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Grapevine cane, an abundant viticultural by-product, contains high levels of stilbenoids and therefore holds promise as a natural antifugal and antimycotoxigenic agent. Produced by a microwave-assisted hydro-ethanolic extraction process, the grapevine cane extract (GCE) was tested for its activity against three mycotoxigenic fungi F. graminearum, A. flavus, and P. expansum. Dose-response assays were performed, based on radial growth and inhibition of specific mycotoxin production. For all fungi, growth inhibition IC50 values clustered between 1.0 and 5.0 g/L, while for specific toxin production, IC50 were lower (≈0.5 g/L) except for patulin, which increased in a dose-dependent manner in the presence of the extract. Specific experiments were designed to highlight the effect of the extracts at various stages of the fungal life cycle (e.g., spore germination, early mycelium, and established colonies). F. graminearum spores’ germination was strongly inhibited (5.0 to 15 g/L), while for other fungi, germination was only delayed. Interestingly, antifungal and especially antimycotoxigenic effects were shown to be persistent after exposure.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB1 when growing in solid and liquid media. A total degradation of AFB1 was achieved by Mycostop® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB1’s genotoxicity.

Bacterial spores are among the most resistant forms of life on Earth. Their exceptional resistance properties rely on various strategies, among them the core singular structure, organization and hydration. By using elastic incoherent neutron scattering, we probed the dynamics of Bacillus subtilis spores to determine whether core macromolecular motions at the sub-nanosecond timescale could also contribute to their resistance to physical stresses. In addition, in order to better specify the role of the various spore components, we used different mutants lacking essential structure such as the coat (PS4150 mutant), or the calcium dipicolinic acid complex (CaDPA) located in the core (FB122 mutant). PS4150 allows to better probe the core’s dynamics, as proteins of the coat represent an important part of spore proteins, and FB122 gives information about the role of the large CaDPA depot for the mobility of core’s components. We show that core’s macromolecular mobility is not particularly constrained at the sub-nanosecond timescale in spite of its low water content as some dynamical characteristics as force constants are very close to those of vegetative bacteria such as Escherichia coli or to those of fully hydrated proteins. Although the force constants of the coatless mutant are similar to the wild-type’s ones, it has lower mean square displacements (MSDs) at high Q showing that core macromolecules are somewhat more constrained than the rest of spore components. However, no behavior reflecting the glassy state regularly evoked in the literature could be drawn from our data. As hydration and macromolecules’ mobility are highly correlated, the previous assumption, that core low water content might explain spores’ exceptional resistance properties seems unlikely. Thus, we confirm recent theories, suggesting that core water is mostly as free as bulk water and proteins/macromolecules are fully hydrated. The germination of spores leads to a much less stable system with a force constant of 0.1 N/m and MSDs ~2.5 times higher at low Q than in the dormant state. DPA has also an influence on core mobility with a slightly lower force constant for the DPA-less mutant than for the wild-type, and MSDs that are ~ 1.8 times higher on average than for the wild-type at low Q. At high Q, germinated and DPA-less spores were very similar to the wild-type ones, showing that DPA and core compact structure might influence large amplitude motions rather than local dynamics of macromolecules.

Results from first neutron scattering experiments on bacterial spores are reported. The elastic intensities and mean square displacements have a non-linear behaviour as function of temperature, which is in agreement with a model presenting more pronounced variations at around 330 K (57[degrees]C) and 400 K (127[degrees]C). Based on the available literature on thermal properties of bacterial spores, mainly referring to differential scanning calorimetry, they are suggested to be associated to main endothermic transitions induced by coat and/or core bacterial response to heat treatment.

Ochratoxin A (OTA) is a secondary metabolite produced by fungal pathogens such as Penicillium verrucosum, which develops in food commodities during storage such as cereals, grapes, and coffee. It represents public health concerns due to its genotoxicity, carcinogenicity, and teratogenicity. The objective of this study was to evaluate the ability of actinobacteria and their metabolites to degrade OTA and/or to decrease its production. Sixty strains of actinobacteria were tested for their ability to prevent OTA formation by in vitro dual culture assays or with cell free extracts (CFEs). In dual culture, 17 strains strongly inhibited fungal growth, although it was generally associated with an increase in OTA specific production. Seventeen strains inhibited OTA specific production up to 4% of the control. Eleven actinobacteria CFEs reduced OTA specific production up to 62% of the control, while no substantial growth inhibition was observed except for two strains up to 72% of the control. Thirty-three strains were able to degrade OTA almost completely in liquid medium whereas only five were able to decrease it on solid medium, and two of them reduced OTA to an undetectable amount. Our results suggest that OTA decrease could be related to different strategies of degradation/metabolization by actinobacteria, through enzyme activities and secretion of secondary metabolites interfering with the OTA biosynthetic pathway. CFEs appeared to be ineffective at degrading OTA, raising interesting questions about the detoxification mechanisms. Common degradation by-products (e.g., OTα or L-β-phenylalanine) were searched by HPLC-MS/MS, however, none of them were found, which implies a different mechanism of detoxification and/or a subsequent degradation into unknown products.

Cocoa fermentation plays a key role in defining chocolate’s flavor, with yeasts being central to this process. This study aimed to explore intraspecific genetic diversity of major indigenous yeasts (i.e., Saccharomyces cerevisiae and Pichia kudriavzevii), and their potential interaction in the cocoa pulp environment. Their metabolic intraspecific diversity was characterized in synthetic cocoa pulp medium. Then, Saccharomyces cerevisiae, Pichia kudriavzevii, and other strains were introduced to each other to evaluate their potential negative interaction. Interesting strain associations were selected to further explore their interaction in synthetic cocoa pulp medium as well as real fresh cocoa pulp. From a fermentation campaign in Ivory Coast, a set of Saccharomyces (S.) cerevisiae and Pichia (P.) kudriavzevii strains were isolated from batches classified according to their chocolate quality (i.e., standard, intermediate, or premium chocolate). Less abundant species (i.e., Torulaspora franciscae, Kluyveromyces marxianus) were also isolated and tested for their potential negative interactions with S. cerevisiae and P. kudriavzevii. A set of strains were selected and cultured in single and in co-culture in a minimal cocoa pulp synthetic medium and in fresh cocoa pulp to highlight potential positive and/or negative interactions regarding fermentative aroma profile (i.e., higher alcohols, acetate esters, medium-chain fatty acids, and ethyl esters). The results highlighted the dominance of S. cerevisiae in fermentation kinetics and medium- to long-chain ester production, contrasted with P. kudriavzevii’s efficiency in short-chain ester synthesis. Intraspecific aroma profile variations can be pointed out. The co-cultures of P. kudriavzevii and S. cerevisiae strains isolated from the premium chocolate batch had a positive impact on the fermented pulp aroma profile. Negative interactions were observed with Torulaspora franciscae, which eliminated P. kudriavzevii’s aroma expression. Finally, the comparison of the data obtained for the minimal cocoa pulp synthetic medium compared to the cocoa pulp allowed us to draw conclusions about the use of synthetic media for studying cocoa fermentation. These findings emphasize the complex microbial interactions in cocoa fermentation that could shape future cocoa bean aroma.

Acetic acid bacteria are involved in many food and beverage fermentation processes. They play an important role in cocoa bean fermentation through their acetic acid production. They initiate the development of some of the flavor precursors that are necessary for the organoleptic quality of cocoa, and for the beans’ color. The development of starter cultures with local strains would enable the preservation of the microbial biodiversity of each country in cocoa-producing areas, and would also control the fermentation. This approach could avoid the standardization of cocoa bean fermentation in the producing countries. One hundred and thirty acetic acid bacteria were isolated from three different cocoa-producing countries, and were identified based on their 16S rRNA gene sequence. The predominate strains were grown in a cocoa pulp simulation medium (CPSM-AAB) in order to compare their physiological traits regarding their specific growth rate, ethanol and lactic acid consumption, acetic acid production, and relative preferences of carbon sources. Finally, the intraspecific diversity of the strains was then assessed through the analysis of their genomic polymorphism by (GTG)5-PCR fingerprinting. Our results showed that Acetobacter pasteurianus was the most recovered species in all of the origins, with 86 isolates out of 130 cultures. A great similarity was observed between the strains according to their physiological characterization and genomic polymorphisms. However, the multi-parametric clustering results in the different groups highlighted some differences in their basic metabolism, such as their efficiency in converting carbon substrates to acetate, and their relative affinity to lactic acid and ethanol. The A. pasteurianus strains showed different behaviors regarding their ability to oxidize ethanol and lactic acid into acetic acid, and in their relative preference for each substrate. The impact of these behaviors on the cocoa quality should be investigated, and should be considered as a criterion for the selection of acetic acid bacteria starters.

Spores are bacteria passed into a metabolically dormant state due to starvation and awaiting better external conditions to germinate and colonize the medium again. Their resistance to numerous stress factors is amazing and the reasons behind are still not completely unveiled. Water seems to play an important role for that, as hydration inside the spores is reduced but paired with a remarkable water mobility. To learn more about these facts, we applied a sophisticated approach: we first analysed neutron scattering spectra of whole spores, then we subtracted spectra corresponding to proteins, lipids and sugars to obtain results as close as possible to the water signal. Our procedure revealed strongly reduced rotational dynamics of the proteome, but a high mobility of water molecules and small molecular subgroups at the sub-nanosecond time scale. Such combination might be key to explain the dormant state of spores which is vigilant to revive in adapted conditions.