Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage.

Dry fermented sausages are produced worldwide by well-controlled fermentation processes involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilot-scale sausage productions using "worst-case" conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage.

Water Microbiota in Greenhouses With Soilless Cultures of Tomato by Metabarcoding and Culture-Dependent Approaches.

Water supply, in hydroponic greenhouses, can originate from groundwater, surface water or rainwater stored in open tanks. To limit contamination of water supply, several methods have been used including active and passive methods such as slow filtration techniques which consist in passing the nutrient solutions slowly through filters. The purpose of this study was to describe the microbiota associated with water sampled before entering greenhouses and in recirculating nutrient solutions, either before or after running through a biofiltration system. Metabarcoding analysis revealed that water ecosystems were unique niches for diverse bacterial and fungal communities. Microbial composition varied greatly across storage conditions (groundwater vs. rainwater) and among greenhouses, suggesting that water microbiota is site- and storage-condition-specific. Nonetheless, we found that microbiota structure in open-stored water (either coming from ground or rain) shared a higher degree of similarity than with water directly pumped out of the ground. Open-stored waters were characterized by predominant taxa, notably those involved in aerobic chemoheterotrophy, such as the Sphingomonadaceae and Hyphomicrobiaceae families. Water directly collected from the ground showed the lowest levels of fungal and bacterial richness while also characterized by a significantly higher level of bacterial equitability and an enrichment in taxa involved in N-cycling. Slow filtration allowed reducing cultivable bacterial loads as well Pythium spp. and Fusarium oxysporum propagules, based on culture-dependent results, without compromising microbiota richness and diversity. Although compositional structure was similar following biofiltration, significant differences in bacterial (but not fungal) taxa abundance were reported, with primarily an enrichment of Chelativorans, Mycobacterium, and Gemmata as well as a depletion of Rhodobacter, Aminobacter, and Ellin329. The exact mechanisms by which such taxa would be favored at the expense of other remained unknown. Besides the accurate description of microbiota found in water at both taxonomical and predicted functional levels, our study allowed comparing the water microbiota between various storage system and following biofiltration. Although preliminary, our results provide a first insight into the potential microbial diversity, which can increase ecosystem functionality.

Isolation, Identification and Enzymatic Activity of Halotolerant and Halophilic Fungi from the Great Sebkha of Oran in Northwestern of Algeria.

The Great Sebkha of Oran is a closed depression located in northwestern of Algeria. Despite the ranking of this sebkha among the wetlands of global importance by Ramsar Convention in 2002, no studies on the fungal community in this area have been carried out. In our study, samples were collected from two different regions. The first region is characterized by halophilic vegetation and cereal crops and the second by a total absence of vegetation. The isolated strains were identified morphologically then by molecular analysis. The biotechnological interest of the strains was evaluated by testing their ability to grow at different concentration of NaCl and to produce extracellular enzymes (i.e., lipase, amylase, protease, and cellulase) on solid medium. The results showed that the soil of sebkha is alkaline, with the exception of the soil of cereal crops that is neutral, and extremely saline. In this work, the species Gymnoascus halophilus, Trichoderma gamsii, the two phytopathogenic fungi, Fusarium brachygibbosum and Penicillium allii, and the teleomorphic form of P. longicatenatum observed for the first time in this species, were isolated for the first time in Algeria. The halotolerance test revealed that the majority of the isolated are halotolerant. Wallemia sp. and two strains of G. halophilus are the only obligate halophilic strains. All strains are capable to secrete at least one of the four tested enzymes. The most interesting species presenting the highest enzymatic index were Aspergillus sp. strain A4, Chaetomium sp. strain H1, P. vinaceum, G. halophilus, Wallemia sp. and Ustilago cynodontis.

Beneficial Protective Role of Endogenous Lactic Acid Bacteria Against Mycotic Contamination of Honeybee Beebread.

The purpose of this article is to reveal the role of the lactic acid bacteria (LAB) in the beebread transformation/preservation, biochemical properties of 25 honeybee endogenous LAB strains, particularly: antifungal, proteolytic, and amylolytic activities putatively expressed in the beebread environment have been studied. Seventeen fungal strains isolated from beebread samples were identified and checked for their ability to grow on simulated beebread substrate (SBS) and then used to study mycotic propagation in the presence of LAB. Fungal strains identified as Aspergillus niger (Po1), Candida sp. (BB01), and Z. rouxii (BB02) were able to grow on SBS. Their growth was partly inhibited when co-cultured with the endogenous honeybee LAB strains studied. No proteolytic or amylolytic activities of the studied LAB were detected using pollen, casein starch based media as substrates. These findings suggest that some honeybee LAB symbionts are involved in maintaining a safe microbiological state in the host honeybee colonies by inhibiting beebread mycotic contaminations, starch, and protein predigestion in beebread by LAB is less probable. Honeybee endogenous LAB use pollen as a growth substrate and in the same time restricts fungal propagation, thus showing host beneficial action preserving larval food. This study also can have an impact on development of novel methods of pollen preservation and its processing as a food ingredient.

Bacterial Shifts in Nutrient Solutions Flowing Through Biofilters Used in Tomato Soilless Culture.

In soilless culture, slow filtration is used to eliminate plant pathogenic microorganisms from nutrient solutions. The present study focused on the characterization and the potential functions of microbial communities colonizing the nutrient solutions recycled on slow filters during a whole cultivation season of 7 months in a tomato growing system. Bacterial microflora colonizing the solutions before and after they flew through the columns were studied. Two filters were amended with Pseudomonas putida (P-filter) or Bacillus cereus strains (B-filter), and a third filter was a control (C-filter). Biological activation of filter unit through bacterial amendment enhanced very significantly filter efficacy against plant potential pathogens Pythium spp. and Fusarium oxysporum. However, numerous bacteria (103-104 CFU/mL) were detected in the effluent solutions. The community-level physiological profiling indicated a temporal shift of bacterial microflora, and the metabolism of nutrient solutions originally oriented towards carbohydrates progressively shifted towards degradation of amino acids and carboxylic acids over the 7-month period of experiment. Single-strand conformation polymorphism fingerprinting profiles showed that a shift between bacterial communities colonizing influent and effluent solutions of slow filters occurred. In comparison with influent, 16S rDNA sequencing revealed that phylotype diversity was low in the effluent of P- and C-filters, but no reduction was observed in the effluent of the B-filter. Suppressive potential of solutions filtered on a natural filter (C-filter), where the proportion of Proteobacteria (α- and ß-) increased, whereas the proportion of uncultured candidate phyla rose in P- and B-filters, is discussed.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

Diversity of spoilage fungi associated with various French dairy products.

Yeasts and molds are responsible for dairy product spoilage, resulting in significant food waste and economic losses. Yet, few studies have investigated the diversity of spoilage fungi encountered in dairy products. In the present study, 175 isolates corresponding to 105 from various spoiled dairy products and 70 originating from dairy production environments, were identified using sequencing of the ITS region, the partial ß-tubulin, calmodulin and/or EFα genes, and the D1-D2 domain of the 26S rRNA gene for filamentous fungi and yeasts, respectively. Among the 41 species found in spoiled products, Penicillium commune and Penicillium bialowiezense were the most common filamentous fungi, representing around 10% each of total isolates while Meyerozyma guilliermondii and Trichosporon asahii were the most common yeasts (4.8% each of total isolates). Several species (e.g. Penicillium antarcticum, Penicillium salamii and Cladosporium phyllophilum) were identified for the first time in dairy products or their environment. In addition, numerous species were identified in both spoiled products and their corresponding dairy production environment suggesting that the latter acts as a primary source of contamination. Secondly, the resistance to chemical preservatives (sodium benzoate, calcium propionate, potassium sorbate and natamycin) of 10 fungal isolates representative of the observed biodiversity was also evaluated. Independently of the fungal species, natamycin had the lowest minimum inhibitory concentration (expressed in gram of preservative/l), followed by potassium sorbate, sodium benzoate and calcium propionate. In the tested conditions, Cladosporium halotolerans and Didymella pinodella were the most sensitive fungi while Yarrowia lipolytica and Candida parapsilosis were the most resistant towards the tested preservatives. This study provides interesting information on the occurrence of fungal contaminants in dairy products and environments that may help developing adequate strategies for fungal spoilage control.

A model for the effect of pH on the growth of chalk yeasts.

Hyphopichia burtonii, Pichia anomala, and Saccharomycopsis fibuligera were isolated from spoiled packaged sliced bread. These chalk yeasts were characterized by a wide range of pH for which growth was almost optimum. Thus, the curve growth vs pH exhibited plateau and sharp profiles close to the minimum and the maximum pH. This study described a chalk yeast model (CYM) for the effect of pH derived from a new germination model for fungi (Dantigny, P., Nanguy, S., P.-M., Judet-Correia, D., and Bensoussan, M. 2011, International Journal of Food Microbiology, 146, 176-181). The CYM is asymmetric, versatile, based on parameters with biological significance, and compatible with the gamma concept. The CYM was compared to the cardinal pH model (CPM) which is widely used to describe the effect of pH on microbial growth. The CYM exhibited RMSE values two fold less than those obtained with the CPM for H. burtonii, and S. fibuligera for which plateaus were clearly observed. For P. anomala, the plateau was less obvious, but the RMSE value obtained with the CYM was similar to that found with the CPM. The CYM could extend its use to represent the effect of pH on mold growth.

Diversity of bacterial communities that colonize the filter units used for controlling plant pathogens in soilless cultures.

In recent years, increasing the level of suppressiveness by the addition of antagonistic bacteria in slow filters has become a promising strategy to control plant pathogens in the recycled solutions used in soilless cultures. However, knowledge about the microflora that colonize the filtering columns is still limited. In order to get information on this issue, the present study was carried out over a 4-year period and includes filters inoculated or not with suppressive bacteria at the start of the filtering process (two or three filters were used each year). After 9 months of filtration, polymerase chain reaction (PCR)-single strand conformation polymorphism analyses point out that, for the same year of experiment, the bacterial communities from control filters were relatively similar but that they were significantly different between the bacteria-amended and control filters. To characterize the changes in bacterial communities within the filters, this microflora was studied by quantitative PCR, community-level physiological profiles, and sequencing 16SrRNA clone libraries (filters used in year 1). Quantitative PCR evidenced a denser bacterial colonization of the P-filter (amended with Pseudomonas putida strains) than control and B-filter (amended with Bacillus cereus strains). Functional analysis focused on the cultivable bacterial communities pointed out that bacteria from the control filter metabolized more carbohydrates than those from the amended filters whose trophic behaviors were more targeted towards carboxylic acids and amino acids. The bacterial communities in P- and B-filters both exhibited significantly more phylotype diversity and markedly distinct phylogenetic compositions than those in the C-filter. Although there were far fewer Proteobacteria in B- and P-filters than in the C-filter (22% and 22% rather than 69% of sequences, respectively), the percentages of Firmicutes was much higher (44% and 55% against 9%, respectively). Many Pseudomonas species were also found in the bacterial communities of the control filter. The persistence of the amended suppressive-bacteria in the filters is discussed with regards to the management of suppressive microflora in soilless culture.

Influence of Pythium oligandrum biocontrol on fungal and oomycete population dynamics in the rhizosphere.

Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.