Bacillus spp. Isolated from Miang as Potential Probiotics in Nile Tilapia Culture-In Vitro Research.

Among 79 Bacillus spp. isolated from Miang, a fermented tea in north Thailand, 17 Bacillus strains were selected with probiotic potential in Nile tilapia culture based on the capabilities of bacteriocin production and associated antimicrobial activities against fish pathogens, Aeromonas hydrophila and Streptococcus agalactiae. However, only six isolates were selected for further extensive studies based on the strength of their antimicrobial activities and their tolerance against simulated gastrointestinal conditions. The molecular identification by 16S rRNA gene sequence analysis revealed that five isolates, K2.1, K6.1, K7.1, K15.4, and K22.6, were Bacillus tequilensis, and the isolate K29.2 was Bacillus siamensis. B. siamensis K29.2 showed complete susceptibility to antibiotics tested in this study, while B. tequilensis K 15.4 showed moderate resistance to some antibiotics; therefore, both strains were selected as potential probiotic bacteria. B. tequilensis K15.4 and B. siamensis K29.2 were capable of the production and secretion of extracellular protease and polysaccharide degrading enzymes, including cellulase, xylanase, and ß-mannanase. The tannin tolerant test also demonstrated their ability to grow on selective agar plates and secrete cellulase and ß-mannanase in the presence of hydrolyzable tannin. In addition, in vitro digestion of commercial fish substrate revealed that the extracellular enzymes produced by both strains efficiently reacted with feed protein and polysaccharides. Based on the results from this study, B. siamensis K29.2 was deemed to have the highest potential multifunctional probiotic qualities for application in Nile tilapia culture, while the antibiotic-resistant gene in B. tequilensis K15.4 must be clarified before field application.

Antibacterial Activities of Phenolic Compounds in Miang Extract: Growth Inhibition and Change in Protein Expression of Extensively Drug-Resistant Klebsiella pneumoniae.

The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25-0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria.

Ultrasonic-assisted enzymatic improvement of polyphenol content, antioxidant potential, and in vitro inhibitory effect on digestive enzymes of Miang extracts.

The aims of this research were to optimize the ultrasonic-assisted enzymatic extraction of polyphenols under Miang and tannase treatment conditions for the improvement of antioxidant activity of Miang extracts via response surface methodology. Miang extracts treated with and without tannase were investigated for their inhibitory effects on digestive enzymes. The optimal conditions for ultrasonic-assisted enzymatic extraction of the highest total polyphenol (TP) (136.91 mg GAE/g dw) and total flavonoid (TF) (5.38 mg QE/g dw) contents were as follows: 1 U/g cellulase, 1 U/g xylanase, 1 U/g pectinase, temperature (74 °C), and time (45 min). The antioxidant activity of this extract was enhanced by the addition of tannase obtained from Sporidiobolus ruineniae A45.2 undergoing ultrasonic treatment and under optimal conditions (360 mU/g dw, 51 °C for 25 min). The ultrasonic-assisted enzymatic extraction selectively promoted the extraction of gallated catechins from Miang. Tannase treatment improved the ABTS and DPPH radical scavenging activities of untreated Miang extracts by 1.3 times. The treated Miang extracts possessed higher IC50 values for porcine pancreatic α-amylase inhibitory activity than those that were untreated. However, it expressed approximately 3 times lower IC50 values for porcine pancreatic lipase (PPL) inhibitory activity indicating a marked improvement in inhibitory activity. The molecular docking results support the contention that epigallocatechin, epicatechin, and catechin obtained via the biotransformation of the Miang extracts played a crucial role in the inhibitory activity of PPL. Overall, the tannase treated Miang extract could serve as a functional food and beneficial ingredient in medicinal products developed for obesity prevention.

Three new yeast species from flowers of Camellia sinensis var. assamica collected in Northern Thailand and their tannin tolerance characterization.

Our recent research study focused on Miang fermentation revealed that tannin-tolerant yeasts and bacteria play vital roles in the Miang production process. A high proportion of yeast species are associated with plants, insects, or both, and nectar is one of the unexplored sources of yeast biodiversity. Therefore, this study aimed to isolate and identify yeasts of tea flowers of Camellia sinensis var. assamica and to investigate their tannin tolerance, which is a property essential to Miang production processes. A total of 82 yeasts were recovered from a total of 53 flower samples in Northern Thailand. It was found that two and eight yeast strains were distinct from all other known species within the genera Metschnikowia and Wickerhamiella, respectively. These yeast strains were described as three new species, namely, Metschnikowia lannaensis, Wickerhamiella camelliae, and W. thailandensis. The identification of these species was based on phenotypic (morphological, biochemical, and physiological characteristics) and phylogenetic analyses of a combination of the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) ribosomal RNA gene. The yeast diversity in tea flowers acquired from Chiang Mai, Lampang, and Nan provinces had a positive correlation with those acquired from Phayao, Chiang Rai, and Phrae, respectively. Wickerhamiella azyma, Candida leandrae, and W. thailandensis were the species uniquely found in tea flowers collected from Nan and Phrae, Chiang Mai, and Lampang provinces, respectively. Some of the tannin-tolerant and/or tannase-producing yeasts were associated with yeasts in the commercial Miang process and those found during Miang production, i.e., C. tropicalis, Hyphopichia burtonii, Meyerozyma caribbica, Pichia manshurica, C. orthopsilosis, Cyberlindnera fabianii, Hanseniaspora uvarum, and Wickerhamomyces anomalus. In conclusion, these studies suggest that floral nectar could support the formation of yeast communities that are beneficial for Miang production.

Assessment of Tannin Tolerant Non-Saccharomyces Yeasts Isolated from Miang for Production of Health-Targeted Beverage Using Miang Processing Byproducts.

This research demonstrated an excellent potential approach for utilizing Miang fermentation broth (MF-broth), a liquid residual byproduct from the Miang fermentation process as a health-targeted beverage. One hundred and twenty yeast strains isolated from Miang samples were screened for their potential to ferment MF-broth and four isolates, P2, P3, P7 and P9 were selected, based on the characteristics of low alcoholic production, probiotic properties, and tannin tolerance. Based on a D1/D2 rDNA sequence analysis, P2 and P7 were identified to be Wikerhamomyces anomalus, while P3 and P9 were Cyberlindnera rhodanensis. Based on the production of unique volatile organic compounds (VOCs), W. anomalus P2 and C. rhodanensis P3 were selected for evaluation of MF-broth fermentation via the single culture fermentation (SF) and co-fermentation (CF) in combination with Saccharomyces cerevisiae TISTR 5088. All selected yeasts showed a capability for growth with 6 to 7 log CFU/mL and the average pH value range of 3.91-4.09. The ethanol content of the fermented MF-broth ranged between 11.56 ± 0.00 and 24.91 ± 0.01 g/L after 120 h fermentation, which is categorized as a low alcoholic beverage. Acetic, citric, glucuronic, lactic, succinic, oxalic and gallic acids slightly increased from initial levels in MF-broth, whereas the bioactive compounds and antioxidant activity were retained. The fermented MF-broth showed distinct VOCs profiles between the yeast groups. High titer of isoamyl alcohol was found in all treatments fermented with S. cerevisiae TISTR 5088 and W. anomalus P2. Meanwhile, C. rhodanensis P3 fermented products showed a higher quantity of ester groups, ethyl acetate and isoamyl acetate in both SF and CF. The results of this study confirmed the high possibilities of utilizing MF-broth residual byproduct in for development of health-targeted beverages using the selected non-Saccharomyces yeast.

Local Wisdom and Diversity of Medicinal Plants in Cha Miang Forest in Mae Kampong Village, Chiang Mai, Thailand, and Their Potential for Use as Osteoprotective Products.

"People-Forest-Miang" communities are villages located in the cultivated area of Camellia sinensis var. assamica, or Cha Miang, in northern Thailand. Cha Miang forests are a form of agriculture relying on forest-rich bioresources. This study focuses on a survey of the diversity of medicinal plants used by "People-Forest-Miang" communities in Mae Kampong Village, Chiang Mai, Thailand. The results demonstrated that 73 species of medicinal plants were used to prevent and treat various ailments. The highest number of species (30.14%) was used for musculoskeletal system disorders, followed by digestive system disorders (21.92%) and unspecified medicinal disorders (15.07%). The alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. The ALP activity of ethanol and deionized water extracts of the nine selected medicinal plants used for musculoskeletal system disorders were examined in the MG63 cell line. The results showed that the numerous water extracts, including MKP1, MKP2, MKP5, MKP6, MKP7, MKP8, and MKP9, and the ethanolic extracts-namely, MKP2, MKP3, MKP7, and MKP9-significantly increased ALP activity in the MG-63 cell line. The findings indicate that some medicinal plants may be further studied for active chemicals and developed as natural active pharmaceutical ingredients for osteoprotective products.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

Development of HPLC Method for Catechins and Related Compounds Determination and Standardization in Miang (Traditional Lanna Fermented Tea Leaf in Northern Thailand).

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.

Comparison of Phenolic Contents and Scavenging Activities of Miang Extracts Derived from Filamentous and Non-Filamentous Fungi-Based Fermentation Processes.

The study investigated the impact of the fermentation process on the phenolic contents and antioxidant and anti-inflammatory activities in extracts of Miang, an ethnic fermented tea product of northern Thailand. The acetone (80%) extraction of Miang samples fermented by a non-filamentous fungi-based process (NFP) and filamentous fungi-based process (FFP) had elevated levels of total polyphenols, total tannins, and condensed tannins compared to young and mature tea leaves. The antioxidant studies also showed better the half-maximal inhibitory concentration (IC50) values for fermented leaves in both 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity assays as well as improved ferric reducing antioxidant power (FRAP) compared to young and mature tea leaves. Extracts of NFP and FFP samples at concentrations of 50 and 100 ppm showed better protective effects against hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species (ROS) production in HT-29 colorectal cells without exerting cytotoxicity. Additionally, lipopolysaccharide (LPS)-induced production of nitric oxide (a proinflammatory mediator as well as a reactive nitrogen species) was also inhibited by these fermented Miang extracts with an IC50 values of 17.15 µg/mL (NFP), 20.17 µg/mL (FFP), 33.96 µg/mL (young tea leaves), and 31.33 µg/mL (mature tea leaves). Therefore, both NFP-Miang and FFP-Miang showed the potential to be targeted as natural bioactive functional ingredients with preventive properties against free radical and inflammatory-mediated diseases.

Acid Stable Yeast Cell-Associated Tannase with High Capability in Gallated Catechin Biotransformation.

Previously, nine tannin-tolerant and tannase-producing yeasts were isolated from Miang; all produced cell-associated tannase (CAT) during growth in tannin substrate. Among which, only CAT from Sporidiobolus ruineniae showed better stability than its purified form. Yet, it is of particular interest to directly characterize CATs from the latter yeasts. In this study, four CATs from yeasts, namely Cyberlindnera rhodanensis A22.3, Candida sp. A39.3, Debaryomyces hansenii A45.1, and Cy. rhodanensis A45.3 were characterized. The results indicate that all CATs were produced within the same production yield (11 mU/mL). Most CATs exhibited similar pH and temperature optima and stabilities, except for CAT from Cy. rhodanensis A22.3. This CAT was assigned as acid-stable tannase due to its unusual optimum pH of 2.0 with pH stability and half-life thermostability in the range of pH 2.0-4.0, and 70 °C, respectively. All CATs demonstrated high substrate specificity toward epigallocatechin gallate and epicatechin gallate, thus forming epigallocatechin and epicatechin, respectively. Moreover, they showed operational stability to repeated use for up to five cycles without loss of the initial activity. Therefore, CATs from these yeasts could be useful for the extraction and biotransformation of tea catechins and related applications.

Validated HPTLC and antioxidant activities for quality control of catechin in a fermented tea (Camellia sinensis var. assamica).

Miang, a Thai traditional fermented tea (Camellia sinensis var. assamica), is exploited as nutraceutical and cosmeceutical ingredients despite limited standardization studies. Thus, this research aimed to develop a simple and rapid method for miang quality control using catechin and high-performance thin-layer chromatography (HPTLC) validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the Association of Official Analytical Collaboration (AOAC). The developing solvent consisting of toluene: ethyl acetate: acetone: formic acid (6:6:6:1 v/v/v/v) showed acceptable specificity with R f value of 0.54 ± 0.02 and linearity with correlation coefficient of 0.9951. The recovery was 98.84%-103.53%, and the RSD of intra- and inter-day precision was 0.70%-3.00% and 1.93%-4.94%, respectively. Miang ethyl acetate fraction is suggested to be attractive ingredient due to rich catechin (25.78 ± 0.53%), prolonged stability at 40 ◦C, and strong antioxidants determined by the assays of ABTS (IC50 = 3.32 ± 0.74 mg/ml), FRAP (89.05 ± 15.49 mg equivalent of FeSO4/g), and inhibition of lipid peroxidation (IC50 = 4.36 ± 0.67 mg/ml).

The Effect of Steaming and Fermentation on Nutritive Values, Antioxidant Activities, and Inhibitory Properties of Tea Leaves.

Fermented tea (Cha-miang in Thai) is a local product made by traditional food preservation processes in Northern Thailand that involve steaming fresh tea leaves followed by fermenting in the dark. Information on changes in nutritive values, bioactive compounds, antioxidant activities, and health properties that occur during the steaming and fermenting processes of tea leaves is, however, limited. Changes in nutritive values, phenolics, antioxidant activities, and in vitro health properties through inhibition of key enzymes that control obesity (lipase), diabetes (α-amylase and α-glucosidase), hypertension (angiotensin-converting enzyme (ACE)), and Alzheimer's disease (cholinesterases (ChEs) and ß-secretase (BACE-1)) of fermented tea were compared to the corresponding fresh and steamed tea leaves. Results showed that energy, carbohydrate, and vitamin B1 increased after steaming, while most nutrients including protein, dietary fiber, vitamins (B2, B3, and C), and minerals (Na, K, Ca, Mg, Fe, and Zn) decreased after the steaming process. After fermentation, energy, fat, sodium, potassium, and iron contents increased, while calcium and vitamins (B1, B2, B3, and C) decreased compared to steamed tea leaves. However, the contents of vitamin B1 and iron were insignificantly different between fresh and fermented tea leaves. Five flavonoids (quercetin, kaempferol, cyanidin, myricetin, and apigenin) and three phenolic acids (gallic acid, caffeic acid, and p-coumaric acid) were identified in the tea samples. Total phenolic content (TPC) and antioxidant activities increased significantly after steaming and fermentation, suggesting structural changes in bioactive compounds during these processes. Steamed tea exhibited high inhibition against lipase, α-amylase, and α-glucosidase, while fermented tea possessed high anti-ChE and anti-ACE activities. Fresh tea exhibited high BACE-1 inhibitory activity. Results suggest that tea preparations (steaming and fermentation) play a significant role in the amounts of nutrients and bioactive compounds, which, in turn, affect the in vitro health properties. Knowledge gained from this research will support future investigations on in vivo health properties of fermented tea, as well as promote future food development of fermented tea as a healthy food.

Culturable Bacterial Community on Leaves of Assam Tea (Camellia sinensis var. assamica) in Thailand and Human Probiotic Potential of Isolated Bacillus spp.

Assam tea plants (Camellia sinensis var. assamica) or Miang are found in plantations and forests of Northern Thailand. Leaf fermentation has been performed for centuries, but little information is available about their associated microbial community. One hundred and fifty-seven bacterial isolates were isolated from 62 Assam tea leaf samples collected from 6 provinces of Northern Thailand and classified within the phyla of Firmicutes, Actinobacteria, and Proteobacteria. Phayao and Phrae provinces exhibited the highest and the lowest bacterial diversities, respectively. The bacterial community structural pattern demonstrated significant differences between the west and the east sides. Since some Bacillus spp. have been reported to be involved in fermented Miang, Bacillus spp. isolated in this study were chosen for further elucidation. Bacillus siamensis ML122-2 exhibited a growth inhibitory effect against Staphylococcus aureus ATCC 25923 and MRSA DMST 20625, and the highest survival ability in simulated gastric and intestinal fluids (32.3 and 99.7%, respectively), autoaggregation (93.2%), cell surface hydrophobicity (50.0%), and bacterial adherence with Vero cells (75.8% of the control Lactiplantibacillusplantarum FM03-1). This B. siamensis ML122-2 is a promising probiotic to be used in the food industry and seems to have potential antibacterial properties relevant for the treatment of antibiotic-resistant infections.

Microbial Community Dynamics During the Non-filamentous Fungi Growth-Based Fermentation Process of Miang, a Traditional Fermented Tea of North Thailand and Their Product Characterizations.

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. ß-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.

Co-production of gallic acid and a novel cell-associated tannase by a pigment-producing yeast, Sporidiobolus ruineniae A45.2.

BACKGROUND: Gallic acid has received a significant amount of interest for its biological properties. Thus, there have been recent attempts to apply this substance in various industries and in particular the feed industry. As opposed to yeasts, fungi and bacteria and their tannases have been well documented for their potential bioconversion and specifically for the biotransformation of tannic acid to gallic acid. In this research, Sporidiobolus ruineniae A45.2 is introduced as a newly pigment-producing and tannase-producing yeast that has gained great interest for its use as an additive in animal feed. However, there is a lack of information on the efficacy of gallic acid production from tannic acid and the relevant tannase properties. The objective of this research study is to optimize the medium composition and conditions for the co-production of gallic acid from tannic acid and tannase with a focus on developing an integrated production strategy for its application as a feed additive. RESULTS: Tannase produced by S. ruineniae A45.2 has been classified as a cell-associated tannase (CAT). Co-production of gallic acid obtained from tannic acid and CAT by S. ruineniae A45.2 was optimized using response surface methodology and then validated with the synthesis of 11.2 g/L gallic acid from 12.3 g/L tannic acid and the production of 31.1 mU/mL CAT after 48 h of cultivation in a 1-L stirred tank fermenter. Tannase was isolated from the cell wall, purified and characterized in comparison with its native form (CAT). The purified enzyme (PT) revealed the same range of pH and temperature optima (pH 7) as CAT but was distinctively less stable. Specifically, CAT was stable at up to 70 °C for 60 min, and active under its optimal conditions (40 °C) at up to 8 runs. CONCLUSION: Co-production of gallic acid and CAT is considered an integrated and green production strategy. S. ruineniae biomass could be promoted as an alternative source of carotenoids and tannase. Thus, the biomass, in combination with gallic acid that was formed in the fermentation medium, could be directly used as a feed additive. On the other hand, gallic acid could be isolated and purified for food and pharmaceutical applications. This paper is the first of its kind to report that the CAT obtained from yeast can be resistant to high temperatures of up to 70 °C.

Nutritional biotransformation in traditional fermented tea (Miang) from north Thailand and its impact on antioxidant and antimicrobial activities.

Miang is a traditional fermented tea made from fermentation of Assam tea leaves with mixed microbial culture involving lactic acid bacteria and yeast. Miang has important bioactive benefits such as antioxidant and antimicrobial activity with relevance to health benefits. Miang is categorized into two processes; filamentous fungi growth-based (FFP) and non-filamentous fungi-based (NFP) process, depending on area of production. Further, Miang is also divided into 2 types; astringent Miang and sour Miang, depending on fermentation time. The aim of this research was to determine the important macronutrient biotransformation of Miang diversity under above processes and types and explore the impact on bioactive compounds relevant to antioxidant and antimicrobial activities. During fermentation, pH, total acid, nutritional components, total polyphenols (TP), total tannins (TT), total flavonoids (TF), total catechins (TC), antioxidant activity and antimicrobial activity were evaluated. Miang when fermented for longer sour Miang process compared to shorter time astringent Miang increased crude protein, fiber, and ash contents whereas soluble carbohydrates decreased. Even though TP, TT, TF and TC of sour Miang was lower, the overall antioxidant activity was higher than astringent Miang. This suggests that in addition to the phenolic compounds, other specific phenolics and substances such as biotransformed protein and fat could contribute to antioxidant properties. Additionally, Miang also contains antimicrobial activities against dental caries pathogenic bacteria Streptococcus mutans, gastrointestinal disease causing Vibrio cholerae and Salmonella enterica serovar Typhimurium through likely effects of organic acids and phenolic compounds.

Expression and biochemical characterization of a new alkaline tannase from Lactobacillus pentosus.

Lactobacillus pentosus BA-7 and L. pentosus QA1-5 are tannin-tolerant lactic acid bacteria that were isolated from Miang, a traditional fermented tea-leaf found in northern Thailand and a tannin-rich substrate. Tannase encoding genes were isolated, cloned and overexpressed in Escherichia coli BL21(DE3). The recombinant tannase was produced with production yields of 40 and 39 KU/L for LpTanBA-7 and LpTanQA1-5, respectively. Both revealed the same molecular weight of 50 kDa as estimated by SDS-PAGE and were optimally active under alkaline pH conditions LpTanQA1-5 revealed optimal temperatures in a range of 37-40 °C as is typically found in lactic acid bacteria, while LpTanBA-7 was active at higher temperatures with an optimum temperature range of 45-55 °C. LpTanBA-7 was found to be more stable within the same range of temperatures than LpTanQA1-5. Furthermore, it was active and stable toward various organic solvents and produced 50 mg/mL of gallic acid from 100 mg/mL tannic acid. Based on the results, LpTanBA-7 is considered a new alkali-moderately thermophilic tannase obtained from lactic acid bacterium that may be capable of a feasible production capacity of gallic acid and its esters. Furthermore, tannase that is active at high temperatures could also be used in tea products in order to develop a sweet aftertaste, as well as to improve levels of antioxidant activity.

Diversity of lactic acid bacteria from Miang, a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract.

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.

Distribution of tannin-'tolerant yeasts isolated from Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) in northern Thailand.

Miang is a fermented food product prepared from the tea leaves of Camellia sinensis var. assamica, and is traditionally produced in mountainous areas of northern Thailand. Although Miang has a long history and reveals deep-rooted cultural involvement with local people in northern Thailand, little is known regarding its microbial diversity. Yeasts were isolated from 47 Miang samples collected from 28 sampling sites, including eight provinces in upper northern Thailand. A hundred and seven yeast isolates were recovered and identified within 14 species based on the comparison of the D1/D2 sequence of the large subunit (LSU) rRNA gene. Candida ethanolica was determined to be the dominant species that was frequently found in Miang together with minor resident yeast species. All yeast isolates demonstrated their tannin-tolerant capability when cultivated on yeast malt agar (YMA) containing 50g/l tannin, but nine isolates displayed clear zones forming around their colonies, e.g., Debaryomyces hansenii, Cyberlindnera rhodanensis, and Sporidiobolus ruineniae. The results obtained from a visual reading method of tannase revealed that all yeast isolates were positive for methyl gallate, indicating that they possess tannase activity. It is assumed that a tannin-tolerant ability is one of the most important factors for developing a yeast community in Miang. This research study is the first report to describe tannin-tolerant yeasts and yeast communities in traditionally fermented tea leaves.

Antibacterial and antioxidant activities of acid and bile resistant strains of lactobacillus fermentum isolated from Miang

Miang is a kind of traditional fermented tea leaves, widely consumed in northern Thailand as a snack. It contains several kinds of Lactobacilli spp. The aim of this study was to isolate strains of Lactobacillus fermentum from miang and to investigate their antibacterial and antioxidant activities. The agar spot and well assays were used for determination of antibacterial power. The antibacterial mechanism was investigated by cell morphologic change under scanning electron microscope (SEM). Antioxidant activity was studied by means of free radical scavenging and ferric reducing power assays. The acid and bile screening tests indicated that L. fermentum FTL2311 and L. fermentum FTL10BR presented antibacterial activity against several pathogenic bacteria: Listeria monocytogenes DMST 17303, Salmonella Typhi DMST 5784, Shigella sonnei DMST 561 (ATCC 11060)and Staphylococcus aureus subsp. aureus DMST 6512 (ATCC 6538Ptm). The results from SEM suggested that the antibacterial action was due to the destruction of cell membrane which consequently caused the pathogenic cell shrinking or cracking. The antioxidant study suggested that both L. fermentum FTL2311 and L. fermentum FTL10BR strains could liberate certain substances that possessed antioxidant activity expressed as trolox equivalent antioxidant capacity (TEAC) and equivalent concentration (EC) values for free radical scavenging and reducing mechanisms, respectively. The supernatant of L. fermentum FTL2311 broth revealed TEAC and EC values of 22.54±0.12 and 20.63±0.17 µM.mg-1 respectively, whereas that of L. fermentum FTL10BR yielded TEAC and EC values of 24.09±0.12 and 21.26±0.17 µM.mg-1 respectively. These two strains isolated from miang present high potential as promising health-promoting probiotics.