Andrea Osimani

Bacteria and yeast microbiota in milk kefir grains from different Italian regions

Kefir grains are a unique symbiotic association of different microrganisms, mainly lactic acid bacteria, yeasts and occasionally acetic acid bacteria, cohabiting in a natural polysaccharide and a protein matrix. The microbial composition of kefir grains can be considered as extremely variable since it is strongly influenced by the geographical origin of the grains and by the sub-culturing method used. The aim of this study was to elucidate the bacteria and yeast species occurring in milk kefir grains collected in some Italian regions by combining the results of scanning electron microscopy analysis, viable counts on selective culture media, PCR-DGGE and pyrosequencing. The main bacterial species found was Lactobacillus kefiranofaciens while Dekkera anomala was the predominant yeast. The presence of sub-dominant species ascribed to Streptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted. In addition, Lc. lactis, Enterococcus sp., Bacillus sp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community. This work further confirms both the importance of combining culture-independent and culture-dependent approaches to study microbial diversity in food and how the combination of multiple 16S rRNA gene targets strengthens taxonomic identification using sequence-based identification approaches.

Investigation of the microbial ecology of Ciauscolo, a traditional Italian salami, by culture-dependent techniques and PCR-DGGE

The microbial ecology of 22 samples of commercially available Ciauscolo salami were investigated using a polyphasic approach, based on culture-dependent and -independent techniques. The viable counts of pathogen and hygiene indicator microorganisms highlighted the adequate application of good manufacturing practices, while the viable counts of the lactic acid bacteria, coagulase negative cocci, and yeasts showed dominance of the first of these microbial groups. Bacterial and fungal DNA were extracted directly from the salami and amplified by PCR, using two primer sets targeting the 16S and 28S rRNA genes, respectively. Denaturing gradient gel electrophoresis (DGGE) and sequencing of selected bands were used to investigate the microbial ecology of these Ciauscolo salami. The most frequently found bacterial species were Lactobacillus sakei and Lb. curvatus, while Debaryomyces hansenii was the prevalent yeast species detected. Cluster analysis of the DGGE profiles and calculation of biodiversity indices allowed the degree of microbial similarity across these salami to be determined.

Unpasteurised commercial boza as a source of microbial diversity

Boza is a cereal-based fermented beverage widely consumed in many countries of the Balkans. The aim of this study was to investigate the microbiota of three Bulgarian boza samples through a combination of culture-dependent and -independent methods with the long-term objective of formulating a multi-strain starter culture specifically destined for the manufacture of new cereal-based drinks. The isolation campaign for lactic acid bacteria (LAB) allowed the identification of Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus coryniformis, Lactobacillus buchneri, Pediococcus parvulus and members of the Lactobacillus casei group. Concerning yeasts, the following isolates were identified: Pichia fermentans, Pichia norvegensis, Pichia guilliermondii (synonym Meyerozyma guilliermondii) and Torulaspora spp. A high intra-species diversity was revealed by Randomly Amplified Polymorphic DNA (RAPD) analysis. In parallel, microbial DNA was directly extracted from the three boza samples, and portions of the rrn operons were analysed through Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The molecular fingerprinting partially confirmed the results of culturing. Among LAB, the species Weissella confusa, Weissella oryzae, Leuconostoc citreum, Lactococcus lactis, Pediococcus parvulus and Pediococcus ethanolidurans were detected together with members of the Lb. casei group. Among the yeasts, the species P. fermentans, M. guilliermondii, Galactomyces geotrichum and Geotrichum fragrans were found. The overall results confirmed boza as having a rich and heterogeneous biodiversity both in terms of species and genetically diverse strains, thus encouraging its exploitation for the isolation and future technological characterisation of cultures to be selected for the manufacture of innovative cereal-based drinks.

An eight-year report on the implementation of HACCP in a university canteen: impact on the microbiological quality of meals

An investigation aimed at assessing the microbiological quality of meals consumed at a university canteen after implementation of the HACCP system and personnel training was carried out. Cooked and warm-served products (74 samples), cooked and cold-served products (92 samples) and cold gastronomy products (63 samples) sampled from 2000 to 2007 underwent microbiological analyses. All the samples were tested for: Samonella spp., Listeria monocytogenes, total mesophilic aerobes, coliforms, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and sulphite-reducing clostridia. The microbiological contamination of work surfaces (tables, tablewares, cutters, ladles, slicing machines, wash-basins, etc.), hands and white coats of members of the canteen staff was also assessed. The microbiological results clearly demonstrated the success of the HACCP plan implementation, through a general improvement of the hygiene conditions of both meals and work surfaces.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

Lactic acid bacteria and yeasts from wheat sourdoughs of the Marche Region

The need for a greater diversification of baked products has given rise to the on-going search for yeast and lactic acid bacteria (LAB) strains with optimal baking potential. Thirty-six yeasts and 118 LAB, isolated from nine type I sourdoughs that were sampled in bakeries located in the Marche region (central Italy), were molecularly and phenotypically characterized. The polyphasic approach used revealed the biodiversity of the microbial communities investigated and two yeasts and ten LAB cultures with the potential to be used in sourdough bread-making processes were identified.

Insight into the proximate composition and microbial diversity of edible insects marketed in the European Union

In recent years, the idea of exploiting edible insects for their industrial production has attracted the attention of media, research institutions and food industry operators, because of the numerous positive factors associated with this food source. Notwithstanding, insects are still underutilized in Western countries. Moreover, edible insects are carriers of natural microorganisms; hence, safety issues may arise from their industrial production. This study was aimed at providing insight into the proximate composition, with a focus in the fatty acid and amino acid composition, and microbial diversity of some processed edible insects marketed in the European Union. A high content of protein and fat was seen, with values ranging from 59.46 to 46.78 and 35.32 to 15.18%, respectively, with nutritionally valuable characteristics in both the lipid fractions and amino acid profiles. Furthermore, a great variation in microbial counts was seen. Both commensal and potential pathogenic microorganisms ascribed to the genera Pediococcus, Weissella, Streptomyces, Acinetobacter, Agrococcus, Arthrobacter, Naxibacter, Planomicrobium, Rufibacter, Bacillus, Clostridium, Vibrio, Desulfovibrio, Loktanella, Escherichia, Tetrapisispora, Aspergillus, Eurotium, Debaryomyces, and Wallemia, were identified by PCR-DGGE. The high diversity in the chemical composition and microbial profile of the marketed edible insects analyzed suggest a role of both the rearing and processing procedures on these variables. The results overall collected encourage the exploitation of edible insects as a valuable large-scale, animal-based commodity.

Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production.

Beer is one of the worlds most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile-filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture-dependent methods and PCR-DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR-DGGE technique coupled with the culture-dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by-products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.

Bacterial dynamics in a raw cow's milk Caciotta cheese manufactured with aqueous extract of Cynara cardunculus dried flowers.

Aims:  To investigate the bacterial dynamics of a Caciotta cheese traditionally manufactured in the Montefeltro area (Central Italy) with raw cow’s milk and an aqueous extract of dried flowers from Cynara cardunculus as a coagulating agent.