Abstract:Fermented foods and beverages are a heterogeneous class of products with a relevant worldwide significance for human economy, nutrition and health for millennia. A huge diversity of microorganisms is associated with the enormous variety in terms of raw materials, fermentative behavior and obtained products. In this wide microbiodiversity it is possible that the presence of microbial pathogens and toxic by-products of microbial origin, including mycotoxins, ethyl carbamate and biogenic amines, are aspects liable to reduce the safety of the consumed product. Together with other approaches (e.g., use of preservatives, respect of specific physico-chemical parameters), starter cultures technology has been conceived to successfully dominate indigenous microflora and to drive fermentation to foresee the desired attributes of the matrix, assuring quality and safety. Recent trends indicate a general return to spontaneous food fermentation. In this review, we point out the potential risks for human health associated with uncontrolled (uninoculated) food fermentation and we discuss biotechnological approaches susceptible to conciliate fermented food safety, with instances of an enhanced contribution of microbes associated to spontaneous fermentation.Keywords: fermented food; fermentation; beverage; safety; risks; spontaneous fermentation; starter cultures; spoilage microbes; pathogens; contaminant Fermented Foods and Beverages: Scientific Dimension, Social Relevance, and Economic SignificanceA large basket of food and beverages is obtained from a microbial-based transformation of food raw materials. Different classes of microorganisms can be involved, mainly yeasts and bacteria, with a certain role of molds. The obtained fermented foods and beverages have ben staple foods for millennia, with a considerable importance in the human diet for reasons of generally enhanced shelf-life, palatability, safety and nutritional quality [1]. In fact, the desired fermentation process consists of protechnological microbial development in the given matrix, with direct and indirect effects of primary and secondary microbial metabolism. Protechnological microorganisms, in order to obtain energy and to sustain their anabolic processes, reduce the content of carbohydrates and other macromolecules available in the raw matrix, accumulating catabolic products (e.g., lactic acid, ethanol). These biological dynamics, together with the possible release of antimicrobial compounds [2], reduce the risks of undesired microbial developments (thus increasing product shelf-life and safety level). On the other hand, both primary and secondary metabolites strongly influence palatability

Lactobacillus casei can metabolize L-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of L-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on L-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for L-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on L-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of L-malic acid and repressed by glucose, whereas TC system expression was induced by L-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5-TTATT(A/T)AA-3] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.The metabolism of L-malic acid by lactic acid bacteria (LAB) has brought about considerable interest because of its relevance in winemaking (24). The degradation of L-malate to L-lactate leads to a reduction of the acidity of wine, and it provides microbiological stability by preventing the secondary growth of LAB after bottling. Most LAB decarboxylate Lmalate to L-lactate by an NAD ϩ -and Mn 2ϩ -dependent malolactic enzyme (MLE) (Fig. 1); nevertheless, a few LAB species can also degrade L-malate to pyruvate by a malic enzyme (ME) (Fig. 1). This pathway was first detected in Enterococcus faecalis (20) and later in Lactobacillus casei (23,33) and Streptococcus bovis (14). In contrast to the utilization of L-malate through MLE, the utilization of the ME pathway enables these organisms to grow with L-malate as a carbon source (22). However, whereas MLE has been the focus of an extensive research effort, the physiological role and the regulation of ME remain largely unknown.L. casei is a facultative heterofermentative lactic acid bacterium frequently used as a cheese starter culture and which is also employed as a probiotic. Extensive research has been carried out on the study of sugar catabolism (28, 39-41); however, the knowledge of the utilization of organic acids has received less attention. As previously indicated, physiological and biochemical studies identified two L-malate dissimilation pathways in L. casei. Furthermore, these studies showed that ME expression...

Among the innovative trends in the wine sector, the continuous exploration of enological properties associated with wine microbial resources represents a cornerstone driver of quality improvement. Since the advent of starter cultures technology, the attention has been focused on intraspecific biodiversity within the primary species responsible for alcoholic fermentation (Saccharomyces cerevisiae) and, subsequently, for the so-called ‘malolactic fermentation’ (Oenococcus oeni). However, in the last decade, a relevant number of studies proposed the enological exploitation of an increasing number of species (e.g., non-Saccharomyces yeasts) associated with spontaneous fermentation in wine. These new species/strains may provide technological solutions to specific problems and/or improve sensory characteristics, such as complexity, mouth-feel and flavors. This review offers an overview of the available information on the enological/protechnological significance of microbial resources associated with winemaking, summarizing the opportunities and the benefits associated with the enological exploitation of this microbial potential. We discuss proposed solutions to improve quality and safety of wines (e.g., alternative starter cultures, multistrains starter cultures) and future perspectives.

: For 15 years, non-Saccharomyces starter cultures represent a new interesting segment in the dynamic field of multinationals and national companies that develop and sell microbial-based biotechnological solutions for the wine sector. Although the diversity and the properties of non- Saccharomyces species/strains have been recently fully reviewed, less attention has been deserved to the commercial starter cultures in term of scientific findings, patents, and their innovative applications. : Considering the potential reservoir of biotechnological innovation, these issues represent an underestimated possible driver of coordination and harmonization of research and development activities in the field of wine microbiology. After a wide survey, we encompassed 26 different commercial yeasts starter cultures formulated in combination with at least one non-Saccharomyces strain. The most recent scientific advances have been explored delving into the oenological significance of these commercial starter cultures. Finally, we propose an examination of patent literature for the main yeasts species commercialised in non-Saccharomyces based products. : We highlight the presence of asymmetries among scientific findings and the number of patents concerning non-Saccharomyces-based commercial products for oenological purposes. Further investigations on these microbial resources might open new perspectives and stimulate attractive innovations in the field of wine-making biotechnologies.

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Microbial starter cultures represent a fundamental level of innovation in the wine sector. Selected yeast strains are routinely used to achieve the needed biomass preparation to accelerate and steer alcoholic fermentation in grape must. The use of starter cultures to induce malolactic fermentation in wine relies on the characterisation and propagation of suitable strains of lactic acid bacteria. Furthermore, the selection of new strains, the renewal of management of microbial resources and new technologies allow continuous improvements in oenology, which may increase the beneficial aspects of wine. In this review, with the aim to stimulate microbial-driven, consumer-oriented advances in the oenological sector, we propose an overview of recent trends in this field that are reported by following the classical separation into 'product innovation' and 'process innovation'. Hence, we shall highlight i) the possible positive innovative impacts of microbial resources on the safety and the sensorial and functional properties of wine (product innovation) and ii) the potential microbial-based improvements allowing the reduction of time/costs and the environmental impacts associated with winemaking (process innovation).

Climate change threatens food systems, with huge repercussions on food security and on the safety and quality of final products. We reviewed the potential of food microbiology as a source of biotechnological solutions to design climate-smart food systems, using wine as a model productive sector. Climate change entails considerable problems for the sustainability of oenology in several geographical regions, also placing at risk the wine typicity. The main weaknesses identified are: (i) The increased undesired microbial proliferation; (ii) the improved sugars and, consequently, ethanol content; (iii) the reduced acidity and increased pH; (iv) the imbalanced perceived sensory properties (e.g., colour, flavour); and (v) the intensified safety issues (e.g., mycotoxins, biogenic amines). In this paper, we offer an overview of the potential microbial-based strategies suitable to cope with the five challenges listed above. In terms of microbial diversity, our principal focus was on microorganisms isolated from grapes/musts/wines and on microbes belonging to the main categories with a recognized positive role in oenological processes, namely Saccharomyces spp. (e.g., Saccharomyces cerevisiae), non-Saccharomyces yeasts (e.g., Metschnikowia pulcherrima, Torulaspora delbrueckii, Lachancea thermotolerans, and Starmerella bacillaris), and malolactic bacteria (e.g., Oenococcus oeni, Lactobacillus plantarum).

This study reports the first application of a next generation sequencing (NGS) analysis. The analysis was designed to monitor the effect of the management of microbial resources associated with alcoholic fermentation on spontaneous malolactic consortium. Together with the analysis of 16S rRNA genes from the metagenome, we monitored the principal parameters linked to MLF (e.g., malic and lactic acid concentration, pH). We encompass seven dissimilar concrete practices to manage microorganisms associated with alcoholic fermentation: Un-inoculated must (UM), pied-de-cuve (PdC), Saccharomyces cerevisiae (SC), S. cerevisiae and Torulaspora delbrueckii co-inoculated and sequentially inoculated, as well as S. cerevisiae and Metschnikowia pulcherrima co-inoculated and sequentially inoculated. Surprisingly, each experimental modes led to different taxonomic composition of the bacterial communities of the malolactic consortia, in terms of prokaryotic phyla and genera. Our findings indicated that, uncontrolled AF (UM, PdC) led to heterogeneous consortia associated with MLF (with a relevant presence of the genera Acetobacter and Gluconobacter), when compared with controlled AF (SC) (showing a clear dominance of the genus Oenococcus). Effectively, the SC trial malic acid was completely degraded in about two weeks after the end of AF, while, on the contrary, malic acid decarboxylation remained uncomplete after 7 weeks in the case of UM and PdC. In addition, for the first time, we demonstrated that both (i) the inoculation of different non-Saccharomyces (T. delbrueckii and M. pulcherrima) and, (ii) the inoculation time of the non-Saccharomyces with respect to S. cerevisiae resources (co-inoculated and sequentially inoculated) influence the composition of the connected MLF consortia, modulating MLF performance. Finally, we demonstrated the first findings of delayed and inhibited MLF when M. pulcherrima, and T. delbrueckii were inoculated, respectively. In addition, as a further control test, we also assessed the effect of the inoculation with Oenococcus oeni and Lactobacillus plantarum at the end of alcoholic fermentation, as MLF starter cultures. Our study suggests the potential interest in the application of NGS analysis, to monitor the effect of alcoholic fermentation on the spontaneous malolactic consortium, in relation to wine.