Influence of Different Types, Utilization Times, and Volumes of Aging Barrels on the Metabolite Profile of Red Wine Revealed by 1H-NMR Metabolomics Approach.

It is well recognized that the aging process is a critical step in winemaking because it induces substantial chemical changes linked to the organoleptic properties and stability of the finished wines. Therefore, this study aimed to investigate the influence of different types, utilization times, and volumes of aging barrels on the metabolite profile of red wines, produced from Thai-grown Shiraz grapes, using a non-targeted proton nuclear magnetic resonance (1H-NMR) metabolomics approach. As a result, 37 non-volatile polar metabolites including alcohols, amino acids, organic acids, carbohydrates and low-molecular-weight phenolics were identified. Chemometric analysis allowed the discrimination of wine metabolite profiles associated with different types of aging containers (oak barrels vs. stainless-steel tanks), as well as the utilization times (2, 6 and >10 years old) and volumes (225, 500 and 2000 L) of the wooden barrels employed. Significant variations in the concentration of formate, fumarate, pyruvate, succinate, citrate, gallate, acetate, tyrosine, phenylalanine, histidine, γ-aminobutyrate, methionine and choline were statistically suggested as indicators accountable for the discrimination of samples aged under different conditions. These feature biomarkers could be applied to manipulate the use of aging containers to achieve the desired wine maturation profiles.

Factors affecting bacterial community dynamics and volatile metabolite profiles of Thai traditional salt fermented fish.

Bacterial diversity of the Thai traditional salt fermented fish with roasted rice bran (Pla-ra) was investigated using classical and molecular approaches. Bacterial population of Pla-ra ranged from 102-106 in solid-state (SSF) and 106-109 CFU/g in submerged (SMF) fermentation types. Halanaerobium spp. and Lentibacillus spp. were the main genera particularly detected when rRNA analysis was applied. Tetragenococcus halophillus were dominant during the final stage in sea salt-recipe samples while Bacillus spp. were found in those rock salt recipes. In contrast, cultural plating demonstrated that Bacillus spp., generally B. amyloliquefaciens, were the dominant genera. In addition, B. pumilus, B. autrophaeus, B.subtilis and B. velezensis shown some relations with rock salt-recipe samples. The main volatile metabolites in all samples were butanoic acid and its derivatives. Key factors affected bacterial profiles and volatile compounds of salt fermented fish were type of salt, addition of roasted rice bran, and fermenting conditions.

Growth kinetics and fermentation properties of autochthonous yeasts in pineapple juice fermentation for starter culture development.

Autochthonous yeasts associated with pineapple fermentation were isolated and their fermentation behaviours were investigated for development of specific culture in pineapple winemaking. Autochthonous yeast isolates, Saccharomycodes ludwigii and Hanseniaspora uvarum, were selected due to their generated products of alcohol and 2-phenylethyl acetate, respectively. The fermentation kinetic parameters of selected autochthonous yeasts as single and co-cultures in chaptalized pineapple juice were investigated comparing to commercial Saccharomyces cerevisiae. The ethanol production rate of S'codes ludwigii (0.104%(v/v)/h) during the initial stage of fermentation was relatively slower compared to those of S. cerevisiae (0.129%(v/v)/h) but increased during middle through final stages with similar ethanol content to the commercial S. cerevisiae (~12%(v/v)). In pineapple juice, fructose was firstly assimilated, S'codes ludwigii (K = 0.405) and S. cerevisiae (K = 0.552), while glucose was secondly used, S'codes ludwigii (K = 0.281) and S. cerevisiae (K = 0.217) for first-order kinetic model. In co-cultures, the two isolated strains displayed synergistic behaviours during fermentation. S'codes ludwigii supported the growth of H. uvarum so that it generated more desirable volatile organic compounds (VOCs) at an early stage. Interestingly, the VOCs could not be produced in co-cultures of H. uvarum with the commercial strains. Then, S'codes ludwigii further completed the alcoholic fermentation through final stage. The fermentation performances of co-cultured autochthonous yeasts demonstrated a new approach for successful pineapple winemaking over S. cerevisiae. In addition, growth kinetics and fermentation behaviour, as observed in this study, could be a key information in development of potential substrates and strains for future alcoholic fermentation.

Synbiotic edible film from konjac glucomannan composed of Lactobacillus casei-01® and Orafti®GR, and its application as coating on bread buns.

BACKGROUND: Konjac glucomannan-based edible films formulated with Lactobacillus casei-01® and chicory-derived inulin Orafti®GR were studied for their properties, stability, and application as coatings on bread buns. RESULTS: Thickness and transparency were variable and dependent on the formulations. Alterations in color properties of all supplemented films were unnoticeable by unaided human eyes, with ΔE less than 3. Lactobacillus casei-01® and Orafti®GR were associated with higher water solubility of the films. Lactobacillus casei-01® decreased the water vapor permeability of the films while Orafti®GR promoted it. The mechanical properties in all combinations remained unchanged, although those with Orafti®GR showed profoundly reduced tensile strength. Scanning electron micrographs and Fourier transform infrared spectra of the films confirmed good homogeneity and intermolecular attraction between the prebiotic and konjac glucomannan. Cell viability in the films stored at room temperature decreased sharply, becoming less than the minimum recommended level after day 4, while viable L. casei-01® in coatings on bread buns gradually decreased, with a reduction of ca. 2 log colony-forming units (CFU) portion-1 over the 7 day storage period at room temperature. CONCLUSION: The synbiotic film and coating developed in this study are a relatively simple strategy for incorporating L. casei-01® and Orafti®GR into bread buns, which are short shelf-life foods. Bread buns with synbiotic coating could diversify functional food choices. Pretreatment, together with other technologies, is required to maintain a desirable number of active probiotic cells for longer. © 2020 Society of Chemical Industry.

Towards semi-synthetic microbial communities: enhancing soy sauce fermentation properties in B. subtilis co-cultures.

BACKGROUND: Many fermented foods and beverages are produced through the action of complex microbial communities. Synthetic biology approaches offer the ability to genetically engineer these communities to improve the properties of these fermented foods. Soy sauce is a fermented condiment with a vast global market. Engineering members of the microbial communities responsible for soy sauce fermentation may therefore lead to the development of improved products. One important property is the colour of soy sauce, with recent evidence pointing to a consumer preference for more lightly-coloured soy sauce products for particular dishes. RESULTS: Here we show that a bacterial member of the natural soy sauce fermentation microbial community, Bacillus, can be engineered to reduce the 'browning' reaction during soy sauce production. We show that two approaches result in 'de-browning': engineered consumption of xylose, an important precursor in the browning reaction, and engineered degradation of melanoidins, the major brown pigments in soy sauce. Lastly, we show that these two strategies work synergistically using co-cultures to result in enhanced de-browning. CONCLUSIONS: Our results demonstrate the potential of using synthetic biology and metabolic engineering methods for fine-tuning the process of soy sauce fermentation and indeed for many other natural food and beverage fermentations for improved products.

Application of Reverse Transcriptase-PCR-DGGE as a rapid method for routine determination of Vibrio spp. in foods.

The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 102 to 105CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 102CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively.

Rapid and simple colorimetric method for the quantification of AI-2 produced from Salmonella Typhimurium.

The aim of this study was to evaluate the feasibility of Fe(III) ion reduction for the simple and rapid quantification of autoinducer-2 (AI-2) produced from bacteria using Salmonella Typhimurium as a model. Since the molecular structure of AI-2 is somewhat similar to ascorbic acid it was expected that AI-2 would also act as a reducing agent and reduce Fe(III) ions in the presence of 1,10-phenanthroline to form the colored [(o-phen)3 Fe(II)]SO4 ferroin complex that could be quantified colorimetrically. In support of this, colony rinses and cell free supernatants from cultures of all tested AI-2 producing strains, but not the AI-2 negative Sinorhizobium meliloti, formed a colored complex with a λmax of 510nm. The OD510 values of these culture supernatants or colony rinses were in broad agreement with the % activity observed in the same samples using the standard Vibrio harveyi bioluminescence assay for AI-2 detection, and with previously reported results. This methodology could potentially be developed as an alternative method for the simple and rapid quantification of AI-2 levels produced in bacterial cultures.

Autochthonous yeasts associated with mature pineapple fruits, freshly crushed juice and their ferments; and the chemical changes during natural fermentation.

This study investigated autochthonous yeasts and their functions in the spontaneous fermentation of freshly crushed pineapple juice samples collected from two different areas of both Thailand and Australia. Hanseniaspora uvarum and Pichia guilliermondii were the main yeast species observed on the fruit skins of Thai samples, and also in the fresh juice and ferments of all samples from both countries. P. guilliermondii was consistently present as the dominant species during the early stage of the fermentation, whereas H. uvarum became more prevalent towards the end of the six-day fermentation period, with populations increasing from an initial level of approximately 5 log CFU/mL to approximately 8 log CFU/mL at the end of fermentation. The ethanol levels in samples from both regions of Thailand were maximal at 2 days of fermentation, reaching approximately 1 to 2% (v/v) but then declined thereafter. In contrast, in the Australian samples ethanol levels continued to increase over the entire six-day fermentation period and reached approximately 3 to 4% (v/v). A significant decrease in citric acid and increase in lactic acid levels were observed throughout the fermentation period in the samples from Thailand, but not in those from Australia where the different acid contents (and pH) were relatively stable. The other wine yeasts and, in particular, Saccharomyces yeasts, were not found in any of sampled fermentation systems that is apparently different from the other fruit juices. These findings suggested that the freshly crushed pineapple juice may possibly have some effects on the other autochthonous yeasts having important role in alcoholic fermentation.

Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes.

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.