Lactic Acid Bacteria Biota and Aroma Profile of Italian Traditional Sourdoughs From the Irpinian Area in Italy

This study identified the lactic acid bacteria (LAB) biota and the volatilome profile of 28 typical sourdoughs of Irpinia—a large area of the Campania region of Southern Italy where numerous breads are produced, even today, following the ancient procedures of sourdough fermentation and for which information on the microbiological and sensory profile is lacking in literature. For this purpose, microbial quality, LAB biodiversity, chemical, and technological characteristics, as well as aroma profile by solid-phase microextraction technique (SPME)–gas chromatography/mass spectrometry (GC/MS) of Irpinian sourdoughs were investigated. The dominant LAB microbiota was examined by both culture-dependent and culture-independent methods Polymerase Chain Reaction/Denaturing Gradient Gel Electrophoresis (PCR-DGGE). Results showed a high biodiversity in LAB community whereas the most frequent lactobacilli species recognized were Lactobacillus plantarum (ca. 22% of total LAB isolates), Lactobacillus sanfranciscensis (11%), Lactobacillus paralimentarius (8%), and Lactobacillus rossiae (6.5%), whereas LAB cocci could be mainly referred to Pediococcus pentosaceus (9.5% of total LAB isolates), Leuconostoc spp. (7.8%), and Weissella cibaria (7.7%). Sourdoughs were characterized by the dominance of one or two LAB species, thus proving that the environment influences the selection and the establishment of few key LAB species and that no specific correlation can be traced between microbial composition and geographical origin of the samples. Furthermore, although sourdoughs were characterized by different qualitative and quantitative volatile organic compound (VOC) compositions, no noticeable correlation between volatilome profile and geographical origin was found. However, it emerged that for more isolated locations, it was possible to find the existence of microbial biotypes and sensory profiles with a strong identity, thus revealing the existence of highly traditional and evocative bread recipes in those geographical contexts

In vivo authentication of Iberian pig feeding regime using faecal volatilome information

Nowadays, people are increasingly interested in the food they consume. Authenticity and natural origin are amongst the most valued issues of food products by society. Although various national and international laws have been created for the regulation of labelling and trade of food, unfortunately, they are often not effective in avoiding food product fraud. The Iberian pig and the cured products obtained with this breed have a great international reputation due to their high quality and added value. However, the authentication of these pigs feeding regime is sometimes difficult. Therefore, the objective of this study was to use faecal volatilome information to differentiate the different feeding regimes which determine the final commercial category of Iberian products. Individual faeces samples were sampled on 10 farms from 133 Iberian pigs to evaluate their volatilome through gas chromatography (GC) coupled to ion mobility spectrometry (IMS). The intensity of GC-IMS plot features were extracted and chemometric tools were employed to develop two different models: one, focused on the discrimination between acorn-fed (completely natural diet grazed) and feed-fed samples, and another one for commercial category classification. Both models were carried out in duplicate, using spectral fingerprint information and a different approach studying specific markers. Good classification rates were obtained in both models: 92,3% and 96,3% were the rates obtained in acorn-fed vs feed-fed model with fingerprint and specific markers information, respectively; and the same classification success was also achieved with both approaches in the second model, focused on commercial category classification. The misclassified samples in both models, which belonged to acorn-fed pigs, may be related to the diet heterogeneity of these animals and the differences in natural resources foraged. The results of the present study highlight GC-IMS as an useful tool to carry out an in vivo authentication of Iberian pig feeding regime and the subsequent commercial category, as well as to avoid labelling fraud. Further studies including larger number of samples are needed in order to obtain more complex models to classify very different samples

Microbial volatilome in food safety. Current status and perspectives in the biocontrol of mycotoxigenic fungi and their metabolites

Fungal infection and mycotoxins contamination in food and feed products cause significant economic losses to the food and agricultural industry. Although the efficacy of synthetic fungicides is unquestionable, there are serious issues associated with their application in agriculture. Improper and/or prolonged application of these products may cause the emergence of resistant fungal populations, carry-over of chemicals in the human diet and adverse effects on non-target species. By contrast, ease in application and negligible effects on the environment makes microbial volatile organic compounds (VOCs) safe and sustainable substitutes to synthetic fungicides. A considerable research investment has highlighted the efficacy and suitability of VOCs emitted by bacteria, yeast and filamentous fungi for application in food crops and stored products. In this review, focus is made on the potential use of microbial VOCs as inhibitors of toxigenic food mycobiota and their mycotoxins. The mode of action of microbial volatile compounds, possible application in different scenarios, limitations and perspectives are discussed.Open Access funding provided by the Qatar National Library. The statements here are solely the responsibility of authors

Determinación de vapores en tiempo real mediante ionización secundaria por electrospray (SESI) acoplada a espectrometría de masas: estudios mecanísticos y aplicaciones bioquímicas

This Doctoral Thesis focuses on the application of Secondary ElectroSpray Ionization (SESI) coupled to High Resolution Mass Spectrometry (HRMS) for the online and noninvasive monitoring of fluctuations in metabolite levels of interest for biochemical applications. Our studies started by getting insights into the mechanism by which volatiles are ionized in their interaction with plumes of electrospray solvents (i.e., SESI). This knowledge could help in the optimization of key parameters in SESI, leading to an enhancement in ionization efficiency. Our observations, using protonated water in the ESI solvent and in the carrier gas, revealed that, in the final stage of SESI, gas-phase ion–molecule reactions lead the mechanism by which electrosprays ionize vapor species. The power of SESI-HRMS technique to measure volatiles was shown for different strains of baker’s yeast during growth to starvation. It was demonstrated that the concentration profiles of 263 volatile metabolites, produced by yeast consuming labeled glucose, can be monitored for at least 48 hours. 111 of these metabolites could be matched by molecular formula in the yeast metabolome database, and 101 additional metabolites were found in the human metabolome database. These results suggest that a large number of these metabolites were not previously reported in the literature nor are their biochemical origin deciphered. Additionally, it was shown how, by replicating the experiment with different strains of yeast, useful information to understand the kinetics and pathways followed by each mutant when processing 13C1-glucose can be obtained. The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathways for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting the metabolism of carbohydrates, fatty acids, and amino acids. In this Doctoral Thesis, we evaluated whether at least the most volatile metabolites of the TCA cycle could be detected in vivo and in real time in exhaled breath. The results provided the first evidence that the TCA cycle can conveniently be monitored in breath, opening new opportunities to study this important metabolic pathway, as stated previously, in vivo and in real time.La Tesis que se presenta se centra en la aplicación de la técnica de ionización secundaria por electrospray (SESI), acoplada a un espectrómetro de masas de alta resolución. El principal objetivo puede definirse como la monitorización de variaciones en las concentraciones de ciertos metabolitos de interés de forma on-line y no invasiva y sus aplicaciones en el campo de la bioquímica. En primer lugar, se llevaron a cabo estudios sobre el mecanismo por el cual los compuestos volátiles se ionizan en SESI al interactuar con el electrospray. Una mejor comprensión de este fenómeno permitiría una mejor optimización de los parámetros claves en SESI y una mejora sustancial en las eficiencias de ionización. Los experimentos realizados con agua pesada tanto como disolvente del electrospray como en el gas de muestra apuntan a que en la etapa final de SESI las reacciones ion-molécula en fase vapor gobiernan el mecanismo mediante el cual se ionizan los compuestos volátiles. Seguidamente, se demostró el potencial de la técnica SESI-HRMS para medir volátiles en diferentes cepas de levadura Saccharomyces cerevisiae durante las fases de crecimiento exponencial y muerte. Se demostró la posibilidad de monitorizar la concentración de hasta 263 metabolitos volátiles, producidos por la levadura alimentada con glucosa marcada isotópicamente, hasta durante 48 horas. La identificación de los metabolitos según su fórmula molecular resultó en 111 metabolitos señalados como candidatos en la “yeast metabolome database”, y otros 101 metabolitos en la “human metabolome database”. Estos resultados demuestran como muchos metabolitos producidos por la levadura, y que pueden ser analizados mediante SESI, son aún desconocidos por la comunidad científica. Adicionalmente, repitiendo el experimento con distintas cepas mutantes de levaduras alimentadas con 13C1-glucosa, se obtuvo información relevante para entender la cinética y rutas metabólicas involucradas en la fermentación alcohólica. El ciclo de los ácidos tricarboxílicos (TCA) es una de las rutas metabólicas más importantes involucradas en la respiración celular de los organismos aeróbicos. TCA provee y recoge subproductos de muchas otras rutas metabólicas interconectándolas y actuando como centro de actividad del metabolismo de carbohidratos, ácidos grasos y aminoácidos. En esta tesis doctoral se ha evaluado la posibilidad de que los compuestos más volátiles del TCA puedan ser detectados en el aliento mediante SESI. Los resultados apuntan a que el TCA podría ser convenientemente monitorizado a través de esta técnica, lo que abre nuevas oportunidades para el estudio de esta importante ruta metabólica de forma no invasiva, en vivo y a tiempo real

Elucidar os efeitos de aldeídos e álcoois saturadosem células de Rhizobium expostas a cádmio: crescimento e resposta bioquímica

Rhizobia are soil bacteria that play significant agronomic and environmental roles and are decisive contributors to soil fertility. However, these microorganisms are affected by environmental stresses such as cadmium contamination, which alters the metabolism of organisms, including bacterial volatilome (a set of metabolites synthesized by an organism). Two groups of compounds that change in cadmium-exposed rhizobia cells are saturated aldehydes and alcohols, yet the effect of these compounds on bacterial cells is poorly known. In this thesis the effects of different aldehydes (hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal and tridecanal) and alcohols (hexanol, heptanol, octanol and nonanol) separately or in combination with cadmium on growth and biochemistry of Rhizobium cells (strain E20-8) are evaluated. Results show that smaller aldehydes (6 to 10 carbons) reduced lipid peroxidation (37% to 50%), while larger aldehydes (11 to 13 carbons) increased it slightly (20% and 30%) or significantly (> 70%), showing the protective function of the smaller aldehydes and the destabilizing effect of the largest ones in membranes. Enzymes with antioxidant activity such as superoxide dismutase decreased in the presence of smaller aldehydes and increased in the presence of larger ones, showing the activation of antioxidant mechanisms in cells exposed to larger aldehydes. Exposure to different aldehydes did not influence Rhizobium cell growth in most of the conditions tested, either in the presence or absence of Cd. However, exposure to 1mM decanal and 10 μM tridecanal caused a significant increase in Cd growth. The comparison of aldehydes with their conjugated alcohols effects on Rhizobium cells exposed or not to Cd allowed to elucidate which of the two types of compounds was more toxic to cells. Results showed the higher toxicity of most aldehydes compared to their conjugated alcohol in the presence of Cd, suggesting that reduction of aldehydes to alcohols may be an effective mechanism of aldehyde toxicity restriction. This thesis presents new information that helps clarify the changes occurring in bacterial cells exposed to Cd and the cell-induced mechanisms to minimize this toxicity. The contribution is especially relevant in influence that these compounds may have in the protection of bacterial communities under environmental stress contexts.Os rizóbios são bactérias do solo que têm um papel agronómico e ambiental significativo, sendo contribuidores decisivos para a fertilidade do solo. No entanto, estes microrganismos são afetados por stresses ambientais como a contaminação por cádmio, o que altera o metabolismo dos organismos, incluindo o volatiloma (conjunto de metabolitos sintetizados por um organismo) bacteriano. Dois grupos de compostos que sofrem alteração em células de Rizóbio expostas ao cádmio são os aldeídos e os álcoois saturados, contudo é pouco conhecido o efeito destes compostos nas células bacterianas. Nesta tese são estudados os efeitos de diferentes aldeídos (hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal e tridecanal) e álcoois (hexanol, heptanol, octanol e nonanol) separadamente ou em combinação com cádmio no crescimento e na bioquímica de células de Rhizobium (estirpe E20-8). Os resultados mostram que aldeídos menores (com 6 a 10 carbonos) reduziram a peroxidação lipídica (37% a 50%), enquanto aldeídos maiores (com 11 a 13 carbonos) aumentaram a peroxidação lipídica ligeiramente (20% e 30%) ou significativamente (>70%), evidenciando a função protetora dos aldeídos menores e desestabilizadora dos maiores nas membranas. As enzimas com atividade antioxidante como a superóxido dismutase diminuiu na presença dos aldeídos menores e aumentou na presença dos maiores, mostrando a ativação de mecanismos antioxidantes em células expostas a aldeídos maiores. Na maioria das condições testadas a exposição aos diferentes aldeídos não influenciou o crescimento das células do Rhizobium, tanto na presença como na ausência de Cd, contudo a exposição a 1mM de decanal e 10 μM tridecanal causaram um aumento significativo no crescimento na condição de Cd. A comparação dos efeitos de aldeídos e dos respetivos álcoois conjugados em células de Rhizobium expostas ou não a Cd permite elucidar qual dos dois tipos de compostos apresenta maior toxicidade para as células. Os resultados evidenciaram a maior toxicidade da maioria dos aldeídos comparativamente ao respetivo álcool conjugado na presença de Cd, sugerindo que a redução de aldeídos a álcoois pode ser um mecanismo efetivo de restrição da toxicidade de aldeídos. Esta tese apresenta informação nova que ajuda a esclarecer as alterações que ocorrem em células bacterianas expostas a Cd e os mecanismos induzidos pelas células para minimizar essa mesma toxicidade. O contributo é especialmente relevante na influência que estes compostos possam ter em contextos de proteção das comunidades bacterianas sob stresse ambiental.Mestrado em Eco-toxicologia e Análise de Risc

PTR-ToF-MS for the online monitoring of alcoholic fermentation in wine: assessment of VOCs variability associated with different combinations of Saccharomyces/non-Saccharomyces as a case-study

7openInternationalItalian coauthor/editorThe management of the alcoholic fermentation (AF) in wine is crucial to shaping product quality. Numerous variables (e.g., grape varieties, yeast species/strains, technological parameters) can affect the performances of this fermentative bioprocess. The fact that these variables are often interdependent, with a high degree of interaction, leads to a huge ‘oenological space’ associated with AF that scientists and professionals have explored to obtain the desired quality standards in wine and to promote innovation. This challenge explains the high interest in approaches tested to monitor this bioprocess including those using volatile organic compounds (VOCs) as target molecules. Among direct injection mass spectrometry approaches, no study has proposed an untargeted online investigation of the diversity of volatiles associated with the wine headspace. This communication proposed the first application of proton-transfer reaction-mass spectrometry coupled to a time-of-flight mass analyzer (PTR-ToF-MS) to follow the progress of AF and evaluate the impact of the different variables of wine quality. As a case study, the assessment of VOC variability associated with different combinations of Saccharomyces/non-Saccharomyces was selected. The different combinations of microbial resources in wine are among the main factors susceptible to influencing the content of VOCs associated with the wine headspaces. In particular, this investigation explored the effect of multiple combinations of two Saccharomyces strains and two non-Saccharomyces strains (belonging to the species Metschnikowia pulcherrima and Torulaspora delbrueckii) on the content of VOCs in wine, inoculated both in commercial grape juice and fresh grape must. The results demonstrated the possible exploitation of non-invasive PTR-ToF-MS monitoring to explore, using VOCs as biomarkers, (i) the huge number of variables influencing AF in wine, and (ii) applications of single/mixed starter cultures in wine. Reported preliminary findings underlined the presence of different behaviors on grape juice and on must, respectively, and confirmed differences among the single yeast strains ‘volatomes’. It was one of the first studies to include the simultaneous inoculation on two non-Saccharomyces species together with a S. cerevisiae strain in terms of VOC contribution. Among the other outcomes, evidence suggests that the addition of M. pulcherrima to the coupled S. cerevisiae/T. delbrueckii can modify the global release of volatiles as a function of the characteristics of the fermented matrixopenBerbegal, C.; Khomenko, I.; Russo, P.; Spano, G.; Fragasso, M.; Biasioli, F.; Capozzi, V.Berbegal, C.; Khomenko, I.; Russo, P.; Spano, G.; Fragasso, M.; Biasioli, F.; Capozzi, V

Lactiplantibacillus plantarum as a model species for VOCs studies in the food sector: VOCs monitoring in a microbiological medium using PTR-ToF-MS

This thesis combines a critical review part of the relevant scientific literature and an experi-mental part, both functional to highlight the interest in mass spectrometry techniques for monitoring and promoting innovation in the fermented products sector. The fermentation of food products brings to-gether complex bioprocesses with a relevant role in the green transition of food systems. Among these processes, lactic acid fermentation carried out by lactic acid bacteria (LAB) represents a transformation of great interest due to its potential impact on quality, safety, and human well-being. Volatile organic compounds (VOCs) represent an interesting and versatile class of chemical markers for monitoring and studying fermentations, as they are metabolites derived from microbial action and offer information on product quality. Direct-injection mass spectrometry (DIMS) techniques allow studies with excellent an-alytical performances, particularly interesting in evaluating fermentation processes. Proton Transfer Re-action - Time of Flight - Mass Spectrometer (PTR-ToF-MS) belongs to DIMS approaches and has var-ious applications in the field of fermentation. The critical review of the literature addressed the issue of analytical methodologies (innovative methodologies) and highlighted the importance of Lactiplanti-bacillus plantarum as a model species among lactic acid bacteria due to the versatility of applications in the field of fermentations of food interest, with an impact on different aspects of the quality of finished products. The overview of the scientific literature was combined with an experimental part on a prelim-inary evaluation of the PTR-ToF-MS in monitoring VOCs associated with the growth of L. plantarum. Six L. plantarum strains were grown separately in a culture medium and the volatiolome has been mon-itored throughout the growth curve. The results provided interesting preliminary evidence on the poten-tial of PTR-ToF-MS for the monitoring of LAB food fermentation, using L. plantarum as a model spe-cies.Esta tese combina uma revisão crítica da literatura científica e uma parte experimental, ambas funcionais para destacar o interesse das técnicas de espectrometria de massa na monitorização e promo-ção da inovação no setor de produtos fermentados. A fermentação de produtos alimentares envolve um conjunto complexo de bioprocessos com um papel relevante na transição ecológica dos sistemas ali-mentares. Entre esses processos, a fermentação láctica realizada por bactérias ácido-lácticas (LAB) re-presenta uma transformação de grande interesse devido ao seu potencial impacto na qualidade, segu-rança e bem-estar humano. Os compostos orgânicos voláteis (COVs) representam uma classe interes-sante e versátil de marcadores químicos na monitorização e estudo das fermentações, uma vez que são metabolitos derivados da ação microbiana e fornecem informações sobre a qualidade do produto. As técnicas de espectrometria de massa de injeção direta (DIMS) permitem estudos com excelentes desem-penhos analíticos, particularmente interessantes para avaliar os processos de fermentação. A Reação de Transferência de Protões (PTR), combinada com Espectrometria de Massa de Tempo de Voo (ToF-MS) (PTR-ToF-MS) pertence às abordagens DIMS e tem várias aplicações no campo da fermentação. A revisão crítica da literatura abordou a questão da metodologia analítica (metodologias inovadoras) e em particular destacou a importância do Lactiplantibacillus plantarum como uma espécie modelo entre as bactérias lácticas devido à versatilidade de aplicações no campo de fermentações de interesse alimentar, com impacto em diferentes aspetos da qualidade dos produtos acabados. A visão geral da literatura científica de referência foi combinada com uma parte experimental na avaliação preliminar do PTR-ToF-MS na monitorização dos COVs associados ao crescimento de L. plantarum. Seis estirpes de L. plantarum foram cultivadas separadamente em meio de cultura e o volatiloma foi monitorizado ao longo da curva de crescimento. Os resultados forneceram evidências interessantes preliminares sobre o poten-cial do PTR-ToF-MS para a monitorização da fermentação ácido-láctica de alimentos, usando L. plan-tarum como espécie modelo

Successful biocontrol of major postharvest and soil-borne plant pathogenic fungi by antagonistic yeasts

Fungal pathogens are the main biotic burden of productivity for economically important crops under field, greenhouse or postharvest conditions. The discovery and development of new environmental-friendly solutions, such as application of living organisms and their derivatives to control plant diseases and pests, are of enormous interest. This study presents the results of a mass screening designed to detect yeast strains with antagonistic activity against postharvest pathogens (Alternaria alternata, Penicillium expansum and Botrytis cinerea) and soil-borne diseases (Verticillium dahliae and Fusarium oxysporum). In fact, this is the first study that focuses on screening the antagonistic potential of a wide variety of yeast genera (13) and species (30) against vascular wilts. The results from in vivo trials demonstrated that fungal infected tomato plants, grown under hydroponic or soil conditions, showed a significant reduction in disease severity after yeast treatment. Wickerhamomyces anomalus Wa-32 was able to antagonise both pathogens and reduce the disease severity up to 40% (V. dahliae) and 50% (F. oxysporum) in soil conditions. In addition, this strain became endophytic in tomato plants. The features of Wa-32 are of enormous interest since no effective antagonistic biocontrol product is available for the simultaneous control of these two fungal pathogens. Postharvest assays with wounded tomato fruits showed that several strains displayed very high biocontrol levels against P. expansum and B. cinerea (up to 86 and 97% reduction in disease severity, respectively) but none of them showed protection against A. alternata. The best protection against B. cinerea was again achieved with W. anomalus Wa-32 and two Metschnikowia pulcherrima strains (Mp-22 and Mp-30). However, the best antagonistic strains of P. expansum were Candida lusitaniae Cl-28, Candida oleophila Co-13, Debaryomyces hansenii Dh-67 and Hypopichia pseudoburtonii Hp-54. These biocontrol effects were also demonstrated in grapes and apples.This work was financed by the Departamento de Desarrollo Económico from the Gobierno de Navarra (Spain): grants 0011-1365-2018-000120 and 0011-1411-2019-000009