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

Diverse bacterial communities are recruited on spores of different arbuscular mycorrhizal fungal isolates

Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbioses with the roots of most food crops, playing a key role in soil fertility and plant nutrition and health. The beneficial activity of AMF may be positively affected by bacterial communities living associated with mycorrhizal roots, spores and extraradical hyphae. Here, we investigated the diversity of bacterial communities associated with the spores of six AMF isolates, belonging to different genera and species and maintained for several generations in pot cultures with the same host plant, under the same environmental conditions and with the same soil. The occurrence of large bacterial communities intimately associated with spores of the AMF isolates was revealed by PCR denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of DGGE bands. Cluster and canonical correspondence analysis showed that the six AMF isolates displayed diverse bacterial community profiles unrelated with their taxonomic position, suggesting that each AMF isolate recruits on its spores a different microbiota. The 48 sequenced fragments were affiliated with Actinomycetales, Bacillales, Pseudomonadales, Burkholderiales, Rhizobiales and with Mollicutes-related endobacteria (Mre). For the first time, we report the occurrence of Mre in Funneliformis coronatum and Rhizophagus intraradices and sequences related to endobacteria of Mortierella elongata in F. coronatum and Funneliformis mosseae. The bacterial species identified are known to possess diverse and specific physiological characteristics and may play multifunctional roles affecting the differential performance of AMF isolates, in terms of infectivity and efficiency

Valorizzazione dei prodotti alimentari del territorio attraverso la loro caratterizzazione salutistica

Valorisation of local food products by the characterization of their health-promoting properties. Typical local food products play a very important economic role, in particular in Italy, where food processes and products are highly diversified, not only at the regional scale, but also within the same province and municipality. The valorisation of such processes and products, often obtained by quality marks, i.e. PDO, PGI, TSG, TAP, can be further prosecuted through their nutritional and nutraceutical characterization. Knowledge of the heath-promoting features of peculiar local foods can be utilized for its diversification and marketing. Tuscany is rich in local and traditional food products, from PDO pecorino cheese to PDO Lunigiana honey, from PDO Garfagnana and Lunigiana chesnut flour to PGI Garfagnana spelt flour IGP, from PGI Sorana bean to Pratomagno “zolfino” bean, from mountain blueberry to white truffle, from PDO Tuscan bread to different PDO extra-virgin olive oils. Here, we discuss the available means for their further characterization from a health-promoting point of view, aimed at pursuing their diversification and valorisation

Multifunctionality and diversity of culturable bacterial communities strictly associated with spores of the plant beneficial symbiont Rhizophagus intraradices

Arbuscular Mycorrhizal Fungi (AMF) live in symbiosis with most crop plants and represent essentialelements of soil fertility and plant nutrition and productivity, facilitating soil mineral nutrient uptakeand protecting plants from biotic and abiotic stresses. These beneficial services may be mediated bythe dense and active spore-associated bacterial communities, which sustain diverse functions, such asthe promotion of mycorrhizal activity, biological control of soilborne diseases, nitrogen fixation, and the supply of nutrients and growth factors. In this work, we utilised culture-dependent methods to isolate and functionally characterize the microbiota strictly associated to Rhizophagus intraradices spores,and molecularly identified the strains with best potential plant growth promoting (PGP) activities by16S rDNA sequence analysis. We isolated in pure culture 374 bacterial strains belonging to different functional groups—actinobacteria, spore-forming, chitinolytic and N2-fixing bacteria—and screened 122 strains for their potential PGP activities. The most common PGP trait was represented by P solubilization from phytate (69.7%), followed by siderophore production (65.6%), mineral P solubilization (49.2%) and IAA production (42.6%). About 76% of actinobacteria and 65% of chitinolytic bacteria displayed multiple PGP activities. Nineteen strains with best potential PGP activities, assigned to Sinorhizobium meliloti, Streptomyces spp., Arthrobacter phenanthrenivorans, Nocardiodes albus, Bacillus sp. pumilus group, Fictibacillus barbaricus and Lysinibacillus fusiformis, showed the ability to produce IAA and siderophores and to solubilize P from mineral phosphate and phytate, representing suitable candidates as biocontrol agents,biofertilisers and bioenhancers, in the perspective of targeted management of beneficial symbionts and their associated bacteria in sustainable food production systems

Facilitation of phosphorus uptake in maize plants by mycorrhizosphere bacteria

A major challenge for agriculture is to provide sufficient plant nutrients such as phosphorus (P) to meet the global food demand. The sufficiency of P is a concern because of it’s essential role in plant growth, the finite availability of P-rock for fertilizer production and the poor plant availability of soil P. This study investigated whether biofertilizers and bioenhancers, such as arbuscular mycorrhizal fungi (AMF) and their associated bacteria could enhance growth and P uptake in maize. Plants were grown with or without mycorrhizas in compartmented pots with radioactive P tracers and were inoculated with each of 10 selected bacteria isolated from AMF spores. Root colonization by AMF produced large plant growth responses, while seven bacterial strains further facilitated root growth and P uptake by promoting the development of AMF extraradical mycelium. Among the tested strains, Streptomyces sp. W94 produced the largest increases in uptake and translocation of 33P, while Streptomyces sp. W77 highly enhanced hyphal length specific uptake of 33P. The positive relationship between AMF-mediated P absorption and shoot P content was significantly influenced by the bacteria inoculants and such results emphasize the potential importance of managing both AMF and their microbiota for improving P acquisition by crops

Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil

In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3′-hydroxylase isoform 1 (CS3′H iso1) genes, 5.7- fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3′H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health

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

Assessment of the Life Cycle Environmental Impact of the Olive Oil Extraction Solid Wastes in the European Union

There is an increasing interest in developing sustainable systems in the European Union (EU) to recover and upgrade the solid wastes of the olive oil extraction process, i.e. wet husk. A Life Cycle Environmental Impact Assessment (LCIA) of wet husk has been carried out aiming at facilitating an appropriate Life Cycle Management of this biomass. Three scenarios have been considered, i.e. combustion for domestic heat, generation of electric power, and composting. The Environmental Product Declaration and the ReCiPe method were used for Life Cycle Impact Assessment. Domestic heating and power generation were the most important impact factors in damaging human health, ecosystems, and natural resources depletion. Composting was 2-4 orders of magnitude less impacting than domestic heat and power generation. Considering human health, the impact of climate change, human toxicity and particulate matter formation represented the main impact categories. Considering ecosystems, climate change and natural land transformation were the main impact categories. Within natural resources, fossil fuel depletion was impacted three orders more than metal depletion. Within domestic heating and power generation scenarios, storage of wet husk along with the extraction by organic solvent, and the waste treatment were the most impacting phases for global warming potential, ozone layer depletion, acidification and non renewable fossil resources depletion. The results obtained for the waste disposal have been comparatively assessed with respect to the environmental impact of the olive oil production chain

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