Exploring the Microbial Community of Traditional Sourdoughs to Select Yeasts and Lactic Acid Bacteria

Sourdoughs represent an awesome example of ecosystem in which yeasts and lactic acid bacteria (LAB) interact with each other, defining the characteristics of the final product in terms of composition, texture, taste and flavor. Therefore, the identification of dominant yeasts and LAB involved in the fermentation process can lead to the selection of starters with suitable fermentation aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (A and B) for Panettone production were collected from an artisan bakery. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 120 isolates were obtained and firstly characterized by conventional microbiological methods. Afterward, genomic DNA was extracted from the cultures, and (GTG)5-PCR fingerprinting analysis was carried out to reduce the redundance among the isolates. Representative yeasts and LAB strains, having a unique profile, were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. The results highlighted the occurrence of Kazachstania humilis and Fructilactobacillus sanfranciscensis in both sourdoughs. Among LAB, also some other strains belonging to Lactobacillus genus were found. Moreover, Saccharomyces cerevisiae and Staphylococcus spp. strains were detected in sample B. In this study, a pool of yeasts and LAB strains for producing starter cultures with specific technological traits for sourdoughs production was obtained

Selection of Promising Exopolysaccharide-Producing Starter Cultures for Gluten-Free Sourdough

Celiac disease is an autoimmune enteropathy that affects the small intestine and is caused by the gliadin fraction of wheat gluten and other alcohol-soluble proteins (prolamines) of barley and rye in genetically predisposed subjects. Nowadays, the only recognized cure for this pathology is the use of gluten-free (GF) products. Although the market for GF products has grown in the last few years, their improvement is still a challenge for the food industry. With regard to GF bakery products, some of them can result in an unappetizing taste with a high fat content. These inconveniences can be minimized or solved by using sourdough fermentation. Sourdough is a stable culture of lactic acid bacteria (LAB) and yeast in a mixture of flour and water. In particular, GF sourdough represents a rich source of naturally occurring LAB and wild yeasts, which play important roles in food fermentation. This is mainly due to their potential in improving functional, technological, and probiotics properties, contributing to safe food with a better taste. Moreover, exopolysaccharide (EPS)-producing strains can also contribute to improving the sensory and rheological quality of the different GF products. This study aims to isolate and characterize the microbial pool from a GF sourdough made with rice flour and to evaluate the ability of selected LAB strains to produce exopolysaccharides (EPS). The yeast and LAB were isolated in selective agar media. A total of 220 isolates (110 yeasts and 110 bacteria) were obtained. They were taxonomically identified and characterized using both traditional and molecular approaches. Sequencing analysis of the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, for yeasts and bacteria, respectively, revealed the occurrence of Saccharomyces cerevisiae as predominant yeasts and Lactiplantibacillus plantarum and Pediococcus pentosaceus as dominant LAB. Regarding the EPS production, 25 strains were able to produce dextran-like EPS with a maximum yield of 2 g/L. This study allowed the isolation and selection of promising EPS-producing starter cultures for the different types of flour sourdough, in particular, GF sourdoug

Anti-Spoilage Activity and Exopolysaccharides Production by Selected Lactic Acid Bacteria

In this study, eight lactic acid bacteria (LAB) strains, previously isolated from traditional and gluten-free sourdoughs, and selected for their potential in improving the sensory and rheological quality of bakery products, were screened against some common spoilage agents. The anti-mould activity was tested using strains of the species Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger. Regarding the antibacterial activity, it was assessed against four strains of the species Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes. Furthermore, LAB strains were evaluated for their ability to produce exopolysaccharides, which are gaining considerable attention for their functional properties and applicability in different food industrial applications. A strain-specific behaviour against the moulds was observed. In particular, F. graminearum ITEM 5356 was completely inhibited by all the LAB strains. Regarding the antibacterial activity, the strains Leuconostoc citreum UMCC 3011, Lactiplantibacillus plantarum UMCC 2996, and Pediococcus pentosaceus UMCC 3010 showed wide activity against the tested pathogens. Moreover, all the LAB strains were able to produce exopolysaccharides, which were preliminarily characterized. The assessed features of the LAB strains allow us to consider them as promising candidates for single or multiple starter cultures for food fermentation processes

Selection of Wine Saccharomyces cerevisiae Strains and Their Screening for the Adsorption Activity of Pigments, Phenolics and Ochratoxin A

Ochratoxin A is a dangerous mycotoxin present in wines and is considered the principal safety hazard in the winemaking process. Several authors have investigated the ochratoxin A adsorption ability of Saccharomyces cerevisiae yeasts, and specifically selected strains for this desired trait. In the present work, a huge selection of wine yeasts was done starting from Portuguese, Spanish and Italian fermenting musts of dierent cultivars. Firstly, 150 isolates were collected, and 99 non-redundant S. cerevisiae strains were identified. Then, the strains were screened following a multi-step approach in order to select those having primary oenological traits, mainly (a) good fermentation performance, (b) low production of H2S and (c) low production of acetic acid. The preselected strains were further investigated for their adsorption activity of pigments, phenolic compounds and ochratoxin A. Finally, 10 strains showed the desired features. The goal of this work was to select the strains capable of absorbing ochratoxin A but not pigments and phenolic compounds in order to improve and valorise both the quality and safety of red wines. The selected strains are considered good candidates for wine starters, moreover, they can be exploited to obtain a further enhancement of the specific adsorption/non-adsorption activity by applying a yeast breeding approach

Valorization of wheat bread waste and cheese whey through cultivation of lactic acid bacteria for bio-preservation of bakery products

In this work, three lactic acid bacteria (LAB) strains, specifically, Lactiplantibacillus plantarum UMCC 2996, Furfurilactobacillus rossiae UMCC 3002, and Pediococcus pentosaceus UMCC 3010, were tested in new bread-whey media composed by wheat bread and sweet cheese whey, designed as an alternative to the conventional MRS medium. The medium resulting from hydrolysis with amylase and neutrase (AN) was considered the best for the growth of all the strains. This medium was particularly optimal for the strain F. rossiae UMCC 3002, which showed an increase in growth of 114% compared to that in MRS medium. Additionally, the bio-preservative ability of the selected LAB was assessed in pectin-based coated sliced bread inoculated with Aspergillus flavus ITEM 7828, Penicillium paneum ITEM 1381, and Aspergillus niger ITEM 7090. Different LAB strain behavior was observed towards the specific molds. A good bio-preservation activity was shown from F. rossiae UMCC 3002 against A. flavus ITEM 7828 with results compared to the protection by ethanol treatment. The results obtained in this study suggest a novel strategy for the cultivation of selected starters with a bio-protection activity by valorizing bread waste and cheese whey by-products, in a circular economy perspective

Studio della diversità e funzionalità di lieviti e batteri lattici isolati da impasti acidi all’intero della Unimore Microbial Collection

Negli ultimi anni, la crescente richiesta da parte dei consumatori di cibi definiti “clean label” e salutari ha influenzato le industrie alimentari e le biotecnologie verso lo sviluppo di colture starter in grado di soddisfare la richiesta di nuovi prodotti. In questo contesto, la riscoperta dei prodotti fermentati, ed in particolare del lievito madre, ha portato alla ricerca di lieviti e batteri lattici con spiccate abilità tecnologiche per la creazione di colture starter innovative. In particolare, questo progetto di dottorato si è concentrato sugli effetti benefici del lievito madre, sul suo pool microbico di lieviti e batteri lattici (LAB) e sulla produzione dei vari metaboliti secondari, con ruolo sia tecnologico che funzionale. Nello specifico, sono stati condotti diversi studi sulla capacità di bio-preservazione dei LAB e la loro abilità di produrre composti ad impatto tecnologico come aromi ed esopolisaccaridi. La prima parte del progetto si è focalizzata sull’ottenimento di ceppi di lieviti e LAB caratterizzati da spiccate abilità tecnologiche e aromatiche, isolati da paste acide per la produzione di Panettoni artigianali e dai relativi impasti. Le varie specie ottenute dai vari isolamenti sono attualmente depositate nella Unimore Microbial Culture Collection (UMCC, www.umcc.unimore.it). Sia i lieviti che i batteri lattici ottenuti risultano tipici colonizzatori degli impasti acidi e sono rappresentati da: Saccharomyces cerevisiae, Kazachstania humilis, Fructilactobacillus sanfranciscensis Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, e Leuconostoc mesenteroides. Nello specifico, sono stati condotti studi di correlazione tra il volatiloma e i vari microrganismi, e successivamente la capacità di produzione di esopolisaccaridi da vari LAB è stata investigata. Questi ultimi infatti, rappresentano un potenziale prodotto microbico per il miglioramento tecnologico e nutrizionale dei prodotti da forno senza glutine (GF), normalmente caratterizzati da assenza di gusto e scarse qualità nutrizionali. Diversi ceppi appartenenti alle specie Lacp. plantarum e P. pentosaceus, isolati da una pasta madre GF ottenuta da farina di riso, hanno mostrato la capacità di produzione di simil-destrani. Inoltre, i LAB ritenuti maggiormente significativi per le per produzione aromatica e/o esopolisaccaridica, sono stati utilizzati in studi per il controllo/eliminazione di egenti deterioranti sia batterici (Escherichia coli, Campylobacter jejuni, Salmonella typhimurium e Listeria monocytogenes) che fungini (Fusarium graminearum, Aspergillus flavus, Penicillium paneum e Aspergillus niger). I risultati ottenuti hanno mostrato una capacità di inibizione ceppo-specifica e, i ceppi L. plantarum UMCC 2996, F. rossiae UMCC 3002, e P. pentosaceus UMCC 3010, ritenuti migliori sono stati impiegati nella realizzazione di coating naturale ad azione bio-preservante e un terreno ottenuto da scarti di pane e sieri di latte. Il terzo ed ultimo anno di dottorato, ulteriori analisi sui ceppi di LAB più promettenti della collezione UMCC e della collezione della Danmarks Tekniske Universitet sono state condotte, con lo scopo di identificare la presenza di eventuali batteriocine e/o composti antifungini. Pertanto, diversi esperimenti a carico di agenti deterioranti e/o patogeni specifici per i prodotti da forno, sono stati condotti. I risultati ottenuti hanno evidenziato la presenza di batteriocine di classe IIb/c e la possibile azione detossificante nei confronti dei metaboliti fungini. In conclusione i risultati ottenuti nel corso del dottorato hanno evidenziato la possibilità di una creazione di colture starter con spiccate abilità tecnologiche.Novel sourdough-based biotechnology applications have recently been raised to address customer demand for natural and healthful food as well as provide new inputs for the food industry. This PhD project has focused on the beneficial effects of sourdough yeasts and lactic acid bacteria (LAB) and the production of various secondary metabolites, with both technological and functional role. Additionally, investigation on the ability of sourdough LAB to produce antifungal and antibacterial compounds to be used as bio-preservatives have been made. The recovery of yeasts and LAB involved in sourdough fermentation was the first step of the project to select strains suitable as starters with optimal technological aptitude and capable of producing desired aromas and/or aromatic precursors. To this aim, yeasts and bacteria strains were isolated from two sourdoughs samples and their derived doughs used for Panettone production. They were identified and deposited in the Unimore Microbial Culture Collection (UMCC, www.umcc.unimore.it). The main species detected were Saccharomyces cerevisiae, Kazachstania humilis, Fructilactobacillus sanfranciscensis Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides. In addition, volatile organic compounds were identified and correlated to the strains. Additionally, the potential of LAB strains to produce exopolysaccharides (EPSs) was assessed. These compounds are gaining popularity due to their useful features and adaptability to a variety of food-related commercial applications such as gluten free (GF) products. EPS-producing LAB strains help to enhance the rheological and sensory qualities of GF products which are often characterized by an unpleasant flavour, and fat-richness. To detect LAB with the potential ability to produce EPSs, several strains were isolated from gluten-free rice sourdoughs. Among them, LAB strains belonging to the species L. plantarum and P. pediococcus were able to produce dextran-like EPS. Moreover, the most promising LAB strains were tested against common spoilage agents including bacteria (Escherichia coli, Campylobacter jejuni, Salmonella typhimurium and Listeria monocytogenes) and moulds (Fusarium graminearum, Aspergillus flavus, Penicillium paneum and Aspergillus niger). A strain-specific behavior against the moulds was detected and a wide activity against bacterial spoilage was proved. Subsequently, the best-chosen strains including L. plantarum UMCC 2996, F. rossiae UMCC 3002, and P. pentosaceus UMCC 3010, were used to design a novel strategy for the cultivation of selected starters with a bio-protection activity by valorizing bread waste and cheese whey by-products. During the third year of PhD, further investigation on the best performer LAB from the UMCC collection and other LAB from Danmarks Tekniske Universitet collection were done to detect specific bacteriocin and antifungal compounds. Different assays against specific spoilage bakery agents Bacillus spp. Lysinobacillus spp. and Paenibacillus spp. were performed showing bacteriostatic activity and genomic finding revealed the presence of functional operons of IIc/b class bacteriocins. Specific bakery moulds were also challenged and GC-MASS and HPLC-DAD were performed along with co-culture and detoxification assays. The obtained results highlighted the specific features of the various strains giving the possibility to built starter culture with specific features

Functional and Healthy Features of Conventional and Non-Conventional Sourdoughs

Sourdough is a composite ecosystem largely characterized by yeasts and lactic acid bacteria which are the main players in the fermentation process. The specific strains involved are influenced by several factors including the chemical and enzyme composition of the flour and the sourdough production technology. For many decades the scientific community has explored the microbiological, biochemical, technological and nutritional potential of sourdoughs. Traditionally, sourdoughs have been used to improve the organoleptic properties, texture, digestibility, palatability, and safety of bread and other kinds of baked products. Recently, novel sourdough-based biotechnological applications have been proposed to meet the demand of consumers for healthier and more natural food and offer new inputs for the food industry. Many researchers have focused on the beneficial effects of specific enzymatic activities or compounds, such as exopolysaccharides, with both technological and functional roles. Additionally, many studies have explored the ability of sourdough lactic acid bacteria to produce antifungal compounds for use as bio-preservatives. This review provides an overview of the fundamental features of sourdoughs and their exploitation to develop high value-added products with beneficial microorganisms and/or their metabolites, which can positively impact human health

Yeasts and Lactic Acid Bacteria for Panettone Production: An Assessment of Candidate Strains

The recovery of yeasts and lactic acid bacteria (LAB) involved in sourdough fermentation is the first step in the selection of starters with suitable technological aptitude and capable of producing desired aromas and/or aromatic precursors. In this work, two sourdoughs samples (MA and MB) and the derived doughs (samples A and B) were collected from a bakery during artisanal Panettone manufacture. Yeasts and bacteria were isolated at different fermentation steps on selective agar media. A total of 77 isolates were obtained and characterized. Representative strains of yeasts and LAB were identified by sequencing the D1/D2 domain of the 26S rRNA and the 16S rRNA genes, respectively. Moreover, the volatile organic compounds (VOCs) produced in the collected samples were detected and correlated to the species found in the same samples. The results highlighted the occurrence of Kazachstania humilis in both samples A and B, while Saccharomyces cerevisiae strains were detected only in samples B. Among LAB, Fructilactobacillus sanfranciscensis was the main species detected in both sourdoughs. Furthermore, strains belonging to the species Lactiplantibacillus plantarum, Furfurilactobacillus rossiae, Lactobacillus parabuchneri, Leuconostoc citreum, and Leuconostoc mesenteroides were assessed in the dough samples