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MARCELLO BRUGNOLI

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Dipartimento di Scienze della Vita

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Pubblicazioni

2024 - Oxidative fermentation of glucose and ethanol in designed media and cooked grape must by acetic acid bacteria [Articolo su rivista]
Brugnoli, M.; Cantadori, E.; Arena, M. P.; Gullo, M.
abstract

In this study, acetic acid bacteria strains were investigated for their ability to oxidize different carbon sources producing the corresponding oxidative products. Bacterial strains were cultivated in seven designed media and their acetification ability was assessed. The most performing strains were further tested to evaluate gluconic acid production in cooked grape must. Organic acids, sugars, and ethanol concentrations were assayed by high-pressure liquid chromatography. Overall, the findings showed high variability amongst strains of the same species, especially amongst Gluconobacter oxydans strains. However, strains ATCC 621H and DSM 3503T resulted to be the highest gluconic acid producers in all tested conditions. This study shows that grape must can be further valorized by selective fermentations for the production of gluconic based products.

2023 - A Microbial Co-Culturing System for Producing Cellulose-Hyaluronic Acid Composites [Articolo su rivista]
Brugnoli, M.; Mazzini, I.; La China, S.; De Vero, L.; Gullo, M.
abstract

In this study, a co-culture system combining bacterial cellulose (BC) producers and hyaluronic acid (HA) producers was developed for four different combinations. AAB of the genus Komagataeibacter sp. and LAB of the Lactocaseibacillus genus were used to produce BC and HA, respectively. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate changes in BC-HA composites chemical and morphological structure. Water absorption, uptake, and antibacterial properties were also tested. Outcomes highlighted a higher bacterial cellulose yield and the incorporation of hyaluronic acid into the composite. The presence of hyaluronic acid increased fiber dimension-nearly doubled for some combinations-which led to a decreased crystallinity of the composites. Different results were observed based on the BC producer and HA producer combination. However, water holding capacity (WHC) in all the samples improved with the presence of HA, while water uptake worsened. A thymol-enriched BC-HA composite showed high antibacterial activity against Escherichia coli DSM 30083(T) and Staphylococcus aureus DSM 20231(T). Results could contribute to opening new applications in the cosmetics or pharmaceutical fields.

2023 - Acetic acid bacteria in agro-wastes: from cheese whey and olive mill wastewater to cellulose [Articolo su rivista]
Brugnoli, Marcello; LA CHINA, Salvatore; Lasagni, Federico; Valeria Romeo, Flora; Pulvirenti, Andrea; Gullo, Maria
abstract

In this study, cheese whey and olive mill wastewater were investigated as potential feedstocks for producing bacterial cellulose by using acetic acid bacteria strains. Organic acids and phenolic compounds composition were assayed by high-pressure liquid chromatography. Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction were used to investigate modifications in bacterial cellulose chemical and morphological structure. Cheese whey was the most efficient feedstock in terms of bacterial cellulose yield (0.300 g of bacterial cellulose/gram of carbon source consumed). Bacterial cellulose produced in olive mill wastewater presented a more well-defined network compared to pellicles produced in cheese whey, resulting in a smaller fiber diameter in most cases. The analysis of bacterial cellulose chemical structure highlighted the presence of different chemical bonds likely to be caused by the adsorption of olive mill wastewater and cheese whey components. The crystallinity ranged from 45.72 to 80.82%. The acetic acid bacteria strains used in this study were characterized by 16S rRNA gene sequencing, allowing to assign them to Komagataeibacter xylinus and Komagataeibacter rhaeticus species. This study proves the suitability to perform sustainable bioprocesses for producing bacterial cellulose, combining the valorisation of agro-wastes with microbial conversions carried out by acetic acid bacteria. The high versatility in terms of yield, morphology, and fiber diameters obtained in cheese whey and olive mill wastewater contribute to set up fundamental criteria for developing customized bioprocesses depending on the final use of the bacterial cellulose.

2023 - Zero- and Low-Alcohol Fermented Beverages: A Perspective for Non-Conventional Healthy and Sustainable Production from Red Fruits [Articolo su rivista]
Brugnoli, Marcello; Cantadori, Elsa; Arena, Mattia Pia; DE VERO, Luciana; Colonello, Andrea; Gullo, Maria
abstract

The growing health consciousness among consumers is leading to an increased presence of functional foods and beverages on the market. Red fruits are rich in bioactive compounds such as anthocyanins with high antioxidant activity. In addition, red fruits contain sugars and are rich in phenolic compounds, vitamin C, dietary fibers, and manganese. Due to these characteristics, they are also suitable substrates for fermentation. Indeed, nowadays, microbial transformation of red fruits is based on alcoholic or lactic fermentation, producing alcoholic and non-alcoholic products, respectively. Although products fermented by acetic acid bacteria (AAB) have been thoroughly studied as a model of health benefits for human beings, little evidence is available on the acetic and gluconic fermentation of red fruits for obtaining functional products. Accordingly, this review aims to explore the potential of different red fruits, namely blackberry, raspberry, and blackcurrant, as raw materials for fermentation processes aimed at producing low- and no-alcohol beverages containing bioactive compounds and no added sugars. AAB are treated with a focus on their ability to produce acetic acid, gluconic acid, and bacterial cellulose, which are compounds of interest for developing fruit-based fermented beverages.

2022 - Better under stress: Improving bacterial cellulose production by Komagataeibacter xylinus K2G30 (UMCC 2756) using adaptive laboratory evolution [Articolo su rivista]
Anguluri, K.; La China, S.; Brugnoli, M.; Cassanelli, S.; Gullo, M.
abstract

Among naturally produced polymers, bacterial cellulose is receiving enormous attention due to remarkable properties, making it suitable for a wide range of industrial applications. However, the low yield, the instability of microbial strains and the limited knowledge of the mechanisms regulating the metabolism of producer strains, limit the large-scale production of bacterial cellulose. In this study, Komagataeibacter xylinus K2G30 was adapted in mannitol based medium, a carbon source that is also available in agri-food wastes. K. xylinus K2G30 was continuously cultured by replacing glucose with mannitol (2% w/v) for 210 days. After a starting lag-phase, in which no changes were observed in the utilization of mannitol and in bacterial cellulose production (cycles 1-25), a constant improvement of the phenotypic performances was observed from cycle 26 to cycle 30, accompanied by an increase in mannitol consumption. At cycle 30, the end-point of the experiment, bacterial cellulose yield increased by 38% in comparision compared to cycle 1. Furthermore, considering the mannitol metabolic pathway, D-fructose is an intermediate in the bioconversion of mannitol to glucose. Based on this consideration, K. xylinus K2G30 was tested in fructose-based medium, obtaining the same trend of bacterial cellulose production observed in mannitol medium. The adaptive laboratory evolution approach used in this study was suitable for the phenotypic improvement of K. xylinus K2G30 in bacterial cellulose production. Metabolic versatility of the strain was confirmed by the increase in bacterial cellulose production from D-fructose-based medium. Moreover, the adaptation on mannitol did not occur at the expense of glucose, confirming the versatility of K2G30 in producing bacterial cellulose from different carbon sources. Results of this study contribute to the knowledge for designing new strategies, as an alternative to the genetic engineering approach, for bacterial cellulose production.

2022 - Candidate Acetic Acid Bacteria Strains for Levan Production [Articolo su rivista]
Anguluri, Kavitha; LA CHINA, Salvatore; Brugnoli, Marcello; DE VERO, Luciana; Pulvirenti, Andrea; Cassanelli, Stefano; Gullo, Maria
abstract

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099 T, Kozakia baliensis DSM 14400 T and Gluconobacter cerinus DSM 9533 T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099 T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400 T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

2022 - Date Fruits as Raw Material for Vinegar and Non-Alcoholic Fermented Beverages [Articolo su rivista]
Cantadori, Elsa; Brugnoli, Marcello; Centola, Marina; Uffredi, Erik; Colonnello, Andrea; Gullo, Maria
abstract

Nowadays foods and beverages with healthy and functional properties, especially those claimed to prevent chronic diseases, are obtaining more and more interest. As a result, numerous foods and beverages have been launched on the market. Among products with enhanced properties, vinegar and fermented beverages, have high potential of growth. Date palm fruits are a versatile raw material, rich in sugars, dietary fibers, minerals, vitamins, and phenolic compounds, thus they are widely used for food production, including date juice, jelly, butter, and fermented bever-ages, such as wine and vinegar. Moreover, their composition makes them suitable for the formu-lation of functional foods and beverages. Microbial transformations of date juice include alco-holic fermentation for producing wine as end product or as a substrate for acetic fermentation. Lactic fermentation is also documented for transforming dates juice and syrup. However, con-sidering acetic acid bacteria, little evidence is available on the exploitation of date juice by acetic and gluconic fermentation for producing beverages. This review presents an overview of date fruit’s composition, the related health benefits for hu-man health, vinegar and date-based fermented non-alcoholic beverages obtained by acetic acid bacteria fermentation.

2021 - Assessing effectiveness of Komagataeibacter strains for producing surface-microstructured cellulose via guided assembly-based biolithography. [Articolo su rivista]
Brugnoli, M.; Robotti, F.; China, La; Anguluri, K.; Haghighi, H.; Bottan, S.; Ferrari, A.; Gullo, M.
abstract

In this study, a medical device made of surface microstructured bacterial cellulose was produced using cellulose‑producing acetic acid bacteria wild‑type strains in combination with guided assembly‑based biolithography. The medical device aims at interfering with the cell’s focal adhesion establishment and maturation around implantable devices placed in soft tissues by the symmetrical array on its surface. A total of 25 Komagataeibacter strains was evaluated over a three‑step selection. In the first step, the ability of strains to produce a suitable bacterial cellulose layer with high production yield was examined, then nine strains, with a uniform and smooth layer of bacterial cellulose, were cultured in a custom‑made silicone bioreactor and finally the characteristics of the symmetrical array of topographic features on the surface were analysed. Selected strains showed high inter and intra species variability in bacterial cellulose production. The devices obtained by K2G30, K1G4, DSM 46590 (Komagataeibacter xylinus), K2A8 (Komagataeibacter sp.) and DSM 15973T (Komagataeibacter sucrofermentas) strains were pouched‑formed with hexagonal surface pattern required for reducing the formation of fibrotic tissue around devices, once they are implanted in soft tissues. Our findings revealed the effectiveness of the selected Komagataeibacter wild‑type strains in producing surface microstructured bacterial cellulose pouches for making biomedical devices.

2021 - Kombucha Tea as a Reservoir of Cellulose Producing Bacteria: Assessing Diversity among Komagataeibacter Isolates [Articolo su rivista]
LA CHINA, Salvatore; DE VERO, Luciana; Anguluri, Kavitha; Brugnoli, Marcello; Mamlouk, Dhouha; Gullo, Maria
abstract

Bacterial cellulose (BC) is receiving a great deal of attention due to its unique properties such as high purity, water retention capacity, high mechanical strength, and biocompatibility. However, the production of BC has been limited because of the associated high costs and low productivity. In light of this, the isolation of new BC producing bacteria and the selection of highly productive strains has become a prominent issue. Kombucha tea is a fermented beverage in which the bacteria fraction of the microbial community is composed mostly of strains belonging to the genus Komagataeibacter. In this study, Kombucha tea production trials were performed starting from a previous batch, and bacterial isolation was conducted along cultivation time. From the whole microbial pool, 46 isolates were tested for their ability to produce BC. The obtained BC yield ranged from 0.59 g/L, for the isolate K2G36, to 23 g/L for K2G30—which used as the reference strain. The genetic intraspecific diversity of the 46 isolates was investigated using two repetitive-sequence-based PCR typing methods: the enterobacterial repetitive intergenic consensus (ERIC) elements and the (GTG)5 sequences, respectively. The results obtained using the two different approaches revealed the suitability of the fingerprint techniques, showing a discrimination power, calculated as the D index, of 0.94 for (GTG)5 rep-PCR and 0.95 for ERIC rep-PCR. In order to improve the sensitivity of the applied method, a combined model for the two genotyping experiments was performed, allowing for the ability to discriminate among strains.