Staphylococcal food poisoning outbreaks occurred in Sicily (Italy) from 2009 to 2016.

Notification of foodborne outbreaks has been mandatory in Europe since 2005, and surveillance is carried out along the entire food chain. Here we report the results obtained from laboratory investigations about four cases of foodborne outbreaks that occurred in Sicily between 2009 and 2016, deemed to be related to staphylococcal enterotoxins (SEs) and coagulase-positive Staphylococci (CPS) by the Local Public Health Authority. Primosale cheese samples were processed by culture methods for enumeration of CPS and immunoenzymatic assays for detection and differentiation of the SEs possibly contained in food samples. In all cases, the mistrusted foods were found to be contaminated by CPS at bacterial loads between 5 and 8 log CFU/g and contained SE type C (SEC). The reported data confirm the risk of staphylococcal food poisoning associated with the consumption of raw milk cheese. SEC is the most commonly occurring SE in goat milk and dairy products and the most represented enterotoxin in Sicilian dairy products. Our results highlighted the need for improving the current monitoring efficiency and implementing the available laboratory methods to collect more faithful epidemiological data on the current prevalence of staphylococcal toxins in the food chain, including SEs currently not detectable by validated analytical methods.

Reduction of PDO Pecorino Siciliano cheese making duration: Microbial dynamics and quality attributes deriving from replacing whey permeate with hot water during cooking.

This work was carried out with the aim to reduce the transformation duration of Protected Designation of Origin (PDO) Pecorino Siciliano cheese. To this purpose, the cooking in hot water (experimental production, EXP) was compared to the traditional cheese cooking under whey permeate (control production, CTR). The microbiological composition of under rind (UR) and core (Co) section of CTR and EXP cheeses was determined by a combined culture-dependent and -independent approach. Total mesophilic microorganisms and lactic acid bacteria (LAB) present in raw ewes' milk (5.0 log CFU/mL) increased during cheese making and reached values of about 8.0 log CFU/g in both sections (UR and Co) of 5-month ripened cheeses of both productions (CTR and EXP) monitored. The identification of the viable LAB populations in ripened cheeses showed that Enterococcus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Limosilactobacillus and Streptococcus dominated UR and Co sections of all cheeses. MiSeq Illumina analysis demonstrated that LAB populations (lactobacilli, lactococci and streptococci) dominated the bacterial community of cheeses at 95.63-98.41 % of relative abundance. The two different cooking operations did not influence the physicochemical characteristics of PDO Pecorino Siciliano cheeses. Sensory evaluation performed by artificial senses analysis and trained panelists confirmed that the modification of PDO Pecorino Siciliano cheese production protocol did not significantly affect product characteristics and overall acceptance. Thus, data of this work confirmed that cooking under hot water allowed to reduce transformation duration and safeguard typicality of PDO Pecorino Siciliano cheese.

Prevalence and antimicrobial resistance profiles of Salmonella spp. in poultry meat.

The spread of multidrug resistant (MDR) Salmonella strains, along the poultry supply chain, can represent a relevant threat to human health. This study aimed to evaluate the prevalence and antimicrobial resistance of Salmonella spp. isolated from poultry meat for human consumption. Between 2019 and 2021, 145 samples were analyzed according to ISO 6579-1:2017. The strains isolated were identified by using biochemical-enzymatic assays and serotyping, according to the Kauffmann-White-Le Minor scheme. The antibiotic susceptibility tests were determined using the Kirby-Bauer method. Forty Salmonella spp. strains were isolated and serotyping showed Salmonella Infantis to be predominant. 80% of the isolated strains were MDR and identified as S. Infantis. This study confirms the circulation of MDR Salmonella isolated from poultry meat and highlights the predominance of the S. Infantis serovar, which represents an emerging risk factor under the One Health holistic approach.

Food risk associated with vegetable consumption, exposure to antimicrobial-resistant strains and pesticide residues.

This preliminary study aimed to detect biological and chemical contaminants in vegetables sold in Sicily for human consumption, assess the spread of antimicrobial-resistant (AMR) strains in these foods, and characterize their antimicrobial-resistance genes. A total of 29 fresh and ready-to-eat samples were analyzed. Microbiological analyses were performed for the detection of Salmonella spp. and the enumeration of Enterococci, Enterobacteriaceae, and Escherichia coli. Antimicrobial resistance was assessed by the Kirby-Bauer method, according to the Clinical and Laboratory Standards Institute guidelines. Pesticides were detected by high-performance liquid chromatography and gas chromatography coupled with mass spectrometry. No samples were contaminated by Salmonella spp., E. coli was detected in 1 sample of fresh lettuce at a low bacterial count (2 log cfu/g). 17.24% of vegetables were contaminated by Enterococci and 65.5% by Enterobacteriaceae (bacterial counts between 1.56 log cfu/g and 5.93 log cfu/g and between 1.6 log cfu/g and 5.48 log cfu/g respectively). From 86.2% of vegetables, 53 AMR strains were isolated, and 10/53 isolates were multidrug resistant. Molecular analysis showed that the blaTEM gene was detected in 12/38 ß-lactam-resistant/intermediate-resistant isolates. Genes conferring tetracycline resistance (tetA, tetB, tetC, tetD, tetW) were detected in 7/10 isolates. The qnrS gene was detected in 1/5 quinolone-resistant isolates, the sulI gene was detected in 1/4 sulfonamide- resistant/intermediate-resistant isolates and the sulIII gene was never detected. Pesticides were detected in 27.3% of samples, all of which were leafy vegetables. Despite the satisfactory hygienic status of samples, the high percentage of AMR bacteria detected stresses the need for an effective monitoring of these foods as well as adequate strategies to counteract the spread of AMR bacteria along the agricultural chain. Also, the chemical contamination of vegetables should not be underestimated, especially considering that leafy vegetables are commonly consumed raw and that no official guidelines about maximum residue limits of pesticides in ready-to-eat vegetables are available.

Food Is Reservoir of MDR Salmonella: Prevalence of ESBLs Profiles and Resistance Genes in Strains Isolated from Food.

Salmonella spp. are among the most frequent causes of foodborne diseases, and the increasing occurrence of MDR strains is an additional cause for concern. In the three-year period 2019-2021, we collected Salmonella spp. strains isolated from different food categories analysed in the context of Regulation (EC) No 2073/2005 in order to assess their antibiotic susceptibility profiles and ESBL production. To determine the susceptibility profiles and identify MDR strains, we used the Kirby-Bauer method to test 17 antibiotics. Double-disc and PCR testing then allowed us to assess the production of ESBLs and the presence of beta-lactamase resistance genes. Phenotypic tests showed that 36 out of 67 strains were MDR and 52.7% of these were ESBL producers. Finally, molecular investigations conducted on ESBL-producing strains revealed the presence of blaSHV, blaCTX-M and blaTEM genes. Our results confirmed the prevalence of S. Infantis, an MDR strain and ESBL producer, in chicken meat. This suggests that further research on the prevalence of antibiotic resistance genes (ARGs) in foodborne strains is needed, especially from a One Health perspective.

Occurrence and Molecular Characterization of Potentially Pathogenic Vibrio spp. in Seafood Collected in Sicily.

Seafood can vehiculate foodborne illnesses from water to humans. Climate changes, increasing water contamination and coastlines anthropization, favor the global spread of Vibrio spp. and the occurrence of antibiotic-resistant isolates. The aim of this study was to evaluate the spread of potentially pathogenic Vibrio spp. in fishery products collected in Sicily and to assess their antibiotic resistance. Bacteriological and molecular methods were applied to 603 seafood samples to detect V. parahaemolyticus, V. cholerae, V. vulnificus, and Vibrio alginolyticus in order to assess their pathogenicity and antimicrobial resistance. About 30% of bivalves and 20% of other fishery products were contaminated by Vibrio spp.; V. parahaemolyticus accounted for 43/165 isolates, 3 of which were carrying either tdh or trh; V. cholerae accounted for 12/165 isolates, all of them non-O1 non-O139 and none carrying virulence genes; and V. vulnificus accounted for 5/165 isolates. The highest rates of resistance were observed for ampicillin, but we also detected strains resistant to antibiotics currently included among the most efficient against Vibrio spp. In spite of their current low incidence, their rise might pose further issues in treating infections; hence, these results stress the need for a continuous monitoring of antimicrobial resistance among fishery products and an effective risk assessment.

Occurrence and Antimicrobial Resistance of Arcobacter spp. Recovered from Aquatic Environments.

Arcobacter spp. are emerging waterborne and foodborne zoonotic pathogens responsible for gastroenteritis in humans. In this work, we evaluated the occurrence and the antimicrobial resistance profile of Arcobacter isolates recovered from different aquatic sources. Besides, we searched for Arcobacter spp. in seaweeds and the corresponding seawater samples. Bacteriological and molecular methods applied to 100 samples led to the isolation of 28 Arcobacter isolates from 27 samples. The highest prevalence was detected in rivers followed by artificial ponds, streams, well waters, and spring waters. Seaweeds contained a higher percentage of Arcobacter than the corresponding seawater samples. The isolates were identified as Arcobacter butzleri (96.4%) and Arcobacter cryaerophilus (3.6%). All the isolates showed a multi-drug resistance profile, being resistant to at least three different classes of antibiotics. Molecular analysis of genetic determinants responsible for tetracycline resistance in nine randomly chosen isolates revealed the presence of tetO and/or tetW. This work confirms the occurrence and the continuous emergence of antibiotic-resistant Arcobacter strains in environmental samples; also, the presence of quinolone-resistant Arcobacter spp. in aquatic sources used for water supply and irrigation represents a potential risk for human health.

Assessment of contamination of Salmonella spp. in imported black pepper and sesame seed and salmonella inactivation by gamma irradiation.

This study shows the frequency of seeds samples contaminated by Salmonella spp. collected randomly from local markets; on 30 black pepper sample no contaminated sample was found while Salmonella spp. was detected in 3 of 36 (8.3%) analyzed sesame samples; three different serotypes were identified: S. Montevideo, S. Stanleyville e S. Tilene. The efficacy of gamma irradiation to inactivate Salmonella Montevideo in black pepper and sesame irradiated between 1 and 5 kGy was evaluated. 3 kGy is sufficient to reduce of 3-4 log CFU/g; whereas 5 kGy have been need to reduce 5.5-6 log CFU/g for samples of black pepper and sesame. No statistically significant differences were found between black pepper and sesame.

Prickly Pear By-Product in the Feeding of Livestock Ruminants: Preliminary Investigation.

In Sicily, the current increasing cultivation of Opuntia ficus-indica corresponds to an availability of prickly pear by-product (PPB) that results from fruit processing for juice extraction. This investigation aims to evaluate the nutritional traits of PPB for ruminant feeding and its stability during a 21-day outdoor storage, using potassium metabisulfite (PMB) as a preservative agent, added to the PPB mass at different doses (0, 50, 100, and 150 g/kg). The fractioning of PPB showed that it included 28% of peel and pulp and 72% of seeds on a dry matter (DM) basis. On the whole, this by-product was low in crude protein (5.32% DM), high in fiber content (51.38%, 41.15% and 14.64% DM for NDFom, ADFom and ADL respectively), non-fiber carbohydrates (NFC, 29.68% DM), and soluble sugars (13.3% DM), with a moderate level of net energy for lactation (4.59 MJ/kg DM). Storage was the main factor of alteration of PPB chemical composition with the exception of ether extract. A decline of NFC and soluble sugars, due to microbial fermentation, was observed with all PMB treatments, especially during the first week of storage, probably due to evolution of both coccus (M17) and rod LAB (MRS), which increased their loads at the seventh day of storage.

Transformation of raw ewes' milk applying "Grana" type pressed cheese technology: Development of extra-hard "Gran Ovino" cheese.

This work was carried out to pursue a double objective: to improve the hygienic safety of cheeses produced from raw ewes' milk; and to produce a new typology of raw ewes' milk through the application of "Grana" technology for which the name "Gran Ovino" was chosen. With this in mind, raw milk from an individual farm was transformed under controlled conditions at a dairy pilot plant. The production technology included the partial skimming of the evening and morning milk mixture by cream surfacing and the addition of a natural whey starter cultures (NWSC) prepared with four selected Streptococcus thermophilus strains (PON6, PON244, PON261 e PON413). Ten microbial groups were investigated by plate counts from raw milk until ripened cheeses. Lactic acid bacteria (LAB) were in the range 104-105 CFU/ml before NWSC addition. After curdling, this group increased by 3 log cycles and was counted at 106 CFU/g after curd cooking. A rapid pH drop (to 6.05) was registered after almost 3 h from NWSC addition. The levels of members of the Enterobacteriaceae family were at about 103 CFU/ml in raw milk and decreased after curd cooking to 1 log cycle. A similar behavior was shown by the other undesired microbial groups and a complete disappearance of staphylococci was registered. The microbiological counts of 9-month ripened cheeses showed the dominance of LAB and undetectable levels of the undesired bacteria. MiSeq Illumina was applied to better investigate the bacterial composition of ripened cheeses and this technique evidenced that the majority of OTUs belonged to Lactobacillus and Streptococcus genera. The final cheeses were characterized by 67.65% dry matter of which 41.85% of fats and 47.02% of proteins. The main cheese fatty acids were palmitic, oleic and myristic acids and the saturated fatty acids/unsaturated fatty acids ratio was 2.17. Forty-one volatile compounds, including acids, esters, ketones, alcohols, aldehydes, phenols and one terpene were emitted from the cheese. Sensory evaluation showed a general appreciation for the new cheese product by judges.

Influence of the early bacterial biofilms developed on vats made with seven wood types on PDO Vastedda della valle del Belìce cheese characteristics.

Early vat bacterial biofilms developed spontaneously through contact with whey have been characterized on seven wood types (Castanea sativa Miller, Cedrus libani, A. Rich., Prunus avium L., Fraxinus ornus L., Juglans regia L., Pinus nigra J.F. Arnold and Populus nigra L.). The present study aimed to evaluate the influence of these biofilms on the microbiological, chemical, physical and sensory characteristics of PDO Vastedda della valle del Belìce (VdB) cheese, processed traditionally from raw ewe's milk using wooden tools. To this purpose, the experimental cheeses after 15 d of refrigerated storage were examined. Lactic acid bacteria (LAB) populations dominated the microbial community of all samples. The species more frequently identified were Lactococcus lactis among starter LAB and Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus fermentum and Pediococcus pentosaceus among non starter LAB. Culture-independent analysis of microbiota diversity was performed by MiSeq Illumina that identified Streptococcus as major group followed by members of Enterobacteriaceae family, Lactococcus and Lactobacillus. Generally, the seven tree species did not negatively affect the physicochemical composition of VdB cheeses. Chestnut (both Sicilian and Calabrian) vats produced cheeses with significant lower hue angle (a*/b*) than other wood types. Among chemical parameters, significant variations were registered for aw, primary and secondary lipid oxidation state (significantly lower for the VdB cheeses produced with poplar wood), and volatile organic compounds (VOCs). The significant differences detected among the VOCs emitted from cheeses were not perceived by the panelists who recognized all cheeses from the different trials as similar. This study confirmed the suitability of cedar, cherry, ash, walnut, black pine and poplar as alternative woods to chestnut for the production of the wooden vats employed in cheese making for the Sicilian traditional dairy productions.

Extra-Intestinal Fluoroquinolone-Resistant Escherichia coli Strains Isolated from Meat.

Extra-intestinal E. coli are emerging as a global threat due to their diffusion as opportunistic pathogens and, above all, to their wide set of antibiotic resistance determinants. There are still many gaps in our knowledge of their origin and spread pathways, although food animals have been adjudicated vehicles for passing mult-drug resistant bacteria to humans. This study analyzed 46 samples of meat purchased from retail stores in Palermo in order to obtain quinolone-resistant E. coli isolates. Strains were screened for their phylogenetic groups, ST131-associated single nucleotide polymorphisms (SNPs), and then typed by ERIC-PCR. Their set of virulence factors, namely, kpsMII, papA, sfaS, focG, iutA, papC, hlyD, and afa genes, were investigated and their fluoroquinolone-resistance determinants evaluated. The data obtained show a dramatically high prevalence of multidrug resistance patterns in the Palermo area, with 28% of the isolates having virulence factor genes typical of ExPEC strains. No B2 group or ST131 strains were detected. Moreover, 20% of our isolates showed positivity to all the plasmid-mediated quinolone resistance (PMQR) determinants, showing a potential to transfer these genes among other bacteria. Therefore, these data underline the possibility that food animals and, specifically, poultry in particular may be a significant source of resistant bacterial strains, posing a potential zoonotic risk.

Detection of Arcobacter spp. in food products collected from Sicilia region: A preliminary study.

The aim of the study was to evaluate the occurrence of Arcobacter spp. in food samples collected from Sicilia region. A total of 91 food products of animal origin (41 meat, 17 fresh milk, 18 shellfish) and 15 samples of fresh vegetables, were examined by cultural method and confirmed by biochemical analysis and PCR methods. The detection of Arcobacter spp. was performed, after selective enrichment, on two selective agar plates: Arcobacter agar and mCCD (modified charcoal cefoperazone deoxycholate) agar supplemented with CAT (Cefoperazone, Amphotericin B and Teicoplanin). Arcobacter species were isolated using the membrane filtration technique. In 13 (14.3%) out of the 91 tested samples, the presence of Arcobacter spp. was found: the isolates were confirmed by multiplex PCR and identified as belonging to the species A. butzleri and A. cryaerophilus. The highest prevalence rate was observed in chicken meat (8.8%) followed by shellfish (3.3%). Negative results have been obtained for raw milks and vegetables samples. The preliminary study highlights the importance of this emerging pathogen and the need for further studies on its prevalence and distribution in different types of food for human consumption.

Retrospective study on the hygienic quality of fresh ricotta cheeses produced in Sicily, Italy.

The present work was carried out to investigate the microbiological profile of Sicilian ewes' ricotta cheeses during fifteen years of investigations (2002-2016). The samples were collected between those conferred to the Istituto Zooprofilattico Sperimentale della Sicilia (IZSSi) Adelmo Mirri, Palermo (Italy), by the competent authority during official control, by food business operator in HACCP systems and in research projects. Enterobacteriaceae, Escherichia coli and coagulase-positive staphylococci (CPS) were found only in some samples. Bacillus cereus was detected in c.a. 16% of samples but the level of contaminations did not reach the threshold that leads to significant toxin production. Pathogenic bacteria such as Listeria monocytogenes, Salmonella spp. and Brucella spp. were never detected. Furthermore, a total of 47 of lactic acid bacteria (LAB) strains were identified at species level by sequencing the 16S rRNA gene, resulting in the identification of 10 species belonging to four genera Enterococcus, Lactobacillus, Lactococcus and Leuconostoc, commonly employed as starter and non starter cultures in different traditional cheese. Results of this study highlighted an improvement of the hygienic conditions of the Sicilian ewes' ricotta cheeses during the last ten years of investigation. This observation was confirmed from reduction of undesired microorganisms such as Enterobacteriaceae, E.coli and CPS, used to define the process hygiene criteria. However, in order to improve the final quality of this product are needed further strategy such as the dairy makers training, with the aim to apply a good hygienic practices during the production.

Formation and Characterization of Early Bacterial Biofilms on Different Wood Typologies Applied in Dairy Production.

The main hypothesis of this work was that Sicilian forestry resources are suitable for the production of equipment to be used in cheese making and indigenous milk lactic acid bacteria (LAB) are able to develop stable biofilms providing starter and nonstarter cultures necessary for curd fermentation and cheese ripening, respectively. Hence, the present work was carried out with deproteinized whey to evaluate LAB biofilm formation on different woods derived from tree species grown in Sicily. Microbiological and scanning electron microscopy analyses showed minimal differences in microbial levels and compositions for the neoformed biofilms. The specific investigation of Salmonella spp., Listeria monocytogenes, Escherichia coli, coagulase-positive staphylococci (CPS), and sulfite-reducing anaerobes did not generate any colony for all vats before and after bacterial adhesion. LAB populations dominated all vat surfaces. The highest levels (7.63 log CFU/cm2) were registered for thermophilic cocci. Different colonies were characterized physiologically, biochemically, and genetically (at strain and species levels). Six species within the genera Enterococcus, Lactobacillus, Lactococcus, and Streptococcus were identified. The species most frequently present were Lactobacillus fermentum and Lactococcus lactis LAB found on the surfaces of the wooden vats in this study showed interesting characteristics important for dairy manufacture. To thoroughly investigate the safety of the wooden vat, a test of artificial contamination on new Calabrian chestnut (control wood) vats was carried out. The results showed that LAB represent efficient barriers to the adhesion of the main dairy pathogens, probably due to their acidity and bacteriocin generation.IMPORTANCE This study highlights the importance of using wooden vats for traditional cheese production and provides evidence for the valorization of the Sicilian forest wood resources via the production of dairy equipment.

Anti-Listeria Activity of Lactic Acid Bacteria in Two Traditional Sicilian Cheeses.

Listeria monocytogenes is a pathogen frequently found in dairy products, and its growth is difficult to control. Bacteriocin-like inhibitory substances (BLIS), produced by lactic acid bacteria (LAB), having proven in vitro anti-Listeria activity, could provide an innovative approach to control L. monocytogenes; however, this application needs to be evaluated in vivo. In this study, twenty LAB strains isolated from different Sicilian dairy environments were tested for control of growth of L. monocytogenes in three different experimental trials. First, raw and UHT milk were inoculated with LAB strains alone, and LAB strains mixed with L. monocytogenes. Second, mini-cheeses containing LAB and/or L. monocytogenes were produced. Third, two traditional Sicilian cheeses inoculated with a multi-strain LAB mixture combined with L. monocytogenes were produced. The addition of BLIS produced by LAB to milk and in mini-cheese production was unable to inhibit the growth of L. monocytogenes. However, an anti-Listeria effect was observed in the Pecorino Siciliano cheeses, where, after 15 days of ripening, the cheeses with added LAB had fewer L. monocytogenes compared to the control cheeses with no added LAB, while in the Vastedda della valle del Belìce cheeses, the multi-strain LAB mixture completely prevented the growth of L. monocytogenes.

Listeria monocytogenes in Smoked Salmon and Other Smoked Fish at Retail in Italy: Frequency of Contamination and Strain Characterization in Products from Different Manufacturers.

Seven hundred seventy-eight samples of packaged smoked fish (774 smoked salmon and 4 smoked swordfish) on sale in Italy, from 50 different manufacturers located in 12 European Union countries, were purchased from the Italian market between May and December 2011. The surface temperatures of the samples on sale ranged from 0 to 13°C (3.4 ± 1.5°C, mean ± SD). Six hundred eighty (87.4%) of 778 samples were stored at ≤4°C. One hundred fifty-seven samples (20.2%, 95% confidence interval 17.5 to 23.1%) were contaminated by Listeria monocytogenes , with 26 samples (3.3%, 95% confidence interval 2.3 to 4.8%) at levels >100 CFU/g. The maximum level of contamination was 1.3 ×106 CFU/g. The differences in the level of contamination of smoked fish between countries (χ2 = 91.54, P < 0.05) and manufacturers (χ2 = 193.22, P < 0.05) were significant. The frequency of detection for products from different manufacturing premises ranged from 0 to 76.9%. Serotyping by serological agglutination revealed that the main serotypes detected were 1/2a (65.3%) and 1/2b (22.4%). Pulsed-field gel electrophoresis typing with restriction enzymes AscI and ApaI yielded 36 pulsotypes from 144 isolates, clustering into 17 groups. Eight main pulsotypes accounted for 70.8% of the isolates. Three of the main pulsotypes were exclusively from products of a single manufacturer. In general, products from the same manufacturer showed genetic homogeneity, with one strongly prevalent pulsotype. Different manufacturers usually showed very different levels of contamination of the final product, confirming the importance of the management of process hygiene for controlling L. monocytogenes contamination.

Valorization of indigenous dairy cattle breed through salami production.

The aim of the research was to produce salami manufactured with meat of three different commercial categories of bovine breed: cow on retirement, beef and young bull. A total of six experimental productions, at small-scale plant, were carried out with and without starter culture inoculums. The evolution of physico-chemical parameters in all trials followed the trend already registered for other fermented meat products. Several LAB species were found during process with different levels of species diversity and frequency of isolation among inoculated (mainly Pediococcus pentosaceus and Staphylococcus xylosus) and uninoculated (mainly Enterococcus devriesei, Lactobacillus curvatus and Lactobacillus sakei) trials. Enterobacteriaceae were found at very low levels during the entire ripening period and no pathogenic bacteria were found in any samples. The multivariate analysis showed that starter inoculums and meat affected significantly the physico-chemical and the microbiological composition of salami. The sensory analysis evidenced the highest overall acceptability was displayed by salami produced with meat from cow on retirement.

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms.

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.