Metagenomics and metabolomics of Toddy, an Indian fermented date palm beverage.

Toddy is a popular fermented palm beverage of India. No scientific information on shotgun metagenomics and metabolomics are available on toddy of India till date. Hence, we choose the fermented date palm beverage, locally called khejur toddy, of West Bengal and Jharkhand states of India, to profile microbial community, their targeted and untargeted metabolites to study the putative bio-functional genes corresponding to regulatory metabolic pathways. Shotgun-based metataxonomic analyses revealed the existence of all domains where bacteria were the most abundant domain (94.48%) followed by eukaryotes (3.38%), viruses (1.53%) and archaea (0.61%). Overall, 54 phyla, 363 families, 1087 genera and 1885 species were observed and identified. Bacillota (49.3%) was the most abundant bacterial phylum. At species level, several species of bacteria and yeasts were detected in toddy samples which included Leuconostoc mesenteroides,Leuconostoc citreum,Lactobacillus helveticus,Lactiplantibacillus plantarum,Lactococcus lactis, Acetobacter malorum, Gluconobacter japonicus, Gluconacetobacter liquefaciens, Fructobacillus durionis, Zymomonas mobilis and yeastsSaccharomyces cerevisiae, Hanseniaspora uvarumandHanseniaspora guilliermondii. Toddy metagenome was also compared with metagenome of pulque, the Mexican fermented fresh sap ofAgave, which was retrieved from NCBI database, and also with metagenomic data of some amplicon-based previous studies on toddy and African fermented palm drink for similarity, dissimilarity and uniqueness among them. Predictive biosynthesis of ethanol, acetic acid, butanoate, linalool, staurosporine, prodigiosin, folic acid, riboflavin, etc. were annotated by KEGG/COG database. Clustered regularly interspaced short palindromic repeats (CRISPR) analysis detected 23 arrays (average length 23.69 bp ± 4.28). Comprehensive Antibiotic Resistance Database (CARD) analysis did not show the presence of any momentous antibiotic resistance gene among the major microbial members. Metabolomics analysis detected many primary and secondary metabolites. We believe this is the first report on complete shotgun metagenomics, and metabolomics of fermented palm drink of India as well as Eastern India.

A special drop: Characterising yeast isolates associated with fermented beverages produced by Australia's indigenous peoples.

Way-a-linah, an alcoholic beverage produced from the fermented sap of Eucalyptus gunnii, and tuba, a fermented drink made from the syrup of Cocos nucifera fructifying bud, are two of several fermented beverages produced by Australian Aboriginal and Torres Strait people. Here we describe the characterisation of yeast isolates from samples associated with the fermentation of way-a-linah and tuba. Microbial isolates were obtained from two different geographical locations in Australia - the Central Plateau in Tasmania, and Erub Island in the Torres Strait. While Hanseniaspora species and Lachancea cidri were the most abundant species in Tasmania, Candida species were the most abundant in Erub Island. Isolates were screened for tolerance to stress conditions found during the production of fermented beverages and for enzyme activities relevant to the appearance, aroma and flavour of these beverages. Based on screening results, eight isolates were evaluated for their volatile profile during the fermentation of wort, apple juice and grape juice. Diverse volatile profiles were observed for beers, ciders and wines fermented with different isolates. These findings reveal the potential of these isolates to produce fermented beverages with unique aroma and flavour profiles and highlight the vast microbial diversity associated with fermented beverages produced by Australia's Indigenous peoples.

Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage.

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.

Ultrasound-assisted fermentation for cider production from Lebanese apples.

The present work studies the impact of low-intensity ultrasound (US) on Hanseniaspora sp. yeast fermentations. The effect of pulse duration and growth phase on US application was first evaluated using a synthetic medium. The optimal conditions were then applied to apple juice US-assisted fermentation. An US treatment chamber was first designed to allow the recycling of the culture medium. The optimal US pulse duration on the yeast growth rate was of 0.5 s followed by 6 s rest period, and during 6 h of both Lag and Log phases. These US parameters led to a faster consumption of glucose in the medium during the fermentation, compared to the untreated culture. The impact of US was also depending on the growth phase, showing higher sensitivity of the yeast to US during the Lag phase rather than the Log phase. US-assisted fermentation of apple juice showed a significant increase in biomass growth and glucose consumption, along with a significant decrease in the ethanol yield. The fastest growth kinetic (by 52%), and the highest ethanol reduction (by 0.55% (v, v)) were obtained for the treatment during the first 12 h of fermentation, thereby, the stationary phase was reached faster, and the maximum biomass growth rate was 10 folds higher compared to the untreated culture. The results obtained in this study demonstrated the promising efficiency of US-assisted fermentation in stimulating the biomass growth and reducing the ethanol content in alcoholic beverages.

An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae.

The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials. One such biomaterial is bacterial cellulose, which is a strong and ultrapure natural polymer used in tissue engineering scaffolds, wound dressings, electronics, food additives, and other products. However, despite the potential of Acetobacteraceae in biotechnology, there has been considerably little effort to fundamentally reprogram the bacteria for enhanced performance. One limiting factor is the lack of a well-characterized, comprehensive toolkit to control expression of genes in biosynthetic pathways and regulatory networks to optimize production and cell viability. Here, we address this shortcoming by building an expanded genetic toolkit for synthetic biology applications in Acetobacteraceae. We characterized the performance of multiple natural and synthetic promoters, ribosome binding sites, terminators, and degradation tags in three different strains, namely, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 53582, and Komagataeibacter rhaeticus iGEM. Our quantitative data revealed strain-specific and common design rules for the precise control of gene expression in these industrially relevant bacterial species. We further applied our tools to synthesize a biodegradable cellulose-chitin copolymer, adjust the structure of the cellulose film produced, and implement CRISPR interference for ready down-regulation of gene expression. Collectively, our genetic parts will enable the efficient engineering of Acetobacteraceae bacteria for the biomanufacturing of cellulose-based materials and other commercially valuable products.

Genomic content of a novel yeast species Hanseniaspora gamundiae sp. nov. from fungal stromata (Cyttaria) associated with a unique fermented beverage in Andean Patagonia, Argentina.

A novel yeast species was isolated from the sugar-rich stromata of Cyttaria hariotii collected from two different Nothofagus tree species in the Andean forests of Patagonia, Argentina. Phylogenetic analyses of the concatenated sequence of the rRNA gene sequences and the protein-coding genes for actin and translational elongation factor-1α indicated that the novel species belongs to the genus Hanseniaspora. De novo genome assembly of the strain CRUB 1928T yielded a 10.2-Mbp genome assembly predicted to encode 4452 protein-coding genes. The genome sequence data were compared to the genomes of other Hanseniaspora species using three different methods, an alignment-free distance measure, Kr, and two model-based estimations of DNA-DNA homology values, of which all provided indicative values to delineate species of Hanseniaspora. Given its potential role in a rare indigenous alcoholic beverage in which yeasts ferment sugars extracted from the stromata of Cytarria sp., we searched for the genes that may suggest adaptation of novel Hanseniaspora species to fermenting communities. The SSU1-like gene encoding a sulfite efflux pump, which, among Hanseniaspora, is present only in close relatives to the new species, was detected and analyzed, suggesting that this gene might be one factor that characterizes this novel species. We also discuss several candidate genes that likely underlie the physiological traits used for traditional taxonomic identification. Based on these results, a novel yeast species with the name Hanseniaspora gamundiae sp. nov. is proposed with CRUB 1928T (ex-types: ZIM 2545T = NRRL Y-63793T = PYCC 7262T; MycoBank number MB 824091) as the type strain. Furthermore, we propose the transfer of the Kloeckera species, K. hatyaiensis, K. lindneri and K. taiwanica to the genus Hanseniaspora as Hanseniaspora hatyaiensis comb. nov. (MB 828569), Hanseniaspora lindneri comb. nov. (MB 828566) and Hanseniaspora taiwanica comb. nov. (MB 828567).

Fourier Transform Near-Infrared Spectroscopy and Chemometrics To Predict Zygosacchromyces rouxii in Apple and Kiwi Fruit Juices.

This study investigated the capability of near-infrared spectroscopy (NIRS) to predict the concentration of Zygosaccharomyces rouxii in apple and kiwi fruit juices. The yeast was inoculated in fresh kiwi fruit juice ( n = 68), reconstituted kiwi juice ( n = 85), and reconstituted apple juice ( n = 64), followed by NIR spectra collection and plate counting. A principal component analysis indicated direct orthogonal signal correction preprocessing was suitable to separate spectral samples. Parameter optimization algorithms increased the performance of support vector machine regression models developed in a single variety juice system and a multiple variety juice system. Single variety juice models achieved accurate prediction of Z. rouxii concentrations, with the limit of quantification at 3 to 15 CFU/mL ( R2 = 0.997 to 0.999), and the method was also feasible for Hanseniaspora uvarum and Candida tropicalis. The best multiple variety juice model obtained had a limit of quantification of 237 CFU/mL ( R2 = 0.961) for Z. rouxii. A Bland-Altman analysis indicated good agreement between the support vector machine regression model and the plate counting method. It suggests that NIRS can be a high-throughput method for prediction of Z. rouxii counts in kiwi fruit and apple juices.

Pineapple (Ananas comosus L. Merr.) wine production in Angola: Characterisation of volatile aroma compounds and yeast native flora.

A pineapple vinification process was conducted through inoculated and spontaneous fermentation to develop a process suitable for a quality beverage during two successive vintages in Huambo, Angola. Wines obtained with the conventional Saccharomyces cerevisiae strain, were analysed by gas chromatography, and a total of 61 volatile constituents were detected in the volatile fraction and 18 as glycosidically bound aroma compounds. Concentration levels of carbonyl and sulphur compounds were in agreement with the limited information reported about pineapple fruits of other regions. We report, for the first time in pineapple wines, the presence of significant concentrations of lactones, ketones, terpenes, norisoprenoids and a variety of volatile phenols. Eight native yeast strains were isolated from spontaneous batches. Further single-strain fermentations allowed us to characterise their suitability for commercial fermentation. Three native strains (Hanseniaspora opuntiae, H. uvarum and Meyerozyma guilliermondii) were selected with sensory potential to ferment pineapple fruits with increased flavour diversity. Results obtained here contribute to a better understanding of quality fermentation alternatives of this tropical fruit in subtropical regions.

Optimization of experimental and modelling parameters for the differentiation of beverage spoiling yeasts by Matrix-Assisted-Laser-Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in response to varying growth conditions.

The growth of spoiling yeasts in beverages results in reduced quality, economic and image losses. Therefore, biochemical and DNA-based identification methods have been developed but are mostly time-consuming and laborious. Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) could deliver discriminative peptide mass fingerprints within minutes and could thus be a rapid and reliable tool for identification and differentiation. However, routine analysis of yeasts by MALDI-TOF MS is yet impaired by low reproducibility and effects of different physiological states of organisms on the reliability of the identification method are still controversial. The aim of this study was to optimize sample preparation and measurement parameterization using three spoilage yeasts (Saccharomyces cerevisiae var. diastaticus, Wickerhamomyces anomalus and Debaryomyces hansenii). The influence of environmental or physiological parameters including oxygen availability, different nutrients, cell density and growth phase were analysed and revealed small differences in mass fingerprints. Yeasts grown in the presence or absence of oxygen were precisely differentiated along these differences in mass fingerprints and a crude classification of growth phase was possible. Cell concentration did not affect the spectra distinctly, neither qualitatively nor quantitatively, and an influence of available nutrients could not be measured in each case. However, core mass peaks remained constant under all tested conditions enabling reliable identification.

Acetobacter malorum and Acetobacter cerevisiae identification and quantification by Real-Time PCR with TaqMan-MGB probes.

The identification and quantification of Acetobacter malorum and Acetobacter cerevisiae in wine and vinegar were performed using the Real-Time PCR (RT-PCR) with two TaqMan-MGB probes designed to amplify the internal transcribed spacer (ITS) region between the 16S-23S rRNA genes. The primers and probes were highly specific, with a detection limit of 10² cells/ml for both species, and the efficiency of the technique was >80%. The RT-PCR technique with these two new TaqMan-MGB probes, together with the five (Acetobacter aceti, Acetobacter pasteurianus, Gluconobacter oxydans, Gluconacetobacter hansenii and Gluconacetobacter europaeus) that are already available (Torija et al., 2010), were validated on known concentrations of Acetic Acid Bacteria (AAB) grown in glucose medium (GY) and in inoculated matrices of wine and vinegar. Furthermore, this technique was applied to evaluate the AAB population in real wine samples collected in the Canary Islands. PCR enrichment performed prior to RT-PCR increased the accuracy of quantification and produced results similar to those detected with SYBR-Green. In real wine samples, the total AAB enumeration ranged from 9 × 10² to 106 cells/ml, and the seven AAB species tested were detected in more than one sample. However, AAB recovery on plates was poor; the isolates obtained on plates were A. malorum, G. oxydans, A. cerevisiae and A. pasteurianus species. RT-PCR with TaqMan-MGB probes is an accurate, specific and fast method for the identification and quantification of AAB species commonly found in wine and vinegar.

Influence of fermentation with Hanseniaspora sp. yeast on the volatile profile of fermented apple.

This study aims to evaluate the aromatic profile of fermented apple, obtained by the action of strains of Hanseniaspora uvarum and Hanseniaspora guilliermondii using two methods of analysis: static headspace and solid phase microextraction (SPME). The results obtained confirm that the strains of the Hanseniaspora genus contributed positively to the aroma profile of fermented apple, producing considerable amounts of esters and higher alcohols. In comparing the methods of analysis of aromatic compounds using headspace and SPME, it was verified that by using the headspace method it was possible to capture amounts that were up to 16 times greater than the value of the volatile compounds obtained by SPME. However, when using SPME, 5 times more compounds were obtained than when using headspace. Even so, in the conditions of this study it was noted that headspace was more efficient in the extraction of the aromatics of fermented apple when taking into consideration the cost effectiveness of both methods.

Determination of the total phenolic and anthocyanin contents and antimicrobial activity of Viburnum opulus fruit juice.

Viburnum opulus is a plant with fruits that are rich in biologically active substances, making it valuable to the food and pharmaceutical industries. Here, we present our study of the total phenolic and anthocyanin contents and antimicrobial activity in the fruit juice of six V. opulus L. accessions. The cultivar 'Krasnaya Grozd' was notable for its exceptionally large amount of total phenolics, 1168 mg/100 g, with anthocyanins comprising 3-5 % of the total phenolic content. The evaluation of the antimicrobial properties confirmed that the juice of V. opulus fruits strongly inhibited the growth of a wide range of human pathogenic bacteria, both Gram-negative (Salmonella typhimurium and S. agona) and Gram-positive (Staphylococcus aureus, Lysteria monocytogenes, and Enterococcus faecalis) organisms. Conversely, the yeasts Debaryomyces hansenii and Torulaspora delbrueckii showed complete resistance to the fruit juice, whereas a low sensitivity was demonstrated by Trichosporon cutaneum, Kluyveromyces marxianus var. lactis, Saccharomyces cerevisiae, S. cerevisiae 12R, and Candida parapsilosis.

Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.

Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.

Yeast species associated with the spontaneous fermentation of cider.

This paper reports the influence of cider-making technology (pneumatic and traditional pressing) on the dynamics of wild yeast populations. Yeast colonies isolated from apple juice before and throughout fermentation at a cider cellar of Asturias (Spain), during two consecutive years were studied. The yeast strains were identified by restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the two flanking internal transcribed sequences (ITS). The musts obtained by pneumatic pressing were dominated by non-Saccharomyces yeasts (Hanseniaspora genus and Metschnikowia pulcherrima) whereas in the apple juices obtained by traditional pressing Saccharomyces together with non-Saccharomyces, were always present. The species Saccharomyces present were S. cerevisiae and S. bayanus. Apparently S. bayanus, was the predominant species at the beginning and the middle fermentation steps of the fermentation process, reaching a percentage of isolation between 33% and 41%, whereas S. cerevisiae took over the process in the final stages of fermentation. During the 2001 harvest, with independence of cider-making technology, the species Hanseniaspora valbyensis was always isolated at the end of fermentations.

Noncarious cervical lesions among a non-toothbrushing population with Hansen's disease (leprosy): initial findings.

OBJECTIVE: The purpose of this preliminary investigation was to examine the presence of noncarious cervical lesions (NCCLs) among a convenience sample of non-toothbrushing subjects with Hansen's disease (leprosy). METHOD AND MATERIALS: A cross-sectional sample of 102 non-toothbrushing subjects (20 to 77 years of age) was examined. The clinical parameter of interest for this study was the presence or absence of NCCLs and their probable etiology as it relates to the subjects' diet, occlusion, and use of medication. Subjects were examined clinically and interviewed according to study protocol. RESULTS: NCCLs were found in 48 subjects (47% of the studied sample). Widespread consumption of acidic foods and beverages acting as corrodents, signs of parafunction, and use of medication that causes xerostomia were also noted. Thus, all may be contributing factors in the etiology of NCCLs in this population. CONCLUSION: This preliminary report suggests that toothbrush/dentifrice abrasion was not a factor in the etiology of NCCLs in the population studied. The authors intend to expand their study among these non-toothbrushing

Pharmacokinetics and relative bioavailability of clofazimine in relation to food, orange juice and antacid.

BACKGROUND: Clofazimine is potentially useful for the treatment of disease due to multidrug resistant Mycobacterium tuberculosis, as well as leprosy and certain chronic skin diseases. Its pharmacokinetics have been incompletely characterized. This study was conducted to explore issues relating to bioavailability in the presence of food, orange juice, and antacid. METHODS: A 5 drug regimen consisting of clofazimine, cycloserine, ethionamide, para-aminosalicyclic acid, and pyridoxime was administered to healthy subjects four times using a four period cross-over design with two weeks washout between treatments. Subjects also received orange juice, a high fat meal, aluminum/magnesium antacid, or only water in random order with the drug regimen. The pharmacokinetics of clofazimine were assessed using individual- and population-based methods and relative bioavailability compared to fasting administration was determined. RESULTS: Clofazimine exhibited a sometimes prolonged and variable lag-time and considerable variability in plasma concentrations. From the population analysis (one-compartment model), the mean oral clearance was 76.7 l/h (CV=74.2%) and mean apparent volume of distribution was 1470 l (CV=36.3%). The first-order absorption rate constant ranged from 0.716 to 1.33 h(-1) (pooled CV=61.7%). Residual (proportional) error was 49.1%. Estimates of bioavailability compared to fasting administration were 145% (90% CI, 107-183%) for administration with high fat food, 82.0% (63.2-101%) for administration with orange juice, and 78.5% (55.1-102%) for administration with antacid. CONCLUSION: Administration of clofazimine with a high fat meal provides the greatest bioavailability, however, bioavailability is associated with high inter- and intra-subject variability. Both orange juice and aluminum-magnesium antacid produced a reduction in mean bioavailability of clofazimine.

Killer toxin of Kluyveromyces phaffii DBVPG 6076 as a biopreservative agent to control apiculate wine Yeasts.

The use of Kluyveromyces phaffii DBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin of K. phaffii DBVPG 6076 showed extensive anti-Hanseniaspora activity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40 degrees C. These biochemical properties would allow the use of K. phaffii killer toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain of Hanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin to H. uvarum cells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that the K. phaffii DBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.