MOLECULAR CHARACTERIZATION, STRESS RESPONSES AND SPECIFIC ENZYMATIC ACTIVITIES OF DEKKERA/BRETTANOMYCES BRUXELLENSIS WINE STRAINS: STRATEGIES OF ANALYSIS AND CONTROL IN THE OENOLOGICAL FIELD.

Among yeasts responsible for wine spoilage, Dekkera/Brettanomyces bruxellensis is the species on which the scientific community has the highest interest. This fact is documented by an increasing in the international publications and by the beginning of the genome sequencing. Recently, it has been traced the evolutional D. bruxellensis lineage by the analysis of promoter sequences, which separated from the Saccharomyces yeasts more than 200 mya. Spoilage caused by D./B. bruxellensis is mainly due to the following issues: \uf0fc this species remains viable and active in beverages preserved by extreme abiotic stress (anaerobiosis, up to 12-13% ethanol (v/v), minimal amounts of fermentable sugars); \uf0fc the adopted treatments (sulphiting, membrane filtration, transfer of wine to sanitized barrels) are not always effective. \uf0fc the off-flavours produced by Brettanomyces include volatile phenols characterised by disagreeable odours. The aim of this thesis was to develop strategies in order to analyse and control the wine spoilage linked to D./B. bruxellensis species. As concern the first issue, spoilage microbial analysis, the present PhD thesis describes: i) the development of new methods for D./B. bruxellensis molecular typing; ii) the phenotypic biodiversity of D./B. bruxellensis species. The topics on the microbial spoilage control were: iii) D./B. bruxellensis response to stress conditions. i) Studies on the natural distribution of D./B. bruxellensis have shown an existing high intraspecies polymorphism degree which is probably due to a fusion event among genomes or to the lacking of a sexual state. Moreover, since D./B. bruxellensis has been mainly associated to fermented beverages that represent mutagenic environments determining the frequent genome rearrangement of D./B. bruxellensis. Genetic variations are usually accumulated with a higher frequency in DNA regions that are not linked to any gene function respect to the coding regions such as introns. Thus, they are considered good indicators in evolutional studies; in S. cerevisiae, the lariat branch point TACTAAC and the 5\u2019 splice site GTATGT (5\u2019ss) are conserved sequences that were used to build primers for the Intron Splice Site amplification analysis (ISS-PCR) described for inter- and intraspecific characterisation of S. cerevisiae. The main goals of this first topic was to develop new methods for D./B. bruxellensis molecular typing. The setting up of a multiplex PCR protocol throughout the use of modified oligonucleotides that targeted 5\u2019ss -GTAAGT- has confirmed a high polymorphism among D. bruxellensis genomes. Thus, a further optimisation of the primers, a simple capillary electrophoresis protocol that can accurately separates the amplified fragments and clear rules for the ISS profiles elaboration were applied. The results points out that the genetic signatures obtained exploiting the ISS as molecular targets are able to show genetic differences that, up to now, only other laborious technique can put in evidence (Karyotyping, PFGE-RFLP, AFLP). The proposed protocol has proved to be reliable and robust. Moreover, considering that a positive correlation between the extent of non-protein-coding DNA and the eukaryotic complexity degree has been observed, the ISS fingerprinting can represents a useful tool to analyse the evolution rate of a yeast species. ii) D./B. bruxellensis yeasts have evolved numerous developmental options in order to adapt and survive the changing status of the environment. Independent studies showed that distinct genetic groups of D./B. bruxellensis can have different physiological characteristics and strong differences in their ability to produce 4-ethylphenols. The main goal of second topic was to characterise D./B. bruxellensis from a phenotipic point of view. In particular, the physiological diversity within D./B. bruxellensis strains was investigated studying the growth and the production of volatile phenols and biogenic amines under a wine model condition. Moreover, the carbon compounds assimilation, H2S production, and vinylphenol reductase (VPR) activity were also analysed. The potential hazard of spoilage when D./B. bruxellensis grows in oenological conditions was confirmed since most of the analysed strains were able to produce volatile phenols or showed a detectable VPR specific activity. Actually metabolic traits, as growth rate and off-flavour production, proved to be related and strain-dependent suggesting that an early detection and identification of \u201cfast-growing yeasts\u201d and \u201cfast volatile phenols producers\u201d could be essential to introduce adequate corrective measures. The experiments on carbon assimilation revealed that about 30% of the analysed yeasts has a own pattern in the utilization of carbonious sources. Three different ranges of VPR activity and H2S production were used to assign the physiological profiles. Combining the results from the three phenotypic experiments, about 70% of the CBS collection can be discriminated at strain level. The present work suggested that the chromosomal rearrangement occurred in this species has generated strains with a high physiological polymorphic state. iii) A goal of the wine industry is to reduce the risk of wine being spoiled by microbial activity. The main aim of the third topic of this research was to study the response to stress conditions in D./B. bruxellensis due to the yeast exposition to an electric current treatment and exogenous SO2. Results indicated that a similar effect occurred on cells after the current treatment in comparison to the SO2 exposition; both treatments resulted in a reduced microbial cell survival in the studied red wine. The kinetics of volatile phenol accumulation confirmed that, the use of an electric field could be adequate to hinder the yeast spoilage. As concern the latter issue, the SO2 resistance, a metabolomic study on the effect of the SO2 addiction to D./B. bruxellensis cultures was carried out too. Results displayed that among the metabolic pathways resulted to be affected by exogenous sulphite concentration, aminoacid metabolisms seem to be involved in the SO2 tolerance. Unlike what was observed in S. cerevisiae, neither adenine nor methionine modified the toxicity level of SO2 under laboratory conditions. The ethanol concentration seems to increase the sensitivity to sulphite suggesting that a membrane system, such as the sulphite efflux pump of S. cerevisiae, could be present in this species. In conclusion, the main research products of this PhD thesis were: \uf0fc a new PCR protocol to typing D./B. bruxellensis that uses specific primers for this yeast species, and a precise and reliable fragment separation protocol by capillary electrophoresis. Actually, this method shows a high reproducibility (94%), it is rapid in comparison to other techniques that in the past allowed a discrimination at strain level of D./B. bruxellensis isolates (Karyotyping, RFLP-PFGE, AFLP, etc.), and it represents a useful tool to monitor the yeast evolution rate; \uf0fc the collection of D./B. bruxellensis phenotypic features that never have been used to evaluate the biodiversity degree in this species, such as the VPR specific activity, H2S production, and the assimilation of carbon compounds different from ones found in wine. The compilation of a database collecting both genetic and phenotypic traits of different D./B. bruxellensis strains is the future perspective to offer an efficient way to counteract this spoilage yeast; \uf0fc a new technology to reduce the survival of D. bruxellensis in wine using a low electric current (LEC) treatment. \uf0fc the understanding of some metabolic mechanisms involved in the SO2 response in D./B. bruxellensis. This step will allow the following upgrade toward the study of molecular mechanisms, and metabolic pathways that this yeast can activate to protect itself against the exposure to high concentration of exogenous sulphur dioxide

Tryptophan-ethylester, the false (unveiled) melatonin isomer in red wine

Among the food plants, the presence of melatonin in grapes (Vitis vinifera L.) deserves particular attention because of the production of wine, an alcoholic beverage of economic relevance and with putative healthy effects. Furthermore, melatonin isomers have been detected in wine too. Recently, one of these isomers has been identified as tryptophan-ethylester, a compound with the same molecular weight of melatonin. In this Commentary, we briefly comment the source(s) of tryptophan-ethylester in wine and the putative nutritional role(s)

Why do grape-based fruit wines could be “super” magic?

In Europe, the global consumption of wine is decreasing and new alternatives of wine have been appearing on the market. The attractiveness of these products rely on the fact that they are inexpensive and easy to drink, with low alcohol content and obtained by mixing wines with fruit juices or flavoring wine. While fruit wines represent an ancient art [1], no studies have investigated the production of beverages that are obtained by the co-fermentation of grape and fruit. The formulation of new mixed-fruit wines could represent the basis for reducing post-harvest fruit losses and contribute to the economy of the existing wine industry. Moreover, throughout the selection of useful yeasts that drive the alcoholic fermentation, the final products could be enriched in novel active bio-functional compounds not found in traditional wines. The research activity has involved the setup of fermentation trials using grape must and fruit juices of strawberry, peach, cherry and kiwi. Must fermentations were carried out inoculating Saccharomyces cerevisiae and Torulaspora delbruekii species in four musts obtained by blending grape must (Chardonnay and Cabernet Sauvignon) and juices at 80:20 and 60:40 grape:fruit juice proportion. The aromatic profile was determined by GC-MS. S. cerevisiae was able to exhaust the available sugars in all trials. In particular, based on the proportion used, alcohol was 3-5% less in wines prepared with red grape and 6-7% (v/v) less in case of white grape. T. delbruekii showed a good fermentation performance as well, suggesting its potential use as starter yeast. Only negligible difference was found for the pH, while the total acidity was higher for mixed-fruit wines obtained with must from white grape and at the 60:40 proportion. In particular, the high level of citric acid detected (up to 8 g/L) in some products could exert a beneficial effect by protecting the wine components, bio-functional compounds included, against oxidation

Corrigendum: Investigating the Effect of Selected Non-Saccharomyces Species on Wine Ecosystem Function and Major Volatiles [Front. Bioeng. Biotechnol., 6, (2018) (169)] DOI: 10.3389/fbioe.2018.00169

In the original article, there was a mistake in Figure 2 as published. The order of the graphs (A-H) is incorrect and does not match the caption nor the in-text citation. The corrected Figure 2 appears below. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated

Melatonin and its derivatives in red wine: contribution of fermenting microorganisms

Melatonin (MEL) is an indoleamine produced mainly by the pineal gland in vertebrates and it has a significant role in regulation of circadian rhythm, mitigation of sleeping disorder and jet lag. It has been found in medicinal plants, plant foods including seeds, fruits and fermented beverages, and, therefore, its occurrence in plants is now ascertained. In grapes and wines, MEL ranges from sub-ng/g to \ub5g/g and from sub-ng/mL to ng/mL, respectively, with varying levels according to both endogenous and exogenous factors. In addition, MEL isomers and tryptophan-ethylester (TEE, a compound with the same molecular weight of MEL) have recently been detected in wine and the fermenting yeast plays an important role for their production [1]. It has been suggested that, in synergy with polyphenols, MEL in wine may contribute to maximize the health-promoting effects of Mediterranean diet. The research aimed to validate an analytical method for the simultaneous detection of MEL, TEE and tryptophan. The sample preparation was developed by means of SPE purification. Purified samples of synthetic wine solution (tartaric acid 5 g/L, ethanol 12% [v/v], pH 3.2) and red wine spiked with the analytes of interest were analyzed by liquid chromatography coupled with both fluorescence and mass spectrometry detectors. MEL, TEE and tryptophan were successfully detected and quantified by both the analytical conditions adopted. The response was linear for all the investigated compounds and it was comparable between synthetic wine solution and red wine. The recovery was higher than 85% and the relative standard deviation was lower than 10%. The developed method was applied for the analysis of red wine samples produced in a cold area of North of Italy. Preliminary results showed an increase of TEE concentrations suggesting the possible positive influence of secondary fermentations other than the alcoholic fermentation

Wine industry’s attitude towards oenological yeasts : Italy as a case study

Yeast inoculation is a widespread practice in winemaking in order to control the must fermentation. However, the use of indigenous wine yeasts can enrich wine quality and differentiate wine styles. Yeast cream preparation (CRY), recently accepted by the International Organization of Vine and Wine, could allow an easier usage of autochthonous yeasts. This work aimed at investigating the actual Italian wine industry\u2019s attitude towards the available formulations of commercial wine yeasts with attention to CRY. Moreover, this study evaluated the perception of wineries toward indigenous yeasts in both winemaking and marketing viewpoints. Data show different levels of knowledge and use about the available yeast formulations. In general, there is not a predominantly positive or negative participants\u2019 opinion regarding the use of indigenous yeasts. Wineries using CRY (4% of the sample) mainly adopt them as a part of the production in order to compare the wines with the ones traditionally obtained with commercial yeasts. CRY is perceived by some interviewees as a potential tool to increase communication and product differentiation. This survey could have anticipated future trends in the use of yeast formulations, determined by the market demands for diversified, unique, and environmentally sustainable products, that can allow an accessible application of precision enology

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier "indolome" to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us "indolome") in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100-1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition

Genotypic Characterization and Biofilm Formation of Shiga-toxin producing Escherichia coli

Shiga toxin producing Escherichia coli (STEC) are recognized as one of the most dangerous food-borne pathogens. The production of Shiga toxins together with intimin protein are among the main virulence factors. However, the ability to form biofilm can protect bacteria against environmental factors (i.e. desiccation, exposure to UV rays, predation, etc) and sanitization procedures (cleaning, rinsing, chlorination), increasing their survival on food products and in manufacturing plants. Forty-five isolates collected from food and fecal samples were genotyped by Pulsed Field Gel Electrophoresis (PFGE) analysis with XbaI restriction enzyme and investigated by searching for toxins (stx1, stx2) and intimin (eae) genes and serogroup (O157, O26, O145, O111, O103 and O104). Afterward, the ability to develop biofilm in microtiter assay and the production of adhesive curli fimbriae and cellulose on agar plates were tested. Our study demonstrated that biofilm formation has a great variability among STEC strains and cannot be related to a specific pulsotype nor even to serogroup or presence of virulence genes

CRISPR/Cas9 system as a valuable genome editing tool for wine yeasts with application to decrease urea production

An extensive repertoire of molecular tools is available for genetic analysis in laboratory strains of S. cerevisiae. Although this has widely contributed to the interpretation of gene functionality within haploid laboratory isolates, the genetics of metabolism in commercially-relevant polyploid yeast strains is still poorly understood. Genetic engineering in industrial yeasts is undergoing major changes due to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) engineering approaches. Here we apply the CRISPR/Cas9 system to two commercial "starter" strains of S. cerevisiae (EC1118, AWRI796), eliminating the CAN1 arginine permease pathway to generate strains with reduced urea production (18.5 and 35.5% for EC1118 and AWRI796, respectively). In a wine-model environment based on two grape musts obtained from Chardonnay and Cabernet Sauvignon cultivars, both S. cerevisiae starter strains and CAN1 mutants completed the must fermentation in 8-12 days. However, recombinant strains carrying the can1 mutation failed to produce urea, suggesting that the genetic modification successfully impaired the arginine metabolism. In conclusion, the reduction of urea production in a wine-model environment confirms that the CRISPR/Cas9 system has been successfully established in S. cerevisiae wine yeasts

A response surface methodology approach to investigate the effect of sulfur dioxide, pH, and ethanol on DbCD and DbVPR gene expression and on the volatile phenol production in Dekkera/Brettanomyces bruxellensis CBS2499

Dekkera/Brettanomyces bruxellensis, the main spoilage yeast in barrel-aged wine, metabolize hydroxycinnamic acids into off-flavors, namely ethylphenols. Recently, both the enzymes involved in this transformation, the cinnamate decarboxylase (DbCD) and the vinylphenol reductase (DbVPR), have been identified. To counteract microbial proliferation in wine, sulfur dioxide (SO2) is used commonly to stabilize the final product, but limiting its use is advised to preserve human health and boost sustainability in winemaking. In the present study, the influence of SO2was investigated in relation with pH and ethanol factors on the expression of DbCD and DbVPR genes and volatile phenol production in D. bruxellensis CBS2499 strain under different model wines throughout a response surface methodology (RSM). In order to ensure an exact quantification of DbCD and DbVPR expression, an appropriate housekeeping gene was sought among DbPDC, DbALD, DbEF, DbACT, and DbTUB genes by GeNorm and Normfinder algorithms. The latter gene showed the highest expression stability and it was chosen as the reference housekeeping gene in qPCR assays. Even though SO2could not be commented as main factor because of its statistical irrelevance on the response of DbCD gene, linear interactions with pH and ethanol concurred to define a significant effect (p < 0.05) on its expression. The DbCD gene was generally downregulated respect to a permissive growth condition (0 mg/L mol. SO2, pH 4.5 and 5% v/v ethanol); the combination of the factor levels that maximizes its expression (0.83-fold change) was calculated at 0.25 mg/L mol. SO2, pH 4.5 and 12.5% (v/v) ethanol. On the contrary, DbVPR expression was not influenced by main factors or by their interactions; however, its expression is maximized (1.80-fold change) at the same conditions calculated for DbCD gene. While no linear interaction between factors influenced the off-flavor synthesis, ethanol and pH produced a significant effect as individual factors. The obtained results can be useful to improve the SO2management at the grape harvesting and during winemaking in order to minimize the D./B. bruxellensis spoilage