Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage.

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.

Impact of sheep wool residues as soil amendments on olive beneficial symbionts and bacterial diversity.

In recent years the use of organic matter soil amendments, such as agricultural by-products, has been implemented with the aim of increasing soil fertility, while minimizing the environmental impact of agriculture. Sheep wool residues (SWR) have shown beneficial effects on plant nutrition and soil properties, while only few works assessed their impact on soil microbial communities. The main aim of this work was to investigate the possible valorization of two SWR types (scoured residues, white wool, WW, and carbonized scoured residues, black wool, BW) as organic soil amendments, in pot-grown olive trees, by evaluating their impact on soil bacterial communities and mycorrhizal symbionts. The two SWR types did not negatively impact on the diversity and composition of soil bacterial communities, as revealed by PCR-denaturating gradient gel electrophoresis (PCR-DGGE) of partial 16S rRNA gene, and on the activity of native arbuscular mycorrhizal fungi (AMF), while positively affecting plant growth. Only the highest doses of one SWR type (2% BW) caused a decrease in bacterial diversity and native AMF ability to colonize olive roots. DGGE bands sequencing allowed the identification of the major bacterial taxa. Sequences corresponding to Ohtaekwangia spp., Beta proteobacterium, Blastocatella sp., Ramlibacter monticola and Massilia frigida/rubra, Dongia sp. and Chloroflexi were mainly represented in SWR-amended soils, while those represented by Chryseolinea soli and Acidobacteria were abundant in control soil. Overall, this work showed that SWR may be valorized as organic soil amendments, as soil bacteria and AMF, representing key factors of biological soil fertility, were not negatively affected, while the activity of bacterial genera and species known for their ability to decompose complex compounds was boosted. Further studies will investigate the biodegradation efficiency of the diverse bacterial taxa developing in SWR-amended soils.

Novel Yeasts Producing High Levels of Conjugated Linoleic Acid and Organic Acids in Fermented Doughs.

Traditional fermented foods are obtained by a complex consortium of autochthonous microorganisms producing a wide variety of bioactive compounds, thus representing a reservoir of strains with new functional properties. Here, doughs obtained using five different wholegrain flours were singly fermented with selected yeast strains, which were evaluated for their functional traits. Lactate, volatile fatty acids and conjugated linoleic acid isomers produced by fermented doughs were detected by HPLC, while dough anti-inflammatory capacity was measured on human peripheral blood mononuclear cells by flow cytometry. Yeast potential probiotic activity was assessed by evaluating their resistance to simulated gastric and intestinal fluids. For the first time we report evidence of yeast strains producing high levels of the conjugated linoleic acid (CLA) isomer CLA 10-12tc and propionic acid, which are known for their specific health benefits. Moreover, such yeast strains showed an anti-inflammatory capacity, as revealed by a significantly decreased production of the strongly pro-inflammatory cytokine IL-1ß. All our Saccharomyces cerevisiae strains were remarkably resistant to simulated gastric and intestinal fluids, as compared to the commercial probiotic strain. The two strains S. cerevisiae IMA D18Y and L10Y showed the best survival percentage. Our novel yeast strains may be exploited as valuable functional starters for the industrial production of cereal-based innovative and health-promoting fermented foods.

Use of chitosan and tannins as alternatives to antibiotics to control mold growth on PDO Pecorino Toscano cheese rind.

The fungal microbiota usually growing on the cheese surface during ripening processes promote rind formation and the development of organoleptic characteristics, imparting positive sensory attributes to cheeses. As cheese contamination may also occur by undesirable molds, specific actions for preventing their growth are usually realized in dairy industries by using the antibiotic natamycin, which may represent a risk factor for human health and environmental sustainability. Here, agroindustrial by-products with natural antimicrobial properties, i.e. tannins and chitosan, were tested in a cheese-making trial producing PDO Tuscan pecorino cheese. Morphological and molecular methods revealed that the main components of rind fungal communities of PDO Tuscan pecorino cheese were represented by P. solitum, P. discolour and P. verrucosum. The use of chitosan on cheese rinds did not significantly affect the composition of rind fungal communities developing during the whole ripening process compared with controls treated with natamycin, whose numbers ranged from 3.4 ± 1.3 × 103 to 3.2 ± 1.8 × 104 and from 6.3 ± 3.5 × 102 to 4.0 ± 1.5 × 104, respectively. Overall, grape marc tannins and chitosan did not significantly affect the number and composition of fungal communities developing during PDO Pecorino Toscano cheese ripening, as well as its physical, chemical and nutritional profiles, showing that they may represent effective alternatives to the antibiotic natamycin.

Characterization and selection of functional yeast strains during sourdough fermentation of different cereal wholegrain flours.

The increasing demand for healthy baked goods boosted studies on sourdough microbiota with beneficial metabolic traits, to be used as potential functional starters. Here, 139 yeasts isolated from cereal-based fermented foods were in vitro characterized for their phytase and antioxidant activities. The molecular characterization at strain level of the best 39 performing isolates showed that they did not derive from cross contamination by baker's yeast. Afterwards, the 39 isolates were in vivo analyzed for their leavening ability, phytase activity and polyphenols content using five different wholegrain flours, obtained from conventional and pigmented common wheat, emmer and hull-less barley. Combining these findings, through multivariate permutation analysis, we identified the 2 best performing strains, which resulted diverse for each flour. Doughs singly inoculated with the selected strains were further analyzed for their antioxidant capacity, phenolic acids, xanthophylls and anthocyanins content. All the selected yeasts significantly increased the total antioxidant activity, the soluble, free and conjugated, forms of phenolic acids and anthocyanins of fermented doughs. This study revealed the importance of a specific selection of yeast strains for wholegrain flours obtained from different cereals or cultivars, in order to enhance the pro-technological, nutritional and nutraceutical traits of fermented doughs.

Beneficial Plant Microorganisms Affect the Endophytic Bacterial Communities of Durum Wheat Roots as Detected by Different Molecular Approaches.

This study aimed at characterising the endophytic bacterial communities living in durum wheat roots, as affected by wheat cultivar and inoculation of the Arbuscular mycorrhizal fungus Funneliformis mosseae IMA1 and the wheat root endophytic bacterium Lactobacillus plantarum B.MD.R.A2. These microorganisms were inoculated, alone or in combination, in durum wheat (cultivars Odisseo and Saragolla). Non-inoculated plants of each cultivar represented the controls. Forty-three days after sowing, roots were deprived of the epiphytic microbiota and subjected to DNA extraction. The DNA was used as template in PCR-DGGE analysis of the 16S rRNA gene (variable region V3-V5) and 16S (region V1-V3) metagenetics. Odisseo and Saragolla root endophytic bacterial biotas differed for number of OTUs and composition. In detail, Pseudomonas was higher in Odisseo than in Saragolla. The inoculation of F. mosseae and L. plantarum increased the abundance of Pseudomonas, some Actinobacteria (e.g., Streptomyces, Microbacterium, two genera including several plant growth promoting (PGP) strains) and Bacteroidetes in both cultivars. However, the endophytic bacterial biota of Saragolla roots inoculated just with lactobacilli did not differ from that of the control. The inoculation of Saragolla with F. mosseae, alone or in combination with lactobacilli, led to higher abundance of Rhodococcus, belonging to Actinobacteria and encompassing PGP strains. First, this work showed that F. mosseae and L. plantarum shape the endophytic bacterial biota of durum wheat roots. Abundance of some OTUs was affected by the microbial inoculation, depending on the cultivar. This result represents a starting point for exploitation of beneficial endophytes of wheat roots.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

Exploitation of autochthonous Tuscan sourdough yeasts as potential starters.

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.

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.

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.