Degradation of Cell Wall Polysaccharides during Traditional and Tank Fermentation of Chinese Liupao Tea.

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides' breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9-49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP.

Screening strategy for predominant phenolic components of digestive enzyme inhibitors in passion fruit peel extracts on simulated gastrointestinal digestion.

BACKGROUND: The targeted biological activity of a natural product is often the result of the combined action of multiple functional components. Screening for predominant contributing components of targeting activity is crucial for quality evaluation. RESULTS: Thirteen and nine phenolic compounds inhibiting α-glucosidase and α-amylase, respectively, were identified in the ethanol extracts of passion fruit peel through liquid chromatography-tandem mass spectrometry and multivariate analysis. Considering the different concentrations of components and their interactions, the role of the semi-inhibitory concentration (IC50 ) in the dose-effect relationship is limited. We proposed the active contribution rate (ACR), which is the ratio of a single component concentration to its IC50 in the whole, to assess the relative activity of each compound. Luteolin, quercetin, and vitexin exhibited a minimum IC50 . Before the simulation of gastrointestinal digestion, quercetin, salicylic acid, and luteolin were identified as the dominant contributors to α-glucosidase inhibition according to ACR, while salicylic acid, 2,3-dihydroxybenzoic acid, and quercetin were identified as dominant contributors to α-amylase inhibition. After simulated digestion, the contents of all polyphenolic compounds decreased by various degrees. Salicylic acid, gentisic acid, and vitexin became the dominant inhibitors of α-glucosidase based on ACR (cumulative 57.96%), while salicylic acid and 2,3-dihydroxybenzoic acid became the dominant inhibitors of α-amylase (cumulative 84.50%). CONCLUSION: Therefore, the ACR evaluation strategy can provide a quantitative reference for screening the predominant contributor components of a specific activity in complex systems. © 2022 Society of Chemical Industry.

Determination of the variations in the metabolic profile and sensory quality of Liupao tea during fermentation through UHPLC-HR-MS metabolomics.

Liupao tea is a dark tea with unique quality. Semi-finished Liupao tea with two different fermentation processes (traditional/tank) was analyzed to explain the chemical characteristics and taste quality. The content change rate of polyphenols, flavonoids, and theabrownin in traditional fermentation was approximately twice that in tank fermentation. Electronic tongue revealed that bitterness and astringency increased, whereas aftertaste-astringency decreased after fermentation. 36 compounds were identified as the biomarkers responsible for the metabolic changes caused by fermentation with significant decrements in catechins, catechin gallate, and α, α-trehalose, and significant increments in gallic acid content (VIP > 3; P < 0.05). In addition, 26 metabolites were identified to distinguish between tank and traditional fermentation, with correlation analysis indicating that catechin gallate, epicatechin and gallic acid accounting for the differences in taste between the two processes. This study provides a comprehensive insight into the chemical composition and sensory quality of different Liupao tea fermentations.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.

Antihyperlipidemic effect and increased antioxidant enzyme levels of aqueous extracts from Liupao tea and green tea in vivo.

Liupao tea (fermented dark tea) may improve the active function of hyperlipidemia. Utilizing a hyperlipidemia Sprague-Dawley model and UPLC-MS/MS metabolomics, we examined how the effect of Liupao and green tea extracts on hyperlipidemia and antoxidant enzyme levels and compared their constituents. The results showed that the two types of tea could reduce the levels of total cholesterol (TC), total triglyceride, and low-density lipoprotein cholesterol (LDL-C); increase the contents of bile acids and cholesterol in feces; and improve catalase and glutathione peroxidase (GSH-Px) activities. Compared with the model control group, Liupao tea effectively reduced TC and LDL-C levels by 39.53% and 58.55% and increased GSH-Px activity in the liver by 67.07%, which was better than the effect of green tea. A total of 93 compounds were identified from two samples; the amounts of alkaloids and fatty acids increased compared with green tea, and ellagic acid, hypoxanthine, and theophylline with relatively high contents in Liupao tea had a significantly positive correlation with antihyperlipidemic and antioxidant effects. Therefore, Liupao tea had better antihyperlipidemic and antioxidant activities in vivo than green tea, which might be related to the relatively high content of some active substances.

Development and evaluation of a qPCR detection method for citrinin in Liupao tea.

Penicillium is universal in dark tea, and Penicillium citrinum can produce a kidney toxin called citrinin (CIT). Determining CIT is difficult because of the complexity of the dark tea substrate and the diversity of CIT-producing fungi. Therefore, this study established a real-time PCR (qPCR) detection method for CIT-related synthetic genes (ctnD, orf1, ctnA, pksCT, orf5, orf7, and ctnG) in Liupao tea and determined the content of CIT in samples at different production stages and the toxin-producing abilities of fungi (Aspergillus oryzae, etc.) in Liupao tea. CIT was found in all samples during the pile-fermentation process of Liupao tea, and CIT was detected in two samples during the aging process. The established method demonstrated good sensitivity and specificity in detecting CIT-related synthetic genes. The reaction efficiency was within the preferred range of 100 ± 10%. CIT was not detected or was below the detection limit when the Ct value of one or more related synthetic genes was greater than 33.5. Therefore, the established qPCR method can effectively predict the production of CIT in Liupao tea, and it is applicable to the judgment of whether fungi produce CIT.

Effect of pile fermentation on the cells of Chinese Liupao tea: The first record of cell wall of Liupao tea on transmission electron microscope.

Fermentation often degrades the cell wall of dark tea, changes the carbohydrate components in the cell wall of tea, and thus affects the quality of tea. However, the lack of ultrastructural details limits our knowledge on the effect of fermentation on tea cell walls. Morphological studies of cell structures are important; thus, the cell wall of Liupao tea was analyzed under transmission electron microscopy for the first time, and the effects of different raw materials and fermentation methods on the cell wall and main carbohydrates of tea were compared. Overall, fermentation degrades the cell wall of Liupao tea under the action of microorganisms. Interestingly, the middle lamella degrades obviously, whereas the primary wall is complete. The decrease in hemicellulose and increase in water-soluble pectin (WSP) were remarkable, whereas the changes in cellulose and WSP were considerably correlated with the increase in tea polysaccharide (TPS). The results suggest that cell wall degradation might be related to the increase in TPS.

Phenolic compounds, bioactivity, and bioaccessibility of ethanol extracts from passion fruit peel based on simulated gastrointestinal digestion.

Passion fruit peel, a potential source of bioactive compounds, has been used as food stabilizing agent. However, the phenolic composition and bioactivity of passion fruit peel have rarely been reported. The effects of simulated gastrointestinal digestion on the bioactive components, bioactivity and bioaccessibility of passion fruit peel ethanol extracts (PFPE) were investigated using high performance liquid chromatography-tandem mass spectrometry analysis (quasi-targeted metabolomics). Phenols (178) were identified, of which 25 inhibited alpha-glucosidase activity. The stabilities of PFPE phenols were significantly affected by pH changes and digestive enzymes during simulated digestion. The 1,1-diphenyl-2-picrylhydrazyl free radical scavenging capacity and ferric ion reducing antioxidant power were decreased by 32% and 30%, respectively, while 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) free radical scavenging capacity increased by 17%. Alpha-glucosidase inhibition decreased with decreased PFPE phenolic content. Therefore, passion fruit peel could be considered a source of natural antioxidants and alpha-glucosidase inhibitors.

Purification, characterization, and bioactivity of Liupao tea polysaccharides before and after fermentation.

The raw tea polysaccharides (RLTPS) and the aged tea polysaccharides (ALTPS) from raw and aged Liupao tea were extracted and purified to afford five refined fractions. Component analysis revealed that the crude polysaccharide content from raw Liupao tea increased from 1.83 ± 0.09 g / 100 g to 3.44 ± 0.28 g / 100 g and the molecular weight decreased after fermentation. Structural analysis indicated that the molar ratio of rhamnose, galactose, and galacturonic acid increased in refined ALTPS. All the refined polysaccharides were glycoprotein complexes contained pyranose ring structure. The thermal stability and asymmetry of refined ALTPS were stronger than refined RLTPS. For activities in vitro, ALTPS had better anticoagulant activity and bile acid binding capacity than RLTPS. Although the activities of the refined ALTPS fractions were lower than ALTPS, they were still higher than the refined RLTPS. Fermentation plays an important role in improving the quality and biological activity of dark tea.

Evaluation of Mycoflora and Citrinin Occurrence in Chinese Liupao Tea.

Citrinin (CIT), a known nephrotoxic mycotoxin, is mainly produced by Penicillium, Aspergillus, and Monascus species. It is a natural contaminant in cereal grains, foods, and feedstuff. Liupao tea (or Liubao tea) is a typical Chinese dark tea obtained via indigenous tea fermentation facilitated by microorganisms. Certain fungi present in Liupao tea that may produce CIT are a potential threat to consumer health. In the present study, various potential toxigenic mycoflora and the natural occurrence of CIT in Liupao tea were surveyed via the culture-dependent method, high performance liquid chromatography-fluorescence detection (HPLC-FLD), and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Total mold counts ranged from 3.5 × 102 CFU/g to 2.1 × 106 CFU/g tea in 28 tea samples. A total of 218 fungal isolates belonging to five genera and 23 species were identified. Some of these strains, such as Aspergillus ochraceus, Aspergillus oryzae, Penicillium citrinum, and Penicillium chrysogenum, may potentially be a CIT-producing species. In addition, 32.7% of 113 Liupao tea samples were contaminated with CIT at concentrations ranging from 7.8 to 206.1 µg/kg. These CIT concentrations in Liupao tea are chiefly attributed to climatic conditions and water activity during storage that favor fungal proliferation and mycotoxin production. However, CIT could not be detected in Liupao tea stored for over 10 years. These results provide the first information about the potential toxigenic mycoflora and natural occurrence of CIT in Liupao tea. Therefore, storage conditions and fungal community must be monitored to ensure the quality of Liupao tea.

Analyses of fungal community by Illumina MiSeq platforms and characterization of Eurotium species on Liupao tea, a distinctive post-fermented tea from China.

Liupao tea is a distinctive Chinese dark tea obtained by indigenous tea fermentation facilitated by the symbiotic association of bacteria and fungi. The composition of fungal community in 4 Liupao tea samples stored for several years under natural microbial fermentation was evaluated by MiSeq sequencing. Taxonomic analysis revealed 3 phyla, 6 families, 8 genera. The genera Eurotium and Aspergillus were dominant fungi in almost all the samples. A total of 85 strains found in 41 other tea samples were species of Eurotium. amstelodami, Eurotium. niveoglaucum, Eurotium. repens, Eurotium. rubrum, Eurotium. tonophilum and Eurotium. cristatum by culture-dependent method. Of these species, E. repens, E. rubrum and E. tonophilum have not been previously associated with Liupao tea. This report is the first to reveal fungal flora composition using Illumina-based sequencing and provide useful information for relevant studies on the isolation of Eurotium species in Liupao tea. The predominant molds are Eurotium species, and the comparison of fungal diversity in dark teas is worth considering. The taxonomic analysis of the microbial community would also aid the further study of functional genes and metabolic pathways of Liupao tea fermentation.

Pro-coagulant activity of phenolic acids isolated from Blumea riparia.

The effects of extracts of the aerial part of Blumea riparia DC. and their phenolic acids on hemostasis were evaluated. The EtOAc fraction showed significantly reduced blood clotting time (CT) and tail bleeding time of transection (BT) of mice in vivo. This fraction contained vanillic acid (1), syringic acid (2), p-coumaric acid (3), caffeic acid (4), and protocatechuic acid (5). Compound 1 reduced prothrombin time (PT), and strengthened mice uterine contractions. Compound 3 reduced CT and the activated partial thromboplastin time (APTT). Compound 5 reduced CT and increased the frequency of mice uterine contraction in a dose-dependent manner. Compound 2 reduced APTT. Compound 4 remarkably strengthened uterine contraction. Taken together, these data suggest that compounds 1, 3, and 5 possess procoagulant activity which jointly synergize blood coagulation via different mechanisms.