefficients were greater than 0.9997. The recovery ranged from 92.17 to 120.08%. The small peptides were successfully determined by this method in LHs and the structures were confirmed by ESI-MS/MS, and the structure–activity relationships were discussed. The results demonstrated that the proposed method could be used to determine the peptides with tyrosine at the C-terminal, and to characterize a widespread detection mode for small peptide analysis in easy operation and low cost. Practical Applications This study is the first to report that marine Laminaria japonica protein hydrolyzed by combined enzymes of the alcalase, trypsin and papain displayed high angiotensin-converting enzyme (ACE) inhibitory activity. An effective and efficient reverse phase (RP) high-performance liquid chromatography method was developed for simultaneous analysis of eight small antihypertensive peptides with tyrosine at the C-terminal. Adopting the proposed method, Laminaria japonica hydrolysates and the ACE inhibitory activities of small peptides will be successfully analyzed. The results also demonstrated that Laminaria japonica proteins would be a good source of hypotensive peptides.