Periodic Reporting for period 1 - SCOBY DO (Symbiotic Culture of Bacteria and Yeast Ecosystems for the development of Probiotic Extracellular Vesicles)
Período documentado: 2023-10-01 hasta 2025-09-30
The SCOBY-DO (Symbiotic Community of Bacteria and Yeast – Discovery and Outreach) project, funded under the Horizon 2022 -MSCA (Marie Skłodowska-Curie Actions), explores microbial communication using kombucha as a model system. Kombucha is a traditional fermented tea produced by a symbiotic culture of bacteria and yeast. This microbial ecosystem is both resilient and dynamic, offering a natural laboratory for studying microbial cooperation, competition, and stability over time.
The project addresses a key scientific challenge: understanding how microbes communicate through extracellular vesicles (EVs). EVs are nanoscale, membrane-bound structures that transport proteins, nucleic acids, and metabolites. Although their role is increasingly recognised in human biology and medicine, little is known about their ecological role in natural microbial consortia.
The main objectives of SCOBY-DO are:
1. Optimize SCOBY kombucha model system, kombucha-derived single cultures and binary co-cultures to produce EVs.
2. Study i.) Kombucha-derived EVs as potential therapeutic agents in gastrointestinal bacterial infection and ii.) the role of SCOBY EVs in SCOBY cross-species communication iii.) to design and synthetize polymers through the molecular imprinting technologies (MITs) for the specific recognition or release of kombucha-derived EVs.
These objectives align closely with MSCA’s priorities by combining excellent science, interdisciplinary training, and societal engagement.
The project addresses a key scientific challenge: understanding how microbes communicate through extracellular vesicles (EVs). EVs are nanoscale, membrane-bound structures that transport proteins, nucleic acids, and metabolites. Although their role is increasingly recognised in human biology and medicine, little is known about their ecological role in natural microbial consortia.
The main objectives of SCOBY-DO are:
1. Optimize SCOBY kombucha model system, kombucha-derived single cultures and binary co-cultures to produce EVs.
2. Study i.) Kombucha-derived EVs as potential therapeutic agents in gastrointestinal bacterial infection and ii.) the role of SCOBY EVs in SCOBY cross-species communication iii.) to design and synthetize polymers through the molecular imprinting technologies (MITs) for the specific recognition or release of kombucha-derived EVs.
These objectives align closely with MSCA’s priorities by combining excellent science, interdisciplinary training, and societal engagement.
SCOBY DO project implemented a range of complementary methods, combining classical microbiology with cutting-edge analytical techniques:
Extracellular Vesicle Isolation and Characterisation
• EVs were isolated and purified from first and second fermentations kombucha cultures (using differential ultra centrifugation (dUC) and tangential flow filtration (TFF).
• The protein and nanoparticle concentrations in EVs were measured using Qubit and interferometric light microscopy (ILM), respectively. A modified vanillin assay was used to quantify total lipids associated with EVs.
• Protein content was assessed through SDS-PAGE profiling, while morphology was confirmed by cryo-TEM.
Fluorescent Labelling and Imaging
• EVs were labelled with lipophilic fluorescent dyes to visualise uptake by bacterial and yeast cells.
• Live-cell microscopy was employed to monitor dynamic EV transfer in co-culture systems.
Microbial dynamic Insights: The microbial genomic DNA were isolated from first and second fermentation kombucha and SCOBY samples. Further, these DNA were used for shotgun sequences to identify the microbial strains and their functions in the kombucha model systems.
Co-Culture Model Development
• Laboratory cultures of Komagataeibacter rhaeticus (cellulose-producing bacteria) and Brettanomyces bruxellensis (osmophilic yeast) were established to study direct interactions.
Public Engagement and Outreach
SCOBY-DO participated in Futuro Remoto 2024 (Naples, Italy), where kombucha fermentation and microbial communication were demonstrated to a wide audience including students, teachers, and the public.
Workshops and interactive demonstrations in schools were piloted to promote microbiology and EVs awareness in the field of probiotics.
This integrated methodology allowed the project to progress simultaneously in research and outreach.
Extracellular Vesicle Isolation and Characterisation
• EVs were isolated and purified from first and second fermentations kombucha cultures (using differential ultra centrifugation (dUC) and tangential flow filtration (TFF).
• The protein and nanoparticle concentrations in EVs were measured using Qubit and interferometric light microscopy (ILM), respectively. A modified vanillin assay was used to quantify total lipids associated with EVs.
• Protein content was assessed through SDS-PAGE profiling, while morphology was confirmed by cryo-TEM.
Fluorescent Labelling and Imaging
• EVs were labelled with lipophilic fluorescent dyes to visualise uptake by bacterial and yeast cells.
• Live-cell microscopy was employed to monitor dynamic EV transfer in co-culture systems.
Microbial dynamic Insights: The microbial genomic DNA were isolated from first and second fermentation kombucha and SCOBY samples. Further, these DNA were used for shotgun sequences to identify the microbial strains and their functions in the kombucha model systems.
Co-Culture Model Development
• Laboratory cultures of Komagataeibacter rhaeticus (cellulose-producing bacteria) and Brettanomyces bruxellensis (osmophilic yeast) were established to study direct interactions.
Public Engagement and Outreach
SCOBY-DO participated in Futuro Remoto 2024 (Naples, Italy), where kombucha fermentation and microbial communication were demonstrated to a wide audience including students, teachers, and the public.
Workshops and interactive demonstrations in schools were piloted to promote microbiology and EVs awareness in the field of probiotics.
This integrated methodology allowed the project to progress simultaneously in research and outreach.
The project has already generated important preliminary scientific results:
1. Successful EV Isolation: Different populations of EVs were successfully isolated from three different commercial sources of kombucha using both primary and secondary fermentations. These nanoparticles were visualized by cryoTEM and ILM, representing one of the first systematic analyses of EVs in a food fermentation context.
Genome sequencing identified the dominant bacterial and yeast strains: The Komagataeibacter rhaeticus (<20%), a cellulose-producing bacteria and Brettanomyces bruxellensis (>60%), an osmophilic yeast strains were identified as dominant strains in the kombucha model systems (both first and second fermentations). Whereas >60% of K. rhaeticus and <10% of B. bruxellensis considered as dominant strains in the SCOBY pellicles.
2. EVs were found to vary in concentration and size distribution across different kombucha and SCOBY samples, suggesting that microbial composition influences EVs production.
3. Protein Diversity: SDS-PAGE profiling revealed that kombucha EVs contain diverse proteins, potentially linked to microbial adaptation, metabolism, and symbiosis.
4. Scientific and technological impact
This research has yielded foundational insights into microbial ecology and the biology of EVs, advancing our understanding of microbial interactions within complex ecosystems such as the microbial community of kombucha. Using fluorescent labelling and advanced microscopy, the study showed that EVs are actively exchanged between bacteria and yeast, supporting their role in cross-kingdom microbial communication. These findings underscore the significance of EVs as mediators of biochemical exchange and signaling across diverse microbial taxa.
Biotechnological Applications: The discovery of functional EVs in kombucha cultures opens promising avenues for biotechnological innovation. These vesicles may be harnessed for the development of functional foods with enhanced health benefits, natural antimicrobial agents, or novel biomaterials with unique properties derived from microbial sources.
Biomedical Relevance: By elucidating the mechanisms of microbial EV production and uptake, the research contributes to a deeper understanding of microbiome–host interactions and the potential of EVs as vehicles for therapeutic delivery. This knowledge may inform future strategies in precision medicine, including microbiome modulation and targeted drug delivery systems.
By the end of the project, SCOBY-DO is expected to deliver the first comprehensive framework of EV-mediated microbial communication in a natural symbiotic consortium. Further studies will include the exploitation of kombucha EVS in precision nutrition and their potential prebiotic activities.
1. Successful EV Isolation: Different populations of EVs were successfully isolated from three different commercial sources of kombucha using both primary and secondary fermentations. These nanoparticles were visualized by cryoTEM and ILM, representing one of the first systematic analyses of EVs in a food fermentation context.
Genome sequencing identified the dominant bacterial and yeast strains: The Komagataeibacter rhaeticus (<20%), a cellulose-producing bacteria and Brettanomyces bruxellensis (>60%), an osmophilic yeast strains were identified as dominant strains in the kombucha model systems (both first and second fermentations). Whereas >60% of K. rhaeticus and <10% of B. bruxellensis considered as dominant strains in the SCOBY pellicles.
2. EVs were found to vary in concentration and size distribution across different kombucha and SCOBY samples, suggesting that microbial composition influences EVs production.
3. Protein Diversity: SDS-PAGE profiling revealed that kombucha EVs contain diverse proteins, potentially linked to microbial adaptation, metabolism, and symbiosis.
4. Scientific and technological impact
This research has yielded foundational insights into microbial ecology and the biology of EVs, advancing our understanding of microbial interactions within complex ecosystems such as the microbial community of kombucha. Using fluorescent labelling and advanced microscopy, the study showed that EVs are actively exchanged between bacteria and yeast, supporting their role in cross-kingdom microbial communication. These findings underscore the significance of EVs as mediators of biochemical exchange and signaling across diverse microbial taxa.
Biotechnological Applications: The discovery of functional EVs in kombucha cultures opens promising avenues for biotechnological innovation. These vesicles may be harnessed for the development of functional foods with enhanced health benefits, natural antimicrobial agents, or novel biomaterials with unique properties derived from microbial sources.
Biomedical Relevance: By elucidating the mechanisms of microbial EV production and uptake, the research contributes to a deeper understanding of microbiome–host interactions and the potential of EVs as vehicles for therapeutic delivery. This knowledge may inform future strategies in precision medicine, including microbiome modulation and targeted drug delivery systems.
By the end of the project, SCOBY-DO is expected to deliver the first comprehensive framework of EV-mediated microbial communication in a natural symbiotic consortium. Further studies will include the exploitation of kombucha EVS in precision nutrition and their potential prebiotic activities.