Brine salt concentration reduction and inoculation with autochthonous consortia: Impact on Protected Designation of Origin Nyons black table olive fermentations.

Nyons table olives, named after the French city where they are processed, are naturally fermented black table olives. Their specificity relies on the use of the "Tanche" olive variety harvested at full maturity and their slow spontaneous fermentation in 10% salt brine driven by yeast populations. This study aimed at investigating the benefit of inoculating autochthonous consortia to produce Nyons table olives by fermentation in 10% salt brine and in reduced salt conditions (8%). Two strategies were evaluated: inoculation with a defined autochthonous consortium and inoculation by spent brine backslopping. To define the consortium, yeasts were selected among 48 autochthonous isolates and key features included high halotolerance, low pectinolytic and proteolytic activities, however none had ß-glucosidase activities. The consortium included eight yeast strains with distinct technological properties belonging to five dominant species, i.e. Citeromyces nyonsensis, Pichia membranifaciens, Wickerhamomyces anomalus, Zygotorulaspora mrakii and Candida atlantica. Fermentation trials were conducted over a year and compared by evaluating microbial community shifts (16S and ITS metagenetics) and volatile profiles (GC-MS). Regarding fermentations with the defined consortium, four out of five species implanted in early stages while one, Pichia membranifaciens, persisted and largely dominated by the end of the fermentation. Altogether, inoculation with the defined consortium did not disrupt microbial shifts compared to traditional fermentations although minor differences were observed in volatile profiles. The backslopping method yielded the highest impact on microbial populations and olive volatile profiles, with higher ester abundances at the end of fermentation. Finally, reduced salt in brine gave very promising results as no deleterious effects on microbial communities, volatile dynamics but also safety criteria of the olives were observed compared to traditional fermented olives.

Use of metabarcoding and source tracking to identify desirable or spoilage autochthonous microorganism sources during black olive fermentations.

This study aimed at investigating the influence of the process environment and raw materials as sources of microorganisms during Nyons black table olive fermentations. Fermented olives and/or brine from spoiled fermentation tanks were analyzed and compared to good quality samples from fermentations collected during 3 consecutive harvest years. Fresh olives, salt and different process environment samples were also analyzed. Microbial diversity of all samples was analyzed using 16S and ITS2 amplicon sequencing and SourceTracker tool was used to investigate links between environment, raw materials and fermentation samples. First, comparison of microbial diversity in control and most spoiled fermentations revealed striking differences in bacterial composition with an overall higher diversity in spoiled fermentations especially for lactic acid bacteria with Lentilactobacillus buchneri, Lentilactobacillus parafarraginis dominating in brine and Pediococcus parvulus, Pediococcus ethanolidurans dominating in olive fruits. Fungal communities were similar in composition although higher abundances of Pichia membranifaciens and Penicillium carneum/roqueforti were observed in spoiled samples. Secondly, process environment samples were characterized by high bacterial and fungal diversity, especially compared to fresh olive fruits. Overall, dominant fungal species in control fermentations were also found in most environmental samples revealing a "house mycobiota". SourceTracker analysis further highlighted the contribution of brine and water from the optical sorter as a source of fungi. Most interestingly, spoilage fungi and most bacteria were retrieved in brine and environmental samples while others such as P. ethanolidurans were only found in environmental samples indicating that the studied spoilage originated from a fermentation deviation rather than a punctual contamination. Taken altogether, these results highlighted the positive and negative influence of the process environment and emphasized the relevance of studying it to better understand microbial vectors occurring during food fermentations, especially natural ones.

Tailor-made microbial consortium for Kombucha fermentation: Microbiota-induced biochemical changes and biofilm formation.

Kombucha is a very distinct naturally fermented sweetened tea that has been produced for thousands of years. Fermentation relies on metabolic activities of the complex autochthonous symbiotic microbiota embedded in a floating biofilm and used as a backslop for successive fermentations. Here, we designed a tailor-made microbial consortium representative of the core Kombucha microbiota to drive this fermentation. Microbial (counts, metagenetics), physico-chemical (pH, density) and biochemical (organic acids, volatile compounds) parameters were monitored as well as biofilm formation by confocal laser scanning microscopy and scanning electron microscopy. While nine species were co-inoculated, four (Dekkera bruxellensis, Hanseniaspora uvarum, Acetobacter okinawensis and Liquorilactobacillus nagelii) largely dominated. Microbial activities led to acetic, lactic, succinic and oxalic acids being produced right from the start of fermentation while gluconic and glucuronic acids progressively increased. A distinct shift in volatile profile was also observed with mainly aldehydes identified early on, then high abundances of fatty acids, ketones and esters at the end. Correlation analyses, combining metabolomic and microbial data also showed a shift in species abundances during fermentation. We also determined distinct bacteria-yeast co-occurence patterns in biofilms by microscopy. Our study provides clear evidence that a tailor-made consortium can be successfully used to drive Kombucha fermentations.

Linking Pélardon artisanal goat cheese microbial communities to aroma compounds during cheese-making and ripening.

Pélardon is an artisanal French raw goat's milk cheese, produced using natural whey as a backslop. The aim of this study was to identify key microbial players involved in the acidification and aroma production of this Protected Designation of Origin cheese. Microbial diversity of samples, collected from the raw milk to 3-month cheese ripening, was determined by culture-dependent (MALDI-TOF analysis of 2877 isolates) and -independent (ITS2 and 16S metabarcoding) approaches and linked to changes in biochemical profiles (volatile compounds and acids). In parallel, potential dominant autochthonous microorganism reservoirs were also investigated by sampling the cheese-factory environment. Complex and increasing microbial diversity was observed by both approaches during ripening although major discrepancies were observed regarding Lactococcus lactis and Lacticaseibacillus paracasei fate. By correlating microbial shifts to biochemical changes, Lactococcus lactis was identified as the main acidifying bacterium, while L. mesenteroides and Geotrichum candidum were prevalent and associated with amino acids catabolism after the acidification step. The three species were dominant in the whey (backslop). In contrast, L. paracasei, Enterococcus faecalis, Penicillium commune and Scopulariopsis brevicaulis, which dominated during ripening, likely originated from the cheese-making environment. All these four species were positively correlated to major volatile compounds responsible for the goaty and earthy Pélardon cheese aroma. Overall, this work highlighted the power of MALDI-TOF and molecular techniques combined with volatilome analyses to dynamically follow and identify microbial communities during cheese-making and successively identify the key-players involved in aroma production and contributing to the typicity of Pélardon cheese.

Deciphering Microbial Community Dynamics and Biochemical Changes During Nyons Black Olive Natural Fermentations.

French PDO Nyons black table olives are produced according to a traditional slow spontaneous fermentation in brine. The manufacture and unique sensorial properties of these olives thus only rely on the autochthonous complex microbiota. This study aimed at unraveling the microbial communities and dynamics of Nyons olives during a 1.5-year-long spontaneous fermentation to determine the main microbial drivers and link microbial species to key metabolites. Fermentations were monitored at a local producer plant at regular time intervals for two harvests and two olive types (organically and conventionally grown) using culture-dependent and metabarcoding (ITS2 for fungi, V3-V4 region for bacteria) approaches. Olives and brines were also sampled for volatiles, organic acids and phenolic compounds. No major differences in microbiota composition were observed according to olive type or harvest period. Throughout the fermentation, yeasts were clearly the most dominant. ITS2 sequencing data revealed complex fungal diversity dominated by Citeromyces nyonsensis, Wickerhamomyces anomalus, Zygotorulaspora mrakii, Candida boidinii and Pichia membranifaciens species. Bacterial communities were dominated by the Celerinatantimonas genus, while lactic acid bacteria remained scarce. Clear shifts in microbial communities and biochemical profiles were observed during fermentation and, by correlating metabolites and microbiota changes, four different phases were distinguished. During the first 7 days, phase I, a fast decrease of filamentous fungal and bacterial populations was observed. Between days 21 and 120, phase II, W. anomalus and C. nyonsensis for fungi and Celerinatantimonas diazotrophica for bacteria dominated the fermentation and were linked to the pH decrease and citric acid production. Phase III, between 120 and 183 days, was characterized by an increase in acids and esters and correlated to increased abundances of Z. mrakii, P. membranifaciens and C. boidinii. During the last months of fermentation, phase IV, microbial communities were dominated by P. membranifaciens and C. boidinii. Both species were strongly correlated to an increase in fruity esters and alcohol abundances. Overall, this study provides an in-depth understanding about microbial species succession and how the microbiota shapes the final distinct olive characteristics. It also constitutes a first step to identify key drivers of this fermentation.

Antifungal activity of fermented dairy ingredients: Identification of antifungal compounds.

Fungi are commonly identified as the cause for dairy food spoilage. This can lead to substantial economic losses for the dairy industry as well as consumer dissatisfaction. In this context, biopreservation of fermented dairy products using lactic acid bacteria, propionibacteria and fungi capable of producing a large range of antifungal metabolites is of major interest. In a previous study, extensive screening was performed in vitro and in situ to select 3 dairy fermentates (derived from Acidipropionibacterium jensenii CIRM-BIA1774, Lactobacillus rhamnosus CIRM-BIA1952 and Mucor lanceolatus UBOCC-A-109193, respectively) with antifungal activity. The aim of the present study was to determine the main compounds responsible for this antifungal activity. Fifty-six known antifungal compounds as well as volatiles were targeted using different analytical methods (conventional LC and GC, GC-MS, LC-QToF). The most abundant antifungal compounds in P. jensenii-, L. rhamnosus- and M. lanceolatus-derived fermentates corresponded to propionic and acetic acids, lactic and acetic acids, and butyric acid, respectively. Many other antifungal compounds (organic acids, free fatty acids, volatile compounds) were identified but at lower levels. In addition, an untargeted approach using nano LC-MS/MS identified a 9-amino acid peptide derived from αs2-casein in the L. rhamnosus-derived fermentate. This peptide inhibited M. racemosus and R. mucilaginosa in vitro. This study provides new insights on the molecules involved in antifungal activities of food-grade microorganism fermentates which could be used as antifungal ingredients in the dairy industry.