Study of Lactobacillus plantarum coated with Tremella polysaccharides to improve its intestinal adhesion.

BACKGROUND: The adhesion of probiotics to the intestine is crucial for their probiotic function. In previous studies, Tremella polysaccharides (TPS) (with sodium casein) have shown the potential to encapsulate probiotics and protect them in a simulated gastrointestinal tract. This study explored the effect of TPS (with sodium casein) on the adhesion of probiotics. RESULTS: Lactobacillus plantarum was coated with TPS and sodium casein in different proportions, and was freeze-dried. The rheological properties of the mixture of probiotics powder and mucin solution were determined by static and dynamic rheological analysis. Aqueous solutions of probiotic powder and mucin mixture exhibited pseudoplastic fluid rheological properties. The higher the proportion of TPS content, the higher the apparent viscosity and yield stress. The mixed bacterial powder and mucin fluid displayed thixotropy and was in accordance with the Herschel-Bulkley model. The TPS increased the bio-adhesive force of the probiotic powder and mucin. When using TPS as the only carbon source, the adhesion of L. plantarum to Caco-2 cells increased by 228% in comparison with glucose in vitro. Twelve adhesive proteins were also detected in the whole-cell proteome of L. plantarum. Among them, ten adhesive proteins occurred abundantly when grown with TPS as a carbon source. CONCLUSION: Tremella polysaccharides therefore possess probiotic properties and can promote the intestinal adhesion of L. plantarum. © 2024 Society of Chemical Industry.

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.

Deciphering the underlying core microorganisms and the marker compounds of Liupao tea during the pile-fermentation process.

BACKGROUND: Pile fermentation is one of the key steps in developing the Liupao tea (LBT) quality and unique characteristics. The complex biochemical profile of LBT results from microorganisms present during the pile-fermentation process. However, the critical underlying microorganisms and the marker compounds still need to be determined. RESULTS: Staphylococcus, Brevibacterium, Kocuria, Aspergillus, and Blastobotrys were the common dominant microorganisms at the end of the pile fermentation of LBT. Staphylococcus, Aspergillus, Blastobotrys, and nine other genera carried by raw tea are the core microorganisms in the LBT during pile fermentation. A total of 29 critical compounds contributed to the metabolic changes caused by the processing of LBT. Of these, gallic acid, adenine, hypoxanthine, uridine, betaine, 3,4-dihydroxybenzaldehyde, and α-linolenic acid could be characterized as potential marker compounds. Correlation analysis showed that the core microorganisms, including Sphingomonas, Staphylococcus, Kocuria, Aureobasidium, Blastobotrys, Debaryomyce, and Trichomonascus, were closely related to major chemical components and differential compounds. Moreover, the mutually promoting Staphylococcus, Kocuria, Blastobotrys, and Trichomonascus were correlated with the enrichment of marker compounds. Integrated molecular networking and metabolic pathways revealed relevant compounds and enzymes that possibly affect the enrichment of marker compounds. CONCLUSION: This study analyzed the LBT fermentation samples by omics analysis to reveal the stable microbial community structure, critical microorganisms, and markers compounds affecting the quality of LBT, which contributes to a better understanding of pile fermentation of LBT and the fermentation theory of dark tea. © 2023 Society of Chemical Industry.

Isolation, identification, and community diversity of microorganisms during tank fermentation of Liupao tea.

Tank fermentation is a novel approach to fermenting teas; however, the species of microorganisms present remain unclear. The microbial community composition of Liupao tea at various stages of tank fermentation was analyzed using high-throughput sequencing. Sphingomonas, Aquabacterium, Pelomonas, Acinetobacter, Blastobotrys, Aspergillus, Debaryomyces, and Aureobasidium were the predominant genera, which is different from pile fermentation. Fifteen genera (including Lactobacillus, Debaryomyces, Candida, Allobaculum, Flavobacterium, Caulobacter, Blastobotrys, Aspergillus, and Rasamsonia) were identified as biomarkers. PICRUSt analysis predicted that the most abundant functional genes were related to metabolism of carbohydrates, amino acids, cofactors, vitamins, and other secondary metabolites. Using the pure culture method, 283 strains were isolated at various stages of fermentation, representing 20 genera and 29 species of bacteria, and 11 genera and 18 species of fungi. Most of the dominant Sphingomonas, Staphylococcus, Aspergillus, and Blastobotrys identified by sequencing were also isolated. Of these, Sphingomonas olei, Aspergillus luchuensis, Aspergillus niger, Aspergillus aculeatus, Aspergillus amstelodami, Blastobotrys adeninivorans, Candida metapsilosis, and Candida blankii were beneficial strains that might be used to ferment Liupao tea. This study provides a basis for the development of processing technologies and utilization of microbial strains in the production of dark teas. PRACTICAL APPLICATION: Microbial diversity in tank-fermented Liupao tea was reported for the first time. 8 microorganisms were the predominant genera. The species, functions and potential risks of microorganisms was revealed. We clarified the differences between tank and pile fermentation.

Structural Characterization and In Vitro Antioxidant, Hypoglycemic and Hypolipemic Activities of a Natural Polysaccharide from Liupao Tea.

This study extracted and purified a natural polysaccharide (TPS-5) that has a molecular weight of 48.289 kDa from Liupao tea, a typical dark tea with many benefits to human health. TPS-5 was characterized as a pectin-type acidic polysaccharide. It has a backbone composed of → 2,4)- α- L-Rhap-(1) → 4)- α- D-GalAp-(1) →, with a branch composed of → 5)- α- L-Ara-(1 → 5,3)- α- L-Ara-(1 → 3)- ß- D-Gal-(1 → 3,6)- ß- D-Galp-(1) →. The in vitro biological activity evaluation illustrated that TPS-5 has free radical scavenging, ferric-ion-reducing, digestive enzyme inhibitory, and bile-salt-binding abilities. These results suggest that TPS-5 from Liupao tea has potential applications in functional foods or medicinal products.

Effects of cross-linking of rice protein with ferulic acid on digestion and absorption of ferulic acid.

Though rice proteins have been applied to improve the stability of phenolic compounds, it is unclear how rice proteins affect phenolic acid's digestion and bioavailability. This study investigated the consequences of protein-ferulic acid interactions in the gastrointestinal environment. Ferulic acid and rice proteins formed complexes at room temperature, both with and without laccase. Rice protein was reported to be able to prevent ferulic acid from degrading in simulated oral fluid and remain stable in gastrointestinal fluids. With the hydrolysis of pepsin and pancreatin, rice protein-ferulic acid complexes degraded and released ferulic acid. While digested ferulic acid's DPPH scavenging activity was dramatically reduced, it was retained for the rice protein-ferulic acid complex. Moreover, the permeability coefficient of ferulic acid was not affected. Thus, rice protein is a promising food matrix to protect ferulic acid in the digestive tract and maintain the antioxidant functions of ferulic acid.

Genome-wide transcriptome analysis uncovers gene networks regulating fruit quality and volatile compounds in mango cultivar 'Tainong' during postharvest.

Mango is one of the most economically important fruit; however, the gene regulatory mechanism associated with ripening and quality changes during storage remains largely unclear. This study explored the relationship between transcriptome changes and postharvest mango quality. Fruit quality patterns and volatile components were obtained using headspace gas chromatography and ion-mobility spectrometry (HS-GC-IMS). The changes in mango peel and pulp transcriptome were analyzed during four stages (pre-harvesting, harvesting, maturity, and overripe stages). Based on the temporal analysis, multiple genes involved in the biosynthesis of secondary metabolites were upregulated in both the peel and pulp during the mango ripening process. Moreover, cysteine and methionine metabolism related to ethylene synthesis were upregulated in the pulp over time. Weighted gene co-expression network analysis (WGCNA) further showed that the pathways of pyruvate metabolism, citrate cycle, propionate metabolism, autophagy, and SNARE interactions in vesicular transport were positively correlated with the ripening process. Finally, a regulatory network of important pathways from pulp to peel was constructed during the postharvest storage of mango fruit. The above findings provide a global insight into the molecular regulation mechanisms of postharvest mango quality and flavor changes.

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.

Two Polysaccharides from Liupao Tea Exert Beneficial Effects in Simulated Digestion and Fermentation Model In Vitro.

Liupao tea is an important dark tea, but few studies on purified Liupao tea polysaccharide (TPS) are reported in the literature. In this study, two TPSs, named TPS2 and TPS5, with molecular weights of 70.5 and 133.9 kDa, respectively, were purified from Liupao tea. TPS2 contained total sugar content (53.73% ± 1.55%) and uronic acid content (35.18% ± 0.96%), while TPS5 was made up of total sugar (51.71% ± 1.1%), uronic acid (40.95% ± 3.12%), polyphenols (0.43% ± 0.03%), and proteins (0.11% ± 0.07%). TPS2 and TPS5 were composed of Man, Rha, GlcA, Glc, Gal, and Ara in the molar ratios of 0.12:0.69:0.20:0.088:1.60:0.37 and 0.090:0.36:0.42:0.07:1.10:0.16, respectively. The effects of TPS2 and TPS5 on digestion and regulation of gut microbiota in hyperlipidemic rats were compared. In simulated digestion, TPS5 was degraded and had good antioxidant effect, whereas TPS2 was not affected. The bile acids binding capacities of TPS2 and TPS5 were 42.79% ± 1.56% and 33.78% ± 0.45%, respectively. During in vitro fermentation, TPS2 could more effectively reduce pH, promote the production of acetic acid and propionic acid, and reduce the ratio of Firmicutes to Bacteroidetes. TPS5 could more effectively promote the production of butyric acid and increase the abundance of genus Bacteroides. Results indicate that polysaccharides without polyphenols and proteins have better antidigestibility and bile acid binding. Meanwhile, polysaccharides with polyphenols and proteins have a better antioxidant property. Both have different effects on the gut microbiota.

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.