A total of 117 Bacillus strains were isolated from Miang, a culture relevant fermented tea of northern Thailand. These strains were collected from 16 sampling sites in north Thailand. In this collection 95 isolates were tannin-tolerant Bacillus capable of growth on nutrient agar supplemented with 0.5% (w/v) total tannins from tea leaves extract (TE). The strains were also positive for pectinase, xylanase and amylase activity, while 91 and 86 isolates were positive for cellulase and β-mannanase, respectively. Only 21 isolates producing extracellular tannase were selected for further characterization. Identification by 16S rRNA gene sequence analysis revealed that more than 50% (11 of 21 isolates) were Bacillus tequilensis, whereas the remaining were B. siamensis (3), B. megaterium (3), B. aryabhattai (3) and B. toyonensis (1). B. tequilensis K34.2 produced the highest extracellular tannase activity of 0.60 U/mL after cultivation at 37 °C for 48 h. In addition, all 21 isolates were resistant to 0.3% (w/v) bile salt, sensitive to gentamicin, erythromycin, vancomycin and kanamycin and also tolerant to acidic condition. Cell hydrophobicity varied from 9.4 to 80.4% and neutralized culture supernatants of some Bacillus isolates showed bacteriocin producing potentiality against Samonella enterica serovar Typhimurium TISTR 292. All tested probiotic properties indicated that B. tequilensis K19.3, B. tequilensis K34.2 and B. siamensis K19.1 had high probiotic potential. This is the first report describing tannin-tolerant Bacillus and their extracellular tannase producing capability in Miang, a traditional fermented tea of Thailand.

Miang, a traditional fermented tea from Northern Thailand, potentially hosts beneficial probiotic bacteria. A total of 133 isolates of lactic acid bacteria (LAB) isolated from Miang were evaluated for probiotic potential. Among them, 5 strains showed high tolerance to bile and acidic conditions and were selected for further evaluation. All selected strains showed inhibitory activity against human pathogens, including Bacillus cereus, Staphylococcus aureus, and Salmonella ser. Typhimurium. Nucleotide sequences analysis of the 16S rRNA gene revealed that 3 isolates were identified as Lactobacillus pentosus; the remaining were L. plantarum and Pediococcus pentosaceus, respectively. All 5 strains showed a high survival rate of more than 90% when exposed to simulated gastrointestinal conditions and were also susceptible to antibiotics such as erythromycin, tetracycline, and gentamycin, and resistant to vancomycin, streptomycin, and polymycin. In addition, the selected isolates exhibited different degrees of cell surface hydrophobicity (58.3–92.9%) and auto-aggregation (38.9–46.0%). The antioxidant activity reflected in DPPH scavenging activities of viable cells and their cell-free culture supernatants (CFCS) were also found in selected LAB isolates. Moreover, selected LAB isolates showed ability to grow on commercial prebiotics (GOS, FOS or XOS). The preliminary study of spray-drying using cyclodextrin as thermoprotectant suggested that all strains can be designed as a powdered formulation. L. pentosus A14-6 was the best strain, with high tolerance against simulated gastrointestinal conditions, high cell surface hydrophobicity, effective response to tested commercial oligosaccharides, especially XOS, and the highest cell antioxidant properties. L. pentosus A14-6 was therefore targeted for further applications in food and synbiotic applications.

Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) consumed in northern Thailand, was simulated in laboratory conditions using non-filamentous fungi process (NFP) and microbial community was periodically investigated for over 6 months of fermentation by both culture-dependent and -independent techniques. The viable cell numbers of lactic acid bacteria (LAB), yeast, and Bacillus enumerated by the culture-dependent technique markedly surged over 3 days of initial fermentation and then smoothly declined by the end of fermentation. LAB were found as the main microbial population throughout the fermentation period followed by yeast and Bacillus. High-throughput sequencing of microbial community during fermentation revealed that Firmicutes (86.9–96.0%) and Proteobacteria (4.0–12.4%) were the dominant bacterial phyla, whereas Ascomycota was found to be the main fungal phylum with an abundance of over 99% in the fungal community. The dominant bacterial family was Lactobacillaceae (39.7–79.5%) followed by Acetobacteraceae, Enterobacteriaceae, Bacillaceae, Aeromonadaceae, Staphylococcaceae, Moraxellaceae, Clostridiaceae, Exiguobacteraceae, Streptococcaceae, and Halomonadaceae. Meanwhile, the main fungal family was incertae sedis Saccharomycetales (75.6–90.5%) followed by Pichiaceae, Pleosporaceae, Botryosphaeriaceae, Davidiellaceae, Mycosphaerellaceae, and Saccharomycodaceae. In addition, Lactobacillus (29.2–77.2%) and Acetobacter (3.8–22.8%), and the unicellular fungi, Candida (72.5–89.0%) and Pichia (8.1–14.9%), were the predominant genera during the fermentation process. The profiles of physical and chemical properties such as Miang texture, pH, organic acids, polysaccharide-degrading enzyme activities, and bioactive compounds have rationally indicated the microbial fermentation involvement. β-Mannanase and pectinase were assumed to be the key microbial enzymes involved in the Miang fermentation process. Total tannin and total polyphenol contents were relatively proportional to the antioxidant activity. Lactic acid and butyric acid reached maximum of 50.9 and 48.9 mg/g dry weight (dw) at 9 and 63 days of fermentation, respectively. This study provided essential information for deeper understanding of the Miang fermentation process based on the chemical and biological changes during production.

High performance liquid chromatography (HPLC) for catechins and related compounds in Miang (traditional Lanna fermented tea leaf) was developed to overcome the matrices during the fermentation process. We investigated a variety of columns and elution conditions to determine seven catechins, namely (+)-catechin, (−)-gallocatechin, (−)-epigallocatechin, (−)-epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, (−)-epicatechin gallate, as well as gallic acid and caffeine, resulting in the development of reproducible systems for analyses that overcome sample matrices. Among the three reversed-phase columns, column C (deactivated, with extra dense bonding, double endcapped monomeric C18, high-purity silica at 3.0 mm × 250 mm and a 5 µm particle size) significantly improved the separation between Miang catechins in the presence of acid in the mobile phase within a shorter analysis time. The validation method showed effective linearity, precision, accuracy, and limits of detection and quantitation. The validated system was adequate for the qualitative and quantitative measurement of seven active catechins, including gallic acid and caffeine in Miang, during the fermentation process and standardization of Miang extracts. The latter contain catechins and related compounds that are further developed into natural active pharmaceutical ingredients (natural APIs) for cosmeceutical and nutraceutical products.