New cultures of lactic acid bacteria for "free-from" food products: challenges, strategies and industrial scale-up

This PhD dissertation addresses one of the most urgent frontiers in modern food biotechnology: the identification, characterization, and application of novel lactic acid bacteria (LAB) cultures specifically suited for the development of “free-from” food products. These products—ranging from allergen-free and gluten-free to fully plant-based alternatives—are the result of a global shift in consumer preferences, driven by heightened awareness of food intolerances, ethical motivations, environmental sustainability goals, and the increasing influence of health-oriented diets. However, removing traditional ingredients such as gluten, dairy proteins, stabilizers, and synthetic preservatives disrupts the structural and functional balance of food matrices, creating technological challenges related to nutritional adequacy, sensory appeal, and microbiological safety. Conventional LAB starter cultures have long been optimized for animal-derived substrates, which differ significantly in composition from the next-generation plant-based or additive-free formulations now in demand. Therefore, this research explores the untapped potential of LAB strains derived from wild fermentation niches—spontaneous fermentations and bio-reserves with low technological pressure—where microbes have naturally developed adaptive traits for thriving in complex, variable environments. he research provides a comprehensive exploration of the global rise of the “free-from” food movement, analyzing its nutritional, environmental, and ethical drivers alongside key regulatory aspects and market dynamics. This background frames the technological challenges and gaps that innovative LAB cultures are designed to address. Central to the work is the examination of alternative ingredients, particularly plant-based proteins, which are evaluated for both their potential and their critical limitations, including specific microbiological risks and the need for integrated risk assessment. Special attention is devoted to the antimicrobial capabilities of LAB-derived compounds—such as bacteriocins and organic acids—and their role as natural protective agents that support modern clean-label food strategies. Building on this perspective, the research emphasizes the technological value of untapped microbial biodiversity. Indigenous LAB strains were isolated and characterized through detailed comparative genomic, phenotypic, and functional analyses. Particular focus was given to Lactiplantibacillus plantarum strains sourced from spontaneous fermentations of both plant and animal origin. Genomic insights highlighted the strong expression of carbohydrate-active enzymes (CAZymes), especially from GH13 and GH1 families, which are key to breaking down complex carbohydrates and improving the nutritional and technological properties of plant-based substrates. Genes associated with antimicrobial compound production further confirmed the potential of these strains as natural bioprotective agents. Together, these traits demonstrate how carefully selected wild LAB can contribute to safer, more appealing, and truly additive-free “free-from” foods. This applied potential was further validated in a dairy context, addressing the well-known late blowing defect (LBD) in hard cheeses caused by Clostridium tyrobutyricum. A novel L. plantarum strain, selected for its antagonistic effect against spoilage clostridia, was tested in pilot-scale cheesemaking trials. The trials demonstrated the strain’s effectiveness as a bioprotective agent, mainly through rapid acidification, competitive exclusion, and metabolic interference, providing a natural alternative to chemical additives and responding to the growing consumer demand for clean-label dairy products. Finally, the findings extend to the innovation of plant-based fermented beverages. The combined use of two carefully selected L. plantarum strains—one producing exopolysaccharides (EPS) and the other overproducing riboflavin—shows how targeted microbial functions can simultaneously enhance nutritional profiles, improve stability, and elevate sensory properties in oat-based fermented drinks. Fermentation trials confirmed improved fermentation kinetics, higher micronutrient content, and better shelf-life performance, demonstrating the feasibility of using LAB as versatile bio-tools for developing the next generation of sustainable, functional, and clean-label beverages. Throughout the dissertation, visual infographics generated with advanced AI-based image tools (e.g., DALL·E, Midjourney, Stable Diffusion) have been incorporated to synthesize complex scientific data into accessible graphical summaries. This integration demonstrates how artificial intelligence can support knowledge transfer, improve data visualization, and strengthen the communicative clarity of modern scientific writing. Collectively, this work advances the state of knowledge at the interface of food microbiology, fermentation biotechnology, and sustainable functional food design. Several parts of the research have been disseminated in high-impact peer-reviewed journals and submitted for patent protection, underlining its technological and industrial relevance. By combining fundamental biodiversity exploration with practical application, this thesis contributes meaningful strategies for bridging clean-label demands, nutritional enhancement, and microbiological safety in “free-from” food systems—laying the groundwork for scalable solutions that respond to evolving consumer expectations and global sustainability targets.

Questa tesi di dottorato affronta una delle frontiere più urgenti della biotecnologia alimentare contemporanea: l’identificazione, la caratterizzazione e l’applicazione di nuove colture di batteri lattici (LAB) specificamente selezionate per lo sviluppo di alimenti “free-from”, ovvero privi di allergeni, glutine o ingredienti di origine animale. Tali prodotti rispondono a un mutamento globale nelle preferenze dei consumatori, sempre più attenti alle intolleranze alimentari, a motivazioni etiche e agli obiettivi di sostenibilità ambientale, in linea con regimi dietetici orientati alla salute. Tuttavia, la rimozione di componenti tradizionali come glutine, proteine del latte, conservanti sintetici e additivi stabilizzanti altera l’equilibrio strutturale e funzionale delle matrici alimentari, generando sfide tecnologiche legate alla sicurezza microbiologica, al valore nutrizionale e all’accettabilità sensoriale. Le colture starter LAB convenzionali sono state storicamente ottimizzate per substrati di origine animale e risultano spesso inadatte a nuove formulazioni plant-based o additive-free. Per questo motivo, la ricerca si è orientata verso ceppi isolati da fermentazioni spontanee e ambienti a bassa pressione tecnologica, dove i microrganismi hanno sviluppato tratti adattativi utili per sopravvivere in contesti complessi e variabili. La tesi è articolata in quattro sezioni interconnesse. La prima analizza l’evoluzione globale del fenomeno “free-from”, approfondendo i fattori nutrizionali, ambientali ed etici, il quadro normativo e le dinamiche di mercato. Viene quindi delineata la cornice scientifica per individuare le lacune tecnologiche colmabili con nuove colture microbiche. I capitoli successivi trattano ingredienti alternativi, illustrandone vantaggi e limiti, i rischi microbiologici specifici delle nuove formulazioni e l’importanza di un’analisi integrata del rischio. Particolare attenzione è dedicata al potenziale antimicrobico di batteriocine e acidi organici prodotti dai LAB, nonché al loro impiego come colture protettive in strategie clean-label. La seconda sezione approfondisce la biodiversità come leva per l’innovazione tecnologica. Ceppi autoctoni vengono isolati e caratterizzati mediante analisi genomiche e fenotipiche: in particolare Lactiplantibacillus plantarum mostra un’ampia espressione di CAZymes (famiglie GH13, GH1) utili alla degradazione di carboidrati complessi, migliorando fermentabilità e valore nutrizionale di substrati vegetali. La presenza di geni legati alla produzione di antimicrobici conferma la loro efficacia come agenti di bioprotezione. Queste proprietà dimostrano la possibilità di selezionare razionalmente LAB “selvatici” per migliorare sicurezza, proprietà sensoriali e profilo clean-label degli alimenti “free-from”. Su queste basi, la terza sezione applica le conoscenze acquisite al settore lattiero-caseario. Si affronta il problema dello “late blowing defect” nei formaggi stagionati, causato da Clostridium tyrobutyricum. Dopo un’analisi critica, uno studio sperimentale testa un ceppo di L. plantarum selezionato per la sua attività antagonista, validandone l’efficacia su scala pilota grazie a meccanismi di acidificazione rapida e esclusione competitiva. La sezione finale trasferisce l’approccio ai prodotti vegetali fermentati. L’impiego combinato di due ceppi di L. plantarum (uno produttore di EPS, l’altro di riboflavina) dimostra come funzioni microbiche mirate possano aumentare valore nutrizionale, stabilità e qualità sensoriale di bevande fermentate a base avena. Infine, l’uso di strumenti di intelligenza artificiale per la generazione di infografiche riassuntive ha permesso di sintetizzare dati complessi in immagini accessibili, migliorando la chiarezza espositiva. Complessivamente, la tesi combina esplorazione della biodiversità e validazione applicativa, proponendo strategie scalabili per alimenti funzionali “free-from” sicuri, nutrienti e sostenibili.