Isolation of catechin-producing endophytic fungi from Camellia sinensis and influence of catechin nanoparticles on gut microbiota

Abstract

Tea or Camellia sinensis (L.) Kuntze is the second most renowned and consumed beverage, after water. Tea has garnered significant interest as a functional food because of its numerous health advantages, especially in combating non-communicable chronic metabolic conditions. The health-promoting properties of tea are mainly ascribed to its bioactive catechins, viz., catechin, epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin-o-gallate (EGCG), and gallocatechin gallate (GCG). Clinical and preclinical investigations have demonstrated that catechin-rich green tea alleviates diet-induced metabolic distress through gut-level mechanisms, involving the attenuation of dysbiosis and strengthening of the intestinal barrier function to minimize endotoxin-induced inflammation along the gut-liver and gut-adipose axis. The drawbacks of plant-based catechin extraction, including lengthy cultivation periods, seasonal availability, climate-dependent yields, and the overharvesting of plant resources that jeopardize biodiversity, necessitate the search for sustainable and effective alternative sources of bioactive catechins due to their increasing demand for metabolic health-promoting purposes. One such alternative source of catechins could be the endophytic fungi that live in tea plant tissues without harming the host plants. Such symbionts represent an underexplored resource of novel bioactive chemicals and can generate a variety of secondary metabolites that are found in the host plants.

In this work, we examined the variety of endophytes from C. sinensis that can produce catechins. Various fungi were isolated from C. sinensis leaves, procured from the northern Himalayan region, India. However, only four isolates (CSPL6, CSPL6’, CSPL4, and CSPL5b) were found to produce catechin (381.48, 81.75, 12.28, and 166.40 μg/mg of extract, respectively) and EGCG (484.41, 67.29, 277.34, and 281.99 μg/mg of extract, respectively), as validated by high-performance liquid chromatography (HPLC). The isolated fungal strains were distinguished based on colony characteristics and molecular approaches as Pseudopestalotiopsis camelliae-sinensis, Aspergillus aculeatus, Phyllosticta capitalensis, and Didymella sinensis. These provide the first evidence of fungal endophytes that were able to synthesize catechins and EGCG from C. sinensis plant leaves. The gas chromatography-mass spectrometry (GC-MS)-based untargeted metabolomics indicated several pharmacologically important phytochemicals, mostly belonging to classes of citrates, tyrosols, pyridoxines, cinnamic acids, fatty acids, aminopyrimidine, and benzenetriol. The isolates that produced catechins had greatly enriched metabolic pathways related to the formation of butanoate, linoleic, and other fatty acids. The isolates were able to scavenge different intracellular free radicals to varying degrees. This study provides valuable insights regarding catechin-producing endophytes from the tea plant and their free-radical scavenging bioactivities, that could potentially serve to alleviate chronic diseases. Although all four isolates demonstrated effective scavenging activity and antioxidant potential against key intracellular free radicals, CSPL5b showed comparatively higher bioactivities than CSPL6, CSPL6’, and CSPL4. All four fungal extracts enhanced the growth of various probiotic Lactobacillus strains: L. sporogenes, L. rhamnosus, L. plantarum, and L. reuteri at low concentrations (1-8 μg/mL), indicating prebiotic effects that are typically linked to catechins.

The catechins are of immense scientific and industrial attention due to their prebiotic and antioxidant applications and have remarkable effects on gut health. But their effectiveness mainly depends on their absorption, bioavailability, stability, and their interaction with gut microbiota. So, to enhance the functions and properties of the catechins, the EGCG-chitosan nanoparticles (EGCG-CNPs) with sodium tripolyphosphate (TPP) were prepared by the ionic cross-linking method. The nanoparticles synthesized under optimum conditions demonstrated a 53% encapsulation effectiveness, an average particle size of 188.14±21.86 nm, polydispersity index (PDI=0.398), and zeta potential (38.15±2.56 mV). The synthesis of the composite nanoparticles and the formation of new hydrogen bonds between EGCG and chitosan were further demonstrated by the findings of the Scanning Electron Microscope (SEM), X-Ray Diffraction (XRD), and Fourier Transform Infrared (FTIR) analyses. EGCG-CNPs were more effective in preventing EGCG from degrading quickly, as validated by HPLC. Compared to its free form, EGCG-CNPs facilitated time-controlled sustained release from 0 to 12 h. The conformation and structure of chitosan may be altered due to the presence of EGCG. In vitro anaerobic fermentation of synthesized particles affected gut microbial composition, abundance, diversity, richness, and metabolic processes. EGCG-CNPs significantly boost microbial diversity and beneficial short-chain fatty acids (SCFAs)-producing commensals (e.g., Lactobacillus and Bifidobacterium), emphasizing the potential health benefits. The GC-MS-based untargeted metabolomics enabled the detection of diverse gut microbial metabolites such as indoles, amino acids, carbohydrates, phenolics, and sugars, which the EGCG-CNPs impacted.

Collectively, this study reports catechin-producing endophytes from tea leaves that not only possess potent bioactivities but can also be utilized as an alternative and sustainable source of bioactive phytochemicals, especially catechins. EGCG-loaded chitosan particles were produced using an ionic gelation technique. These nanoparticles can act as a unique delivery system for catechins, have great potential to improve the stability of EGCG, protect from gut microbiota-dependent metabolism, and boost the beneficial gut microbiota.