Identification and characterization of lactic acid bacteria and yeasts of PDO Tuscan bread sourdough by culture dependent and independent methods

Sourdough fermentation has been increasingly used worldwide, in accordance with the demand of consumers for tasty, natural and healthy food. The high diversity of lactic acid bacteria (LAB) and yeast species, detected in sourdoughs all over the world, may affect nutritional, organoleptic and technological traits of leavened baked goods. A wide regional variety of traditional sourdough breads, over 200 types, has been recorded in Italy, including special types selected as worthy of either Protected Geographical Indication (PGI) or Protected Designation of Origin (PDO), whose sourdough microbiota has been functionally and molecularly characterized. As, due to the very recent designation, the microbiota of Tuscan bread sourdough has not been investigated so far, the aim of the present work was to isolate and characterize the species composition of LAB and yeasts of PDO Tuscan bread sourdough by culture-independent and dependent methods. A total of 130 yeasts from WLN medium and 193 LAB from both mMRS and SDB media were isolated and maintained to constitute the germplasm bank of PDO Tuscan bread. Ninety six LAB from mMRS medium and 68 yeasts from WLN medium were randomly selected and molecularly identified by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and PCR-RFLP analysis of the ITS region, respectively, and sequencing. The yeast identity was confirmed by 26S D1/D2 sequencing. All bacterial isolates showed 99% identity with Lactobacillus sanfranciscensis, 65 yeast isolates were identified as Candida milleri, and 3 as Saccharomyces cerevisiae. Molecular characterization of PDO Tuscan bread sourdough by PCR-DGGE confirmed such data. The distinctive tripartite species association, detected as the microbiota characterizing the sourdough used to produce PDO Tuscan bread, encompassed a large number of L. sanfranciscensis and C. milleri strains, along with a few of S. cerevisiae. The relative composition and specific physiological characteristics of such microbiota could potentially affect the nutritional features of PDO Tuscan bread, as suggested by the qualitative functional characterization of the isolates. Investigations on the differential functional traits of such LAB and yeast isolates could lead to the selection of the most effective single strains and of the best performing strain combinations to be used as starters for the production of baked goods

Quorum sensing in rhizobia isolated from the spores of the mycorrhizal symbiont Rhizophagus intraradices

Most beneficial services provided by arbuscular mycorrhizal fungi (AMF), encompassing improved crop performance and soil resource availability, are mediated by AMF-associated bacteria, showing key-plant growth-promoting (PGP) traits, i.e., the production of indole acetic acid, siderophores and antibiotics, and activities increasing the availability of plant nutrients by nitrogen fixation and phosphate mobilization. Such functions may be affected by the ability of AMF-associated bacteria to communicate through the production and secretion of extracellular small diffusible chemical signals, N-acyl homoserine lactone signal molecules (AHLs), that regulate bacterial behavior at the community level (quorum sensing, QS). This work investigated the occurrence and extent of QS among rhizobia isolated from AMF spores, using two different QS reporter strains, Agrobacterium tumefaciens NTL4 pZRL4 and Chromobacterium violaceum CV026. We also assessed the quorum quenching (QQ) activity among Bacillus isolated from the same AMF spores. Most rhizobia were found to be quorum-signaling positive, including six isolates producing very high levels of AHLs. The results were confirmed by microtiter plate assay, which detected 65% of the tested bacteria as medium/high AHL producers. A 16S rDNA sequence analysis grouped the rhizobia into two clusters, consistent with the QS phenotype. None of the tested bacteria showed QQ activity able to disrupt the QS signaling, suggesting the absence of antagonism among bacteria living in AMF sporosphere. Our results provide the first evidence of the ability of AMF-associated rhizobia to communicate through QS, suggesting further studies on the potential importance of such a behavior in association with key-plant growth-promoting functions

Health-Promoting Properties of Plant Products: The Role of Mycorrhizal Fungi and Associated Bacteria

The concept of food quality, traditionally based on nutritional and sensory properties, has recently acquired an additional meaning, referring to the health-promoting properties of plant products, that are ascribed to plant secondary metabolites called phytochemicals, primarily represented by polyphenolic compounds and glucosinolates. The diversity and content of phytochemicals in plant products are affected by different variables, such as plant genotype, agronomic factors, and arbuscular mycorrhizal fungi (AMF), which establish mycorrhizal symbioses with most crops, including cereals, legumes, vegetables, fruit trees, sunflower, cotton, and sugarcane. AMF and associated bacteria enhance plant growth and health, and affect the production of polyphenols and carotenoids, and the activity of antioxidant enzymes. The production of health-promoting phytochemicals was shown to be differentially modulated by different AMF isolates and bacterial strains, in several food plants, i.e., tomato, lettuce, strawberry, artichoke, maize, grapevine, sunflower. Here, we provide an overview of recent studies concerning the multiple roles played by AMF and associated bacteria in the modulation of the biosynthesis of plant secondary metabolites with health-promoting activity, and discuss the development of designed multifunctional consortia to be used in sustainable agriculture

Microbially-enhanced composting of olive mill solid waste (wet husk): Bacterial and fungal community dynamics at industrial pilot and farm level.

Bacterial and fungal community dynamics during microbially-enhanced composting of olive mill solid waste (wet husk), used as a sole raw material, were analysed in a process carried out at industrial pilot and at farm level by the PCR-DGGE profiling of the 16 and 26S rRNA genes. The use of microbial starters enhanced the biotransformation process leading to an earlier and increased level of bacterial diversity. The bacterial community showed a change within 15 days during the first phases of composting. Without microbial starters bacterial biodiversity increased within 60 days. Moreover, the thermophilic phase was characterized by the highest bacterial biodiversity. By contrast, the biodiversity of fungal communities in the piles composted with the starters decreased during the thermophilic phase. The biodiversity of the microbial populations, along with physico-chemical traits, evolved similarly at industrial pilot and farm level, showing different maturation times

Arbuscular Mycorrhizal Fungi and Associated Microbiota as Plant Biostimulants: Research Strategies for the Selection of the Best Performing Inocula

Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms establishing mutualistic symbioses with the roots of the most important food crops and playing key roles in the maintenance of long-term soil fertility and health. The great inter- and intra-specific AMF diversity can be fully exploited by selecting AMF inocula on the basis of their colonization ability and efficiency, which are aaffected by fungal and plant genotypes and diverse environmental variables. The multiple services provided by AMF are the result of the synergistic activities of the bacterial communities living in the mycorrhizosphere, encompassing nitrogen fixation, P solubilization, and the production of phytohormones, siderophores, and antibiotics. The tripartite association among host plants, mycorrhizal symbionts, and associated bacteria show beneficial emerging properties which could be efficiently exploited in sustainable agriculture. Further in-depth studies, both in microcosms and in the field, performed on different AMF species and isolates, should evaluate their colonization ability, efficiency, and resilience. Transcriptomic studies can reveal the expression levels of nutrient transporter genes in fungal absorbing hyphae in the presence of selected bacterial strains. Eventually, newly designed multifunctional microbial consortia can be utilized as biofertilizers and biostimulants in sustainable and innovative production systems

Species diversity and community composition of native arbuscular mycorrhizal fungi in apple roots are affected by site and orchard management

Arbuscular mycorrhizal fungi (AMF) are beneficial microrganisms which establish mutualistic symbioses with the roots of most food crops, improving plant performance, nutrient uptake and tolerance to biotic and abiotic stresses. A better understanding of the factors affecting AMF occurrence and diversity is fundamental to implement sustainable agricultural managements effectively profiting from beneficial plant symbionts. Here, we investigated AMF occurrence, diversity and community composition in the roots of apple trees from 21 orchards in South Tyrol, as affected by location, management (organic vs integrated) and altitude, by PCR cloning and sequencing and PCR-DGGE of partial 18S rRNA gene. The screening of 448 clones from 21 clone libraries allowed the identification of 6 native AMF at the species level: Glomus indicum, Sclerocystis sinuosa, Funneliformis mosseae, Rhizoglomus irregulare, Septoglomus constrictus and Claroideoglomus lamellosum. The most abundant genera were represented by Glomus (29.7% of the sequences), Paraglomus (19.4%), Claroideoglomus (17.2%), Sclerocystis (16.1%) and Rhizoglomus (12.3%). Septoglomus, Diversispora and Funneliformis sequences corresponded to less than 4% of total sequences. Although the degree of root colonization was unaffected by treatments, ANOSIM analysis of PCR-DGGE clusters revealed significant differences in apple root AMF diversity between sites and agricultural managements. Species richness was significantly higher in organically managed orchards than in integrated ones. Our findings provide insights into important factors affecting native AMF communities of apple trees, which could be exploited in sustainable fruit production systems, where beneficial soil biota boost biogeochemical cycles, energy fluxes and crop productivity

Composition of health-promoting phenolic compounds in two extra-virgin olive oils and diversity of associated yeasts

Extra virgin olive oil (EVOO), a basic component of the Mediterranean diet, is an important functional food, for its content in health-promoting compounds, showing antioxidant, antiinflammatory and antiproliferative activities. Here, two Tuscan EVOOs were analyzed for the occurrence and concentrations of health-promoting phenols, such as tyrosol and hydroxytyrosol and the secoiridoid derivatives, oleocanthal and oleacein. Independently of the milling period, the two EVOOs showed different contents of oleocanthal and oleacein. During storage, the contents of oleocanthal and oleacein decreased, while those of simple phenols increased. In all oil samples oleacein displayed a higher rate of reduction than oleocanthal. Multivariate analyses of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles, revealed the occurrence and diversity of oil-borne yeast communities, which differed in the two EVOOs. Sequences of excised DGGE bands identified Candida adriatica, Eremothecium coryli and Lachancea fermentati as the main components of the oil-borne yeast community. Our work detected, for the first time, differences in the content of tyrosol, hydroxytyrosol, oleocanthal and oleacein between the two Tuscan EVOOs analyzed, consistent with the differences found in yeast community composition. Further studies could confirm whether oil-borne yeasts may affect the composition of health-promoting oil phenolic compounds

Exploitation of autochthonous Tuscan sourdough yeasts as potential starters.

Abstract The increasing demand for healthy baked goods boosted studies on sourdough microbiota with beneficial metabolic traits, to be used as potential functional starters. Here, yeast populations of traditional sourdoughs collected from four Tuscan bakeries were investigated. Among 200 isolated strains, 78 were randomly selected and molecularly characterized. Saccharomyces cerevisiae was dominant, representing the only species detected in three out of the four sourdoughs. The fourth one harbored also Kazachstania humilis. Inter-delta regions analysis revealed a high intraspecific polymorphism discriminating 16 biotypes of S. cerevisiae isolates, which clustered based on their origin. Representative isolates from each biotype group were individually used to ferment soft and durum wheat flour, aiming at evaluating their pro-technological, nutritional and functional features. During fermentation under standardized conditions, all strains were able to grow of ca. 2 log cycles, but only S. cerevisiae L10Y, D18Y and D20Y had a significantly shorter latency phase in both flours. Overall, the highest volumes were reached after 16 h of fermentation in both soft and durum fermented dough. S. cerevisiae D2Y produced the highest dough volume increase. K. humilis G23Y was the only strain able to increase the total free amino acids concentration of the doughs. Overall, values of phytase activity were significantly higher in durum compared to the corresponding soft fermented dough. K. humilis G23Y and S. cerevisiae D20Y, D24Y showed a threefold higher phytase activity than spontaneously fermented control, and the highest concentration of total phenols. Almost all the strains led to increases of antioxidant activity, without significant differences among them. Investigations on the resistance of the strains to simulated gastric and intestinal conditions, that is considered a pre-requisite for the selection of probiotics, revealed the ability to survive in vitro by many of the strains considered. This study proposed the best performing yeast strains selected among autochthonous sourdough yeasts based on their pro-technological, nutritional and functional traits to be used as starters for making sourdough baked goods or functional cereal-based beverages. Although some yeast strains combined several technological and nutritional traits, the association of more selected strains seemed to be a requisite to get optimal sourdough characteristics

Effects of pavements on diversity and activity of mycorrhizal symbionts associated with urban trees

This study investigated the impact of different types of soil sealing on the communities of a group of beneficial plant symbionts, arbuscular mycorrhizal fungi (AMF), colonizing the roots of two shade trees, Celtis australis and Fraxinus ornus, frequently grown in urbanized sites. Such plants were grown in an experimental site, in northern Italy, established in November 2011 and subjected to four different pavement treatments: impermeable monolithic asphalt, permeable pavers, permeable concrete and unpaved soil. The diversity and composition of root AMF communities were assessed by PCR denaturating gradient gel electrophoresis of partial 18S rRNA gene, AMF taxa were identified by amplicon sequencing and mycorrhizal colonization was evaluated after root clearing and staining. For the first time, our molecular work revealed that impermeable pavements induced shifts in the composition of AMF communities associated to the roots of C. australis and F. ornus and impacted on the percentage of mycorrhizal root length. When the root-zone was covered with permeable pavements, a similar AMF community as that observed in the unpaved soil was detected, providing novel information to be utilised for reducing the disturbance caused by specific types of soil sealing on AMF symbionts, which play a key role in plant nutrition and health. A total of 45 AMF sequence types were detected, with Sclerocystis and Septoglomus as the most abundant phylotypes, accounting for 84% of the sequences. The predominance of Sclerocystis species in the roots of both tree species under impermeable pavements indicated their high and unforeseen tolerance towards harsh environmental conditions. Such species could be utilized as AMF inocula specifically selected for their proven resilience in paved sites, in order to exploit their ability to boost biogeochemical processes fundamental for energy fluxes and plant nutrition and health

Evoluzione delle popolazioni microbiche durante il compostaggio di sanse d'oliva Taggiasca e Pignola ottenuto con l'uso di starter microbici

L’area mediterranea è una regione caratterizzata da una rilevante produzione di olio di oliva, detiene infatti il 94,1% della produzione mondiale di olio. L’elevata concentrazione produttiva in spazi e tempi molto limitati causa un inasprimento dei problemi derivanti dalla gestione dei residui produttivi: acque di vegetazione e residui solidi (sanse). Essi, infatti, contengono molecole come fenoli e polifenoli, che hanno un elevato impatto ambientale se smaltiti tal quali sul terreno. Una valida alternativa a tale pratica è il compostaggio, un processo economico che minimizza l’impatto ambientale dei residui dell’estrazione olearia, trasformandoli in compost. Presso la Cooperativa Olivicola di Arnasco (SV) è stata svolta una prova di compostaggio di sanse vergini prodotte dall’estrazione dell’olio d’oliva Pignola e Taggiasca, inoculate con uno starter microbico specifico. I microrganismi starter, infatti, sono in grado di detossificare e/o degradare le sostanze anti-microbiche presenti nelle sanse vergini, favorendo lo sviluppo dei microrganismi coinvolti nel processo di compostaggio. Inoltre, le sanse vergini sono state inoculate con il microrganismo antagonista Trichoderma atroviride in vista di una possibile valutazione di effetti specifici sulla pianta. Nell'ambito di questa tesi sono state monitorate la temperatura e la diversità microbica presente nelle sanse durante le diverse fasi del compostaggio, sia mediante tecniche microbiologiche coltura-dipendenti, che mediante tecniche molecolari coltura-indipendenti (tecnica PCR-DGGE). In particolare, gli amplificati della regione V3-V5 del rDNA 16S batterico e della regione D1-D2 del rDNA 26S eucariotico degli estratti dei campioni analizzati, sono stati separati mediante elettroforesi su gel di poliacrilammide con gradiente denaturante. I risultati ottenuti dalle analisi microbiologiche hanno permesso di individuare le fasi che caratterizzano il processo di compostaggio, evidenziando le diverse tipologie microbiche proprie di ciascuna fase. L'analisi dei profili DGGE ha consentito di evidenziare che la comunità microbica, sia batterica che fungina, presente nelle sanse subisce una graduale stabilizzazione nel tempo, parallela alle modificazioni del substrato durante le diverse fasi del processo