Candida albicans biofilm formation and growth optimization for functional studies using response surface methodology.

AIM: Optimization of Candida albicans growth and biofilm formation is essential for understanding the recalcitrance of this pathogen to advance functional analysis on hospital tools and material surfaces. Optimization and quantification of biofilm have always been a challenge using the conventional one variable at a time (OVAT) method. The present study uses central composite design-based response surface methodology for optimization of conditions to induce growth and biofilm formation in Candida albicans on polystyrene microtiter plates. METHODS AND RESULTS: Statistical software package, Stat Soft®, STASTICA version 12.6 was used for data analysis. The variables considered in the design matrix were media pH, temperature, incubation period, shaker speed and inoculum size. A four-pronged quantification approach with XTT assay (cell viability), crystal violet assay (biofilm), calcofluor white assay and wet/dry weight measurements (cell mass) was used to understand different aspects of biofilm formation. Cell viability and cell mass were inversely related; however, biofilm was independent of these two factors. The study also highlighted the fact that foetal bovine serum does not significantly contribute to cell adhesion and in turn in vitro biofilm formation in some of the cultures. CONCLUSIONS: A high-throughput optimization of C. albicans growth and biofilm formation on polystyrene microplate has been developed and validated. SIGNIFICANCE AND IMPACT OF STUDY: This is a first time approach to optimize the interaction of parameters for C. albicans biofilm formation using RSM. Heterogeneity in growth conditions for local strains of C. albicans clinical isolates was observed. This microtiter plate-based method can be used for future screening of therapeutics for the control of C. albicans.

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.

Evaluation of phenolic antioxidant-linked in vitro bioactivity of Peruvian corn (Zea mays L.) diversity targeting for potential management of hyperglycemia and obesity.

Peruvian corn biodiversity is one of the highest in the world and may represent an important natural source of health relevant phenolic bioactive compounds whose potential needs to be investigated. This study investigated twenty-two Peruvian corn samples corresponding to five corn races (Arequipeño, Cabanita, Kculli, Granada and Coruca) in relation to their total phenolic contents (TPC), anthocyanin contents, Ultra-Performance Liquid Chromatography (UPLC) phenolic profiles and antioxidant capacity (ABTS and ORAC methods). Subsequently using both free and cell-wall bound phenolic fractions their health relevance targeting hyperglycemia (α-glucosidase and α-amylase inhibition) and obesity (lipase inhibition) potentials was evaluated using in vitro assay models. Antioxidant capacity and TPC were high in bound fractions from yellow-colored races in contrast to the purple-colored race (Kculli) which had high TPC (mainly anthocyanins) and antioxidant capacity in the free form. The major phenolic acids detected by UPLC were ferulic and p-coumaric acids. High α-glucosidase (32.5-76.1%, 25 mg sample dose) and moderate α-amylase inhibitory activities (13.6-29.0%, 250 mg sample dose) were found in all free fractions, but only samples from the Kculli race had lipase inhibitory activity (58.45-92.16%, 12.5 mg sample dose). Principal component analysis revealed that the variability of data was affected by the race and the α-glucosidase and lipase inhibitory activities positively correlated with anthocyanins and antioxidant capacity. Some accessions of Kculli, Granada and Cabanita races are promising for future breeding strategies focused on the development of improved corn varieties targeted for the design of functional foods relevant for hyperglycemia and obesity prevention.

Natural preservatives for superficial scald reduction and enhancement of protective phenolic-linked antioxidant responses in apple during post-harvest storage.

Superficial scald during post-harvest storage is a serious problem for long-term preservation and shelf-life of some apple and pear cultivars. Development of superficial scald and related physiological disorders such as enzymatic and non-enzymatic browning are associated in part with oxidative breakdown and redox imbalance. Therefore, targeting natural antioxidants from food-grade sources as post-harvest treatment to reduce superficial scald has merit. Such natural antioxidants can potentially counter oxidation-linked damages associated with superficial scald through stimulation of antioxidant enzyme responses and biosynthesis of less-oxidized phenolics involving protective redox-linked pathway such as proline-associated pentose phosphate pathway. Based on this rationale, bioprocessed food-grade oregano extract (OX) and soluble chitosan oligosaccharide (COS) were targeted as post-harvest treatment (2 and 4 g L-1) and were compared with diphenylamine (DPA) (1 and 2 g L-1) to reduce superficial scald and to improve protective phenolic-linked antioxidant responses in "Cortland" cultivar stored at 4 °C for 15 weeks. Overall, significant reduction of superficial scald and conjugated triene was observed with DPA and OX (2 g L-1) post-harvest treatments. Furthermore, stimulation of antioxidant enzyme responses such as increases in superoxide dismutase and guaiacol peroxidase activity was also observed, but was more evident with DPA and COS treatment. Overall, results of this study indicated that critical balance of less-oxidized phenolics and antioxidant enzymes and associated anabolic PPP-linked redox regulation is essential for improving post-harvest preservation and reduction of superficial scald in apple.

Changes in physico-chemical, astringency, volatile compounds and antioxidant activity of fresh and concentrated cashew apple juice fermented with Lactobacillus plantarum.

Changes in physico-chemical qualities (pH, total acidity, total and reducing sugar, total phenolic and vitamin C), astringency compounds (condensed and hydrolysable tannin), antioxidant activities [2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical] and flavor volatile compounds in Lactobacillus plantarum-fermented cashew-apple-juice (CAJ) and 11.4 °Bx concentrated-cashew-apple-juice (CCAJ) was investigated. Total phenolics remained unchanged throughout fermentation period, whereas condensed tannins increased and hydrolysable tannins decreased indicating reduced astringency compounds. Antioxidant activity based on both DPPH and ABTS radical scavenging activities marginally declined in some stages but overall were sustained during fermentation. Although the DPPH· radical based antioxidant activity of fermented CAJ was greater than that of fermented 11.4 °Bx CCAJ, a higher ABTS·+ radical scavenging activity was found in fermented 11.4 °Bx CCAJ, reflecting higher water soluble antioxidants. Results also indicated that DPPH· radical scavenging activity was positively correlated to vitamin-C and condensed tannins but not hydrolysable tannins. ABTS·+ radical scavenging activity was also positively correlated to condensed tannins and not hydrolysable tannins. The vitamin-C that increased during initial 12 h fermentation, decreased from 2516 to 2150 mg AAE/L at the end of 72 h fermentation. Fermented CAJ had a remarkable sweet aroma with a fruity note of two major compounds; 3-methyl-1-butanol (14.20 × 107) and 2,6-dimethyl-4-heptanol (14.76 × 107). The high phytochemicals and volatile compounds in fermented CAJ indicated that it could serve as a functional beverage with potential health benefits with reduced astringency due to lower hydrolysable tannins.

Rapid Screening of Natural Plant Extracts with Calcium Diacetate for Differential Effects Against Foodborne Pathogens and a Probiotic Bacterium.

This study focused on advancing a rapid turbidimetric bioassay to screen antimicrobials using specific cocktails of targeted foodborne bacterial pathogens. Specifically, to show the relevance of this rapid screening tool, the antimicrobial potential of generally recognized as safe calcium diacetate (DAX) and blends with cranberry (NC) and oregano (OX) natural extracts was evaluated. Furthermore, the same extracts were evaluated against beneficial lactic acid bacteria. The targeted foodborne pathogens evaluated were Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus using optimized initial cocktails (∼108 colony-forming unit/mL) containing strains isolated from human food outbreaks. Of all extracts evaluated, 0.51% (w/v) DAX in ethanol was the most effective against all four pathogens. However, DAX when reduced to 0.26% and with added blends from ethanol extractions consisting of DAX:OX (3:1), slightly outperformed or was equal to same levels of DAX alone. Subculture of wells in which no growth occurred after 1 week indicated that all water and ethanol extracts were bacteriostatic against the pathogens tested. All the targeted antimicrobials had no effect on the probiotic organism Lactobacillus plantarum. The use of such rapid screening methods combined with the use of multistrain cocktails of targeted foodborne pathogens from outbreaks will allow rapid large-scale screening of antimicrobials and enable further detailed studies in targeted model food systems.

Improving phenolic bioactive-linked anti-hyperglycemic functions of dark germinated barley sprouts (Hordeum vulgare L.) using seed elicitation strategy.

Sprouts of cereal grains, such as barley (Hordeum vulgare L.), are a good source of beneficial phenolic bioactives. Such health relevant phenolic bioactives of cereal sprouts can be targeted to manage chronic hyperglycemia and oxidative stress commonly associated with type 2 diabetes (T2D). Therefore improving phenolic bioactives by stimulating plant endogenous defense responses such as protective pentose phosphate pathway (PPP) during sprouting has significant merit. Based on this metabolic rationale, this study aimed to enhance phenolic bioactives and associated antioxidant and anti-hyperglycemic functions in dark germinated barley sprouts using exogenous elicitor treatments. Dark-germinated sprouts of two malting barley cultivars (Pinnacle and Celebration), treated with chitosan oligosaccharide (COS) and marine protein hydrolysate (GP), were evaluated. Total soluble phenolic content (TSP), phenolic acid profiles, total antioxidant activity (TA) and in vitro inhibitory activities of hyperglycemia relevant α-amylase and α-glucosidase enzymes of the dark germinated barley sprouts were evaluated at day 2, 4, and 6 post elicitor treatments. Overall, TSP content, TA, and α-amylase inhibitory activity of dark germinated barley sprouts decreased, while α-glucosidase inhibitory activity and gallic acid content increased from day 2 to day 6. Among barley cultivars, high phenolic antioxidant-linked anti-hyperglycemic bioactives were observed in Celebration. Furthermore, GP and COS seed elicitor treatments in selective doses improved T2D relevant phenolic-linked anti-hyperglycemic bioactives of barley spouts at day 6. Therefore, such seed elicitation approach can be strategically used to develop bioactive enriched functional food ingredients from cereal sprouts targeting chronic hyperglycemia and oxidative stress linked to T2D.

Phytochemicals-linked food safety and human health protective benefits of the selected food-based botanicals.

Phytochemicals-rich food-based botanicals including traditional or under-utilized plant-based ingredients can serve a dual functional role to help counter food contamination of bacterial origin, while also addressing the rise of diet-linked non-communicable chronic diseases (NCDs) such as type 2 diabetes, chronic hypertension and the associated oxidative stress. Hence the screening of these food-based botanicals for their phenolic content and profile, as well as antimicrobial, antioxidant, anti-hyperglycemic and anti-hypertensive properties has relevant merit. Using in vitro assay models, hot water extracts of different forms (slice, pickle, or powder) of amla (Phyllanthus emblica), clove (Syzygium aromaticum), kokum (Garcinia indica), and garlic (Allium sativum) were analyzed for their total soluble phenolic content (TSP) and phenolic profile as well as antimicrobial activity against strains of Salmonella Enteritidis, Listeria monocytogenes, and Escherichia coli that are associated with food-borne disease outbreaks. In addition, the antioxidant, anti-hyperglycemic and anti-hypertensive activity of the extracts were also determined using in vitro assay models, with the goal of establishing a dual functional role of the food safety and health protective benefits of these botanicals. A high baseline TSP content was observed in all the extracts and the major phenolic phytochemicals detected were gallic, cinnamic, ellagic, benzoic, dihydroxybenzoic, protocatechuic, and p-coumaric acid along with catechin and rutin. All extracts displayed significant antimicrobial activity against most of the bacterial strains tested and the antimicrobial activity was specific for each strain targeted in this study. Furthermore, significant antioxidant, anti-hyperglycemic and antihypertensive activity were observed among the botanical extracts, especially among the amla and kokum extracts. These results indicate that phytochemicals enriched botanicals, including amla and kokum, can be integrated into modern-day food preservation and dietary support strategies aimed at improving the food safety and health protective benefits of the food matrix.

Natural elicitors enhanced suberin polyphenolic accumulation in wounded potato tuber tissues.

Introduction: Unintended wounding or bruising during harvest or postharvest handling leads to significant tuber loss and imposes economic burden to potato industry. Therefore, finding effective strategies to mitigate wound-related tuber losses is very important from industry perspectives. Formation of protective barrier through accumulation of suberin polyphenolics (SPP) is a natural and initial response of potato tuber tissues to wounding. Materials and methods: In this study, efficacy of two natural elicitors, such as chitosan oligosaccharide (COS 0.125 g L-1) and cranberry pomace residue (Nutri-Cran 0.125 g L-1) was investigated using a mechanically wounded tuber tissue model and by histological determination of SPP formation in five agronomically relevant and red-skin potato cultivars (Chieftain, Dakota Rose, Dakota Ruby, Red LaSoda, Red Norland). Furthermore, the potential role of stress protective metabolic regulation involving phenolic metabolites, proline, and antioxidant enzymes in tuber WH processes were also investigated during 0-9 days after wounding. Results and discussion: Exogenous treatments of both COS and Nutri-Cran resulted into enhanced SPP formation in wounded surface, but the impact was more rapid with Nutri-Cran treatment in select cultivars. Greater contents of total soluble phenolic, ferulic acid, chlorogenic acid, total antioxidant activity, and superoxide dismutase activity were also observed in elicitor treated tuber tissues at different time points after wounding. Nutri-Cran treatment also reduced the activity of succinate dehydrogenase in Red Norland and Dakota Ruby at 3 d, indicating a suppression in respiration rate. Collectively, these results suggest that Nutri-Cran can be potentially utilized as an effective WH treatment to potato tubers for minimizing wound-related losses.

Relatively Low Lecithin Inclusion Improved Gelling Characteristics and Oxidative Stability of Single-Washed Mackerel (Auxis thazard) Surimi.

The effect of lecithin addition on the gelling characteristics and oxidative stability of single-washed mackerel (Auxis thazard) surimi was investigated in this study. Surimi was chopped in the presence of 2.5% (w/w) NaCl with different concentrations of lecithin (0, 0.1, 0.5, 1, and 1.5 g/100 g surimi). The rheological behavior, gel-forming ability, microstructure, and lipid oxidation of lecithin-added surimi varied significantly depending on lecithin content. When compared to the control, lecithin at 0.1, 0.5, and 1 g/100 g improved the breaking force of the gel (p < 0.05). The breaking force of the gel decreased significantly as lecithin concentration increased (up to 1.5 g/100 g) (p < 0.05). Deformation, on the other hand, reacted differently to the lecithin than it did to the breaking force. At a lecithin level of 0.1 g/100 g, the surimi gel displayed improved deformation (p < 0.05). Nonetheless, at higher doses (0.5-1.5 g/100 g), lecithin considerably reduced surimi gel deformation (p < 0.05), and the gel containing lecithin at 1.5 g/100 g showed significantly decreased deformation. Surimi with 0.1 g/100 g lecithin had the lowest expressible drip (p < 0.05). In general, lecithin at concentrations ranging from 0.1 to 1 g/100 g reduced expressible drip (p < 0.05), but not at 1.5 g/100 g, which was equivalent to the control (p > 0.05). Adding lecithin to mackerel surimi improved its whiteness slightly, regardless of concentration. Lecithin impacted the microstructures of surimi gel in a concentration-dependent manner. Lecithin at a concentration of 0.1 g/100 g produced a densely packed network with small, jointed clusters and minimal holes within the gel. Joined clusters in the gel were reduced by 0.5-1.5 g/100 g lecithin, and continuous aggregates predominated. Surprisingly, at higher doses of lecithin, notably 1.5 g/100 g, porous structures with continuous voids were perceived. Surimi gels treated with various lecithin doses had lower thiobarbituric acid reactive substances (TBARS) levels than the control (p < 0.05). Overall, lecithin at a low concentration of 0.1 g/100 g was most effective at improving the texture, increasing water-holding capacity, lightening the color, and delaying lipid oxidation of single-washed mackerel surimi.

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.

Microbial Population and Physicochemical Properties of Miang Fermented in Bamboo Tubes by the Luar Ethnic Group in Lao PDR.

Miang is a traditional fermented food made from Assam tea leaves and consumed as a snack. This study investigated the underground Miang fermentation process practiced by the Luar ethnic group in Laos, specifically examining the nutritional composition and microbial dynamics. Lactic acid bacteria and yeast were dominant in the fermentation process, reaching 8.43 and 8.50 log CFU/g after one week before gradually declining, while the coliform bacterial count was at 5.31 log CFU/g in the initial week but became undetectable in the later stages of fermentation. Next-generation sequencing identified Firmicutes (75.02%) and Proteobacteria (23.51%) as the primary phyla. Bacterial genera included Lactobacillus (73.36%) and Acetobacter (21.06%), with fungi mainly represented by Pichia (85.52%) and Candida (13.45%). Fundamental microbes such as Lactobacillus and Acetobacter were predominantly present, alongside Pichia and Candida, in the fungal communities. Microbial activities played a crucial role in generating essential enzymes for Miang's transformation. The nutritional transformation appears to be complete at 5 weeks of fermentation. The moisture content in the final products was approximately 74% and correlated with a change in nitrogen-free extract (NFE) and crude fiber. The fat content showed a slight increase from 1.3% to 2.52%, but protein content slightly declined from 17.21% to 16.05%, whereas ash content did not change significantly. Key polysaccharide-degrading enzymes, particularly pectinase and ß-mannanase, were revealed and peaked at 48.32 and 25.32 U/g Miang, respectively. The total polyphenols increased from 103.54 mg/g dry Miang to 144.19-155.52 mg/g during fermentation. The lowered IC50 value indicated an increase in antioxidant activity. A fermentation period of at least 3 weeks proved to be optimal for enhancing antioxidant properties and bioactive compounds, and mitigating the risk of coliform bacteria.

Phenolic and Metabolic Profiles, Antioxidant Activities, Glycemic Control, and Anti-Inflammatory Activity of Three Thai Papaya Cultivar Leaves.

This study thoroughly examined the proximate composition, bioactive composition, and in vitro biological activities of three different cultivars of papaya leaf extracts (PLEs) as potential functional ingredients and nutraceuticals. The dark green leaves of three papaya cultivars, Khaek Dam (KD), Holland (H), and Thai Local (L), were used in this study. The protein content of the leaves ranged from 25.96 to 32.18%, the fat content ranged from 7.34 to 11.66%, the carbohydrate content ranged from 5.80 to 17.91%, the moisture content ranged from 6.02 to 6.49%, the ash content ranged from 11.23 to 12.40%, and the fiber content ranged from 23.24 to 38.48%. The L cultivar possessed significantly higher protein and carbohydrate contents, whereas the H cultivar had the highest ash content (p < 0.05). The total phenolic content (TPC) ranged from 113.94 to 173.69 mg GAE/g extract, with the KD cultivar having the highest TPC (p < 0.05). Several metabolic compounds such as phenolic compounds (particularly kaempferol, isorhamnetin, quercetin, ferulic acid, isoferulic acid, salicylic acid, sinapic acid, syringic acid, and vanillin), terpenoids (such as eucalyptol), glycosides, and indole were identified. The PLE from the KD cultivar had the highest levels of DPPH• inhibition, metal chelation, reducing power, and antidiabetic activity (p < 0.05), suggesting superior biological activity. All three PLEs reduced the proliferation of RAW 264.7 cells in a dose-dependent manner with low nitric oxide formation. These results indicate that the papaya leaf, particularly from the KD cultivar, could be a promising source of functional food ingredients.

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.

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.

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.

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.

Phenolic-linked biochemical rationale for the anti-diabetic properties of Swertia chirayita (Roxb. ex Flem.) Karst.

The crude extract of Swertia chirayita, an important medicinal plant of Nepal, is locally used for many diseases including type 2 diabetes. In this study, crude aqueous and 12% ethanol solution extracts of S. chirayita collected from nine districts of Nepal were analyzed for anti-diabetic-linked anti-hyperglycemia potential using in vitro biochemical assays. There was moderate-to-high positive correlation between antioxidant activity and total phenolic content of both extracts and moderate-to-high α-glucosidase inhibitory activity. Although the anti-diabetic property of S. chirayita is mainly attributed to the phytochemical swerchirin present in its hexane fraction, we propose that the crude extract of this plant used in local healing also has anti-hyperglycemia potential. The crude extracts indicated the presence of three main phytochemicals mainly mangiferin, swertiamarin, and amarogentin and their derivatives. Among the standard compounds (mangiferin, swertiamarin, and amarogentin), mangiferin showed α-glucosidase and 2,2-diphenyl-1-picrylhydrazyl radical inhibitory activity indicating anti-hyperglycemia potential.

Aqueous spice extracts as alternative antimycotics to control highly drug resistant extensive biofilm forming clinical isolates of Candida albicans.

Candida albicans form biofilm by associating with biotic and abiotic surfaces. Biofilm formation by C. albicans is relevant and significant as the organisms residing within, gain resistance to conventional antimycotics and are therefore difficult to treat. This study targeted the potential of spice-based antimycotics to control C. albicans biofilms. Ten clinical isolates of C. albicans along with a standard culture MTCC-3017 (ATCC-90028) were screened for their biofilm-forming ability. C. albicans M-207 and C. albicans S-470 were identified as high biofilm formers by point inoculation on Trypticase Soy Agar (TSA) medium as they formed a lawn within 16 h and exhibited resistance to fluconazole and caspofungin at 25 mcg and 8 mcg respectively. Aqueous and organic spice extracts were screened for their antimycotic activity against C. albicans M-207 and S-470 by agar and disc diffusion and a Zone of Inhibition was observed. Minimal Inhibitory Concentration was determined based on growth absorbance and cell viability measurements. The whole aqueous extract of garlic inhibited biofilms of C. albicans M-207, whereas whole aqueous extracts of garlic, clove, and Indian gooseberry were effective in controlling C. albicans S-470 biofilm within 12 h of incubation. The presence of allicin, ellagic acid, and gallic acid as dominant compounds in the aqueous extracts of garlic, clove, and Indian gooseberry respectively was determined by High-Performance Thin Layer Chromatography and Liquid Chromatography-Mass Spectrometry. The morphology of C. albicans biofilm at different growth periods was also determined through bright field microscopy, phase contrast microscopy, and fluorescence microscopy. The results of this study indicated that the alternate approach in controlling high biofilm-forming, multi-drug resistant clinical isolates of C. albicans M-207 and S-470 using whole aqueous extracts of garlic, clove, and Indian gooseberry is a safe, potential, and cost-effective one that can benefit the health care needs with additional effective therapeutics to treat biofilm infections.

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.