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.

Tannin-Tolerant Saccharomyces cerevisiae Isolated from Traditional Fermented Tea Leaf (Miang) and Application in Fruit Wine Fermentation Using Longan Juice Mixed with Seed Extract as Substrate.

This study focused on isolating tannin-tolerant yeasts from Miang, a fermented tea leaf product collected from northern Laos PDR, and investigating related food applications. From 43 Miang samples, six yeast isolates capable of ethanol production were obtained, with five isolates showing growth on YPD agar containing 4% (w/v) tannic acid. Molecular identification revealed three isolates as Saccharomyces cerevisiae (B5-1, B5-2, and C6-3), along with Candida tropicalis and Kazachstania humilis. Due to safety considerations, only Saccharomyces spp. were selected for further tannic acid tolerance study to advance food applications. Tannic acid at 1% (w/v) significantly influenced ethanol fermentation in all S. cerevisiae isolates. Notably, B5-2 and C6-3 showed high ethanol fermentation efficiency (2.5% w/v), while others were strongly inhibited. The application of tannin-tolerant yeasts in longan fruit wine (LFW) fermentation with longan seed extract (LSE) supplementation as a source of tannin revealed that C6-3 had the best efficacy for LFW fermentation. C6-3 showed promising efficacy, particularly with LSE supplementation, enhancing phenolic compounds, antioxidant activity, and inhibiting α-glucosidase activity, indicating potential antidiabetic properties. These findings underscore the potential of tannin-tolerant S. cerevisiae C6-3 for fermenting beverages from tannin-rich substrates like LSE, with implications for functional foods and nutraceuticals promoting health benefits.

Antibacterial activities of Miang extracts against selected pathogens and the potential of the tannin-free extracts in the growth inhibition of Streptococcus mutans.

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC50 values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products.

Valorization of Cashew Apple Waste into a Low-Alcohol, Healthy Drink Using a Co-Culture of Cyberlindnera rhodanensis DK and Lactobacillus pentosus A14-6.

This study investigated the potential of microbial fermentative transforming processes in valorizing the cashew apple by-product into a low-alcohol, health-benefiting beverage. We particularly investigated the use of a non-Saccharomyces yeast, Cyberlindnera rhodanensis DK, as the main targeted microbe. At 30 °C without agitation, C. rhodanensis DK caused changes in key parameters during the fermentation of cashew apple juice (CAJ) in terms of varied pH values and initial sugar concentrations. This result indicated that pure CAJ, with pH adjusted to 6 and with the original 6.85% (w/v) total sugar content, was the most feasible condition, as glucose and fructose were mostly consumed at 12 days of fermentation. A co-culture approach with either Saccharomyces cerevisiae TISTR 5088 or Lactobacillus pentosus A14-6 was investigated to improve both physicochemical and fermentation characteristics. Co-fermentation with S. cerevisiae TISTR 5088 resulted in significantly increased ethanol accumulation to 33.61 ± 0.11 g/L, but diminished bioactive compounds, antioxidant activity, and antidiabetic potential. In contrast, co-fermentation with L. pentosus A14-6 demonstrated excellent outcomes, as it significantly increased sugar consumption and finally remained at only 4.95 g/L compared to C. rhodanensis DK alone, produced lower levels of ethanol at only 19.47 ± 0.06 g/L, and higher total titratable acid (TTA), resulting in a final pH of 3.6. In addition, co-fermentation with this lactic acid bacterium significantly enhanced bioactive compounds and antioxidant activity and also retained potential antidiabetic properties. These findings highlight the feasibility of using tailored microbial fermentation strategies to produce low-alcohol beverages with enhanced health-promoting properties from CAJ; however, product-development processes following health food regulations and sensory evaluation are necessary.

A Thermotolerant Yeast Cyberlindnera rhodanensis DK Isolated from Laphet-so Capable of Extracellular Thermostable ß-Glucosidase Production.

This study aims to utilize the microbial resources found within Laphet-so, a traditional fermented tea in Myanmar. A total of 18 isolates of thermotolerant yeasts were obtained from eight samples of Laphet-so collected from southern Shan state, Myanmar. All isolates demonstrated the tannin tolerance, and six isolates were resistant to 5% (w/v) tannin concentration. All 18 isolates were capable of carboxy-methyl cellulose (CMC) degrading, but only the isolate DK showed ethanol production at 45 °C noticed by gas formation. This ethanol producing yeast was identified to be Cyberlindnera rhodanensis based on the sequence analysis of the D1/D2 domain on rRNA gene. C. rhodanensis DK produced 1.70 ± 0.01 U of thermostable extracellular ß-glucosidase when cultured at 37 °C for 24 h using 0.5% (w/v) CMC as a carbon source. The best two carbon sources for extracellular ß-glucosidase production were found to be either xylose or xylan, with ß-glucosidase activity of 3.07-3.08 U/mL when the yeast was cultivated in the yeast malt extract (YM) broth containing either 1% (w/v) xylose or xylan as a sole carbon source at 37 °C for 48 h. The optimal medium compositions for enzyme production predicted by Plackett-Burman design and central composite design (CCD) was composed of yeast extract 5.83 g/L, peptone 10.81 g/L and xylose 20.20 g/L, resulting in a production of 7.96 U/mL, while the medium composed (g/L) of yeast extract 5.79, peptone 13.68 and xylan 20.16 gave 9.45 ± 0.03 U/mL for 48 h cultivation at 37 °C. Crude ß-glucosidase exhibited a remarkable stability of 100%, 88% and 75% stable for 3 h at 35, 45 and 55 °C, respectively.

Purification and characterization of crude fructooligosaccharides extracted from red onion (Allium cepa var. viviparum) by yeast treatment.

BACKGROUND: Yeast treatment has been used for purification of fructooligosaccharides (FOSs). However, the main drawback of this approach is that yeast can only partially remove sucrose from crude FOSs. The main objective of this research was to screen yeast strains for the capability of selectively consuming unwanted sugars, namely fructose, glucose, and sucrose, in crude FOSs extracted from red onion (Allium cepa var. viviparum) with minimal effect on FOS content. RESULTS: Among 43 yeast species isolated from Miang, ethnic fermented tea leaves, and Assam tea flowers, Candida orthopsilosis FLA44.2 and Priceomyces melissophilus FLA44.8 exhibited the greatest potential to specifically consume these unwanted sugars. In a shake flask, direct cultivation of C. orthopsilosis FLA44.2 was achieved in the original crude FOSs containing an initial FOSs concentration of 88.3 ± 1.2 g/L and 52.9 ± 1.2 g/L of the total contents of fructose, glucose, and sucrose. This was successful with 93.7% purity and 97.8% recovery after 24 h of cultivation. On the other hand, P. melissophilus FLA48 was limited by initial carbohydrate concentration of crude FOSs in terms of growth and sugar utilization. However, it could directly purify two-fold diluted crude FOSs to 95.2% purity with 92.2% recovery after 72 h of cultivation. Purification of crude FOSs in 1-L fermenter gave similar results to the samples purified in a shake flask. Extracellular ß-fructosidase was assumed to play a key role in the effective removal of sucrose. Both Candida orthopsilosis FLA44.2 and P. melissophilus FLA44.8 showed γ-hemolytic activity, while their culture broth had no cytotoxic effect on viability of small intestinal epithelial cells, preliminarily indicating their safety for food processing. The culture broth obtained from yeast treatment was passed through an activated charcoal column for decolorization and deodorization. After being freeze dried, the final purified FOSs appeared as a white granular powder similar to refined sugar and was odorless since the main sulfur-containing volatile compounds, including dimethyl disulfide and dipropyl trisulfide, were almost completely removed. CONCLUSION: The present purification process is considered simple and straight forward, and provides new and beneficial insight into utilization of alternative yeast species for purification of FOSs.

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.

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.

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.

Isolation of Efficient Xylooligosaccharides-Fermenting Probiotic Lactic Acid Bacteria from Ethnic Pickled Bamboo Shoot Products.

Xylooligosaccharides (XOSs) are produced from xylan, which is a component of the hemicellulose that can be found in bamboo shoots. Naw Mai Dong, an ethnic pickled bamboo shoot product of northern Thailand, is generally characterized as acidic and has a sour taste. It can be considered a potential source of probiotic lactic acid bacteria (LAB). This study aimed to isolate efficient XOSs-fermenting probiotic LAB from ethnic pickled bamboo shoot products. A total of 51 XOSs-fermenting LAB were recovered from 24 samples of Naw Mai Dong, while 17 strains exhibited luxuriant growth in xylose and XOSs. Among these, seven strains belonging to Levicaseibacillus brevis and Pediococcus acidilactici exhibited similar growth in glucose, xylose, and XOSs, while the rest showed a weaker degree of growth in xylose and XOSs than glucose. Sixteen strains exhibited resistance under gastrointestinal tract conditions and displayed antimicrobial activity against foodborne pathogens. Notably, Lv. brevis FS2.1 possessed the greatest probiotic properties, with the highest %hydrophobicity index and %auto-aggregation. Effective degradation and utilization of XOSs by probiotic strains are dependent upon xylanase and ß-xylosidase production, as well as xylose metabolism. It can be concluded that pickled bamboo shoot products can be a beneficial source of XOSs-fermenting probiotic LAB.

Enterococci as Dominant Xylose Utilizing Lactic Acid Bacteria in Eri Silkworm Midgut and the Potential Use of Enterococcus hirae as Probiotic for Eri Culture.

A total of 51 pentose utilizing lactic acid bacteria (LAB) were isolated from acid-forming bacteria in the midgut of healthy mature Eri silkworm using de Man, Rogosa and Sharpe (MRS) agar containing 10 g/L xylose (MRS-xylose) as the carbon source supplemented with 0.04% (w/v) bromocresol purple. Further analysis of 16S rRNA gene sequences revealed the highest prevalence of up to 35 enterococci isolates, which included 20 isolates of Enterococcus mundtii, followed by Entercoccus faecalis (eight isolates), Weissella cibaria (four isolates), Enterococcus hirae (two isolates), Enterococcus lactis (one isolate), and Enterococcus faecium (one isolate). All 51 LAB isolates showed positive growth on MRS containing a range of polysaccharides as the sole carbon source. All isolates were able to grow and form clear zones on MRS supplemented with 1 g/L xylose, while E. faecalis SC1, E. faecalis SCT2, and E. hirae SX2 showed tannin tolerance ability up to 5 g/L. Moreover, five isolates showed antimicrobial activity against Eri silkworm pathogens, including Bacillus cereus, Staphylococcus aureus, and Proteus vulgaris, with E. hirae SX2 having the highest inhibitory effect. Supplementation of live E. hirae SX2 on castor leaves significantly improved the weight and reduced the silkworm mortality when compared with the control group (p < 0.05). This cocci LAB can be considered as the new probiotic for Eri culture. Additionally, this finding presented the perspective of non-mulberry silkworm that could also be used as the model for further applying to new trends of the sericulture industry.

Wickerhamiella nakhonpathomensis f.a. sp. nov., a novel ascomycetous yeast species isolated from a mushroom and a flower in Thailand.

Strains SU22T (TBRC 14875T) and FLA11.5, representing a novel anamorphic yeast species, were respectively isolated from a fruiting body of a Coprinus species and an inflorescence of a Coffea species collected in Thailand. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed that the two strains differed by two nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and were identical in the ITS regions. Wickerhamiella drosophilae CBS 8459T was the most closely related species, but with 24-26 nucleotide substitutions in the D1/D2 domains of the LSU rRNA gene and 24 nucleotide substitutions in the ITS regions. A phylogenetic analysis, based on the sequences of the D1/D2 domains, indicated that the two strains represented a species in the genus Wickerhamiella which was distinct from other recognized species of the genus. Therefore, the two strains were assigned as a novel species, for which we propose the name Wickerhamiella nakhonpathomensis f.a. sp. nov. The holotype is TBRC 14875T (isotype PYCC 8914T). The MycoBank number of the novel species is MB 840833.

Protective Effect of Probiotics Isolated from Traditional Fermented Tea Leaves (Miang) from Northern Thailand and Role of Synbiotics in Ameliorating Experimental Ulcerative Colitis in Mice.

This study aimed to investigate the protective effect of probiotics and synbiotics from traditional Thai fermented tea leaves (Miang) on dextran sulfate sodium (DSS)-induced colitis in mice, in comparison to sulfasalazine. C57BL/6 mice were treated with probiotics L. pentosus A14-6, CMY46 and synbiotics, L. pentosus A14-6 combined with XOS, and L. pentosus CMY46 combined with GOS for 21 days. Colitis was induced with 2% DSS administration for seven days during the last seven days of the experimental period. The positive group was treated with sulfasalazine. At the end of the experiment, clinical symptoms, pathohistological changes, intestinal barrier integrity, and inflammatory markers were analyzed. The probiotics and synbiotics from Miang ameliorated DSS-induced colitis by protecting body weight loss, decreasing disease activity index, restoring the colon length, and reducing pathohistological damages. Furthermore, treatment with probiotics and synbiotics improved intestinal barrier integrity, accompanied by lowing colonic and systemic inflammation. In addition, synbiotics CMY46 combined with GOS remarkedly elevated the expression of IL-10. These results suggested that synbiotics isolated from Miang had more effectiveness than sulfasalazine. Thereby, they could represent a novel potential natural agent against colonic inflammation.

Potential of Inulin-Fructooligosaccharides Extract Produced from Red Onion (Allium cepa var. viviparum (Metz) Mansf.) as an Alternative Prebiotic Product.

Red onion is a popular ingredient in many Thai dishes and has recently been promoted for commercial cultivation. In this study, inulin-fructooligosaccharides (inulin-FOSs) were extracted from red onions in a simplified extraction method. The extract contained 24.00 ± 0.38 g/L free glucose, fructose and sucrose, while the level of FOSs was recorded at 74.0 ± 2.80 g/L with a degree of polymerization of 4.1. The extract was resistant to simulated gastrointestinal conditions, while selectively promoting probiotic lactobacilli. These outcomes resulted in inhibitory effects against various pathogenic bacteria. The in vitro batch culture fermentation of the extract by natural mixed culture indicated that an unknown sugar identified as neokestose was more rapidly fermented than 1-kestose and other longer-chain inulin-FOSs. Notably, neokestose selectively encouraged a bifidogenic effect, specifically in terms of the growth of Bifidobacteirum breve, which is an infant-type probiotic bacterium. This is the first report to state that neokestose could selectively enhance the bifidogenic effect. In summary, inulin-FOSs extract should be recognized as a multifunctional ingredient that can offer benefits in food and pharmaceutical applications.

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.

In-vitro biotransformation of tea using tannase produced by Enterobacter cloacae 41.

Tannase is a widely used enzyme that improves the quality of tea by facilitating the release of water-soluble polyphenolic compounds, as well as reduces the formation of tea creams. The microbial tannase enzymes are often employed for tea biotransformation by hydrolyses esters of phenolic acids, including the gallated polyphenols found in blacks teas. The study was focused to investigate the tannase enzyme mediated biotransformation of black tea such as CTC-(Crush, tear, curl) & Kangra orthodox which are commonly used by the south Indian peoples. HPLC spectral analysis revealed that tannase treatment on tea cream formation (CTC & Kangra orthodox tea) allows the hydrolysis of the EGC, GA, ECG, and EGCG. A significant reduction in the formation of tea cream and increased antioxidant activity has been observed in the CTC (1.62 fold) and Kangra orthodox (1.55 fold). The results revealed that tannase treatment helps to improve the quality of black tea infusions with respect to cream formation, the intensity of colour, and sensory characteristics of tea. The result of this study indicates that E. cloacae 41 produced tannase can be used to improve the quality of both tea samples.

Efficient Secretion and Recombinant Production of a Lactobacillal α-amylase in Lactiplantibacillus plantarum WCFS1: Analysis and Comparison of the Secretion Using Different Signal Peptides.

Lactic acid bacteria (LAB) have been used as starter cultures and producers of enzymes, antimicrobial peptides or metabolites that contribute to the flavor, texture and safety of food products. Lactiplantibacillus plantarum, one of the best-studied LAB, is considered as safe and effective cell factory for food applications. In this study, our aim was to use L. plantarum as the producer for high levels of a food-grade lactobacillal α-amylase, which has potential applications in food, fermentation and feed industries. The native form of an α-amylase (AmyL) from L. plantarum S21, an amylolytic LAB isolated from Thai fermented rice noodles, was expressed in L. plantarum WCFS1 using the pSIP expression system. The secretion of the α-amylase was driven by the native signal peptides of the α-amylases from L. plantarum S21 (SP_AmyL) and Lactobacillus amylovorus NRRL B-4549 (SP_AmyA), as well as by three Sec-type signal peptides derived from L. plantarum WCFS1; Lp_2145, Lp_3050, and Lp_0373. Among the tested signal peptides, Lp_2145 appears to be the best signal peptide giving the highest total and extracellular enzymatic activities of α-amylase AmyL from L. plantarum S21, which were 13.1 and 8.1 kU/L of fermentation, respectively. These yields were significantly higher than the expression and secretion in L. plantarum WCFS1 using the native signal peptide SP_AmyL, resulting in 6.2- and 5.4-fold increase in total and extracellular activities of AmyL, respectively. In terms of secretion efficiency, Lp_0373 was observed as the most efficient signal peptide among non-cognate signal peptides for the secretion of AmyL. Real-time reverse-transcriptase quantitative PCR (RT-qPCR) was used to estimate the mRNA levels of α-amylase transcript in each recombinant strain. Relative quantification by RT-qPCR indicated that the strain with the Lp_2145 signal peptide-containing construct had the highest mRNA levels and that the exchange of the signal peptide led to a change in the transcript level of the target gene.

Characterization of Sporidiobolus ruineniae A45.2 Cultivated in Tannin Substrate for Use as a Potential Multifunctional Probiotic Yeast in Aquaculture.

At present, few yeast species have been evaluated for their beneficial capabilities as probiotics. Sporidiobolus ruineniae A45.2, a carotenoid-producing yeast, was able to co-produce cell-associated tannase (CAT), gallic acid and viable cells with antioxidant activity when grown in a tannic acid substrate. The aim of this research study was to identify the potential uses of S. ruineniae A45.2 obtained from a co-production system as a potential feed additive for aquaculture. S. ruineniae A45.2 and its CAT displayed high tolerance in pH 2.0, pepsin, bile salts and pancreatin. Furthermore, its viable cells were characterized by moderate hydrophobicity, high auto-aggregation and moderate co-aggregation with Staphylococcus aureus, Salmonella ser. Thyphimurium and Streptococcus agalactiae. These attributes promoted S. ruineniae A45.2 as a multifunctional probiotic yeast. In addition, the intact cells possessed antioxidant activities in a 100-150 µg gallic acid equivalent (GAE)/mL culture. Remarkably, the fermentation broth demonstrated higher antioxidant activity of 9.2 ± 1.8, 9.0 ± 0.9, and 9.8 ± 0.7 mg GAE/mL culture after FRAP, DPPH and ABTS assays, respectively. Furthermore, higher antimicrobial activity was observed against Bacillus cereus, Staphylococcus aureus and Strep. agalactiae. Therefore, cultivation of S. ruineniae A45.2 with a tannic acid substrate displayed significant potential as an effective multifunctional feed additive.