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BACKGROUND: Five probiotic lactobacilli were tested, alone or in combination with two commercial starters, to select the most suitable strain for a probiotic bovine salami production. Lactobacillus plantarum 299v was used with both starters, to make salami according to a traditional recipe. Salami obtained by using just the starters and by spontaneous fermentation, served as control. Microbial dynamics, as well as the main physico-chemical parameters, were monitored throughout ripening. The survival of probiotic 299v was confirmed by strains' tracking by means of RAPD-PCR coupled to a culture-independent approach PCR-DGGE-based. RESULTS: The results showed a remarkable viability of the probiotic strain even after 60 days of storage. Experimental salami exhibited the same level of sensory acceptance of control salami, were hygienically safe, and characterised by pH, weight loss and microbiological loads within the ranges conventionally advocated for optimal fermented sausages. CONCLUSION: Outcomes indicate the workable possibility of using second-quality beef cuts for probiotic salami production. © 2017 Society of Chemical Industry.

Staphylococcal enterotoxins, produced by Staphylococcus aureusand few other species of coagulase positive staphylococci (CPS), are the etiological agents of the staphylococcal food poisoning, one of the most widespread foodborne intoxications. In this book chapter, we will provide an overview on the staphylococcal food poisoning and its causative agents. We will describe the most used culture-dependent and -independent methods to quantitatively detect CPS, or even the most attractive for their future exploitation, providing the reader with new insights on how and when they can be exploited to ensure the identification, quantitative detection and typing of these important pathogenic bacteria. Moreover, classical and advanced methods to quantitatively detect staphylococcal enterotoxins will be provided. Finally, the most promising strategies to mitigate the relevant microbiological risk will be reviewed.

7.1. Introduction 7.2. Established culture media for Lactobacilli 7.2.1. Rogosa agar 7.2.2. MRS medium 7.2.3. Skim milk and whey agar 7.3. M17 medium for selection and enumeration of lactococci and streptococci 7.3.1. Streptococcus thermophilus agar (ST agar) 7.4. Selective media for lactobacilli 7.4.1. MRS vancomycin (MRS-V): vancomycin resistance in Lactobacilli and LAB species 7.4.2. Additional selective agents 7.4.3. MRSV + selective agents for Lb. casei group enumeration 7.4.4 MRS-salicin, MRS-sorbitol, MRS-ribose, MRS gluconate agar 7. 4.5. MRS-clindamycin-ciprofloxacin (MRS-CC) agar. 7.4.6. MMV medium and Lc agar for L. casei group enumeration 7.4.7. MRS containing fructose (MRSF) 7.4.8. mMRS-BPB 7.4.9. MRS-NNLP agar and chromogenic agars for complex communities 7.4.10. Homofermentative-heterofermentative differential (HHD) medium 7.5. Media for their isolation of Bifidobacteria 7.5.1. MRS-NNLP agar 7.5.2. BSM, WSP, TOS-MUP 7.5.3. MRS-ABC 7.6. Phenotyping 7.7. Conclusions

Aims: The positive influence of two selected extremely halophilic archaea strains in the production of salted anchovies (Engraulis encrasicolus, L., 1758) was highlighted. Methods and Results: Anchovies produced with salt artificially contaminated with halophiles exhibited lower loads of staphylococci, Enterobacteriaceae and lactic acid bacteria, and a reduced content of histamine as well as an improved organoleptic acceptance. Conclusions: The findings of this survey are expected to enhance the safety of salted anchovies, with regard to the histamine formation during ripening, and to improve the sensory attributes of this product. Significance and Impact of the Study: This study represents the first report on the positive influence of halophilic archaea in traditional salted anchovies production, thus suggesting new perspectives about a conscious employment of properly selected haloarchaea strains in this traditional manufacture

Lysozyme is a natural enzyme with muramidase activity which can work against a range of lactic acid bacteria (LAB), including Oenococcus, Pediococcus and Lactobacillus spp., that can affect wine stability (Cunningham et al., 1991). For such reason, over the last decade, there has been a growing interest in lysozyme as a supplement to sulphur dioxide for bacterial inhibition (Sonni et al., 2009). Since, lysozyme applications proved to be enough specific to require technical knowledge and expertise to be efficient, an experimental winemaking was performed to evaluate the efficiency of this enzyme application to produce a Sicilian wine from “organic grapes”. Specifically, two experimental conditions were considered by using SO2 or lysozyme, alternatively, according to a defined industrial winemaking scheme, with periodically samplings in order to: i) control organic acid content, ii) control unwanted LAB and iii) to prevent malolactic fermentation. Musts to be employed derived by grapes of “Catarratto”, an ancient white variety, whose wine presented susceptibility to oxidation phenomena. The evaluation during time of lactic microflora revealed an appreciable microbial reduction following to lysozyme addition, even if this drop took a longer time (about 48 h) if compared to the production with SO2 (about 2 h). In addition, in the wine added with lysozime the content of acetic acid, after 30 days of the end of alcoholic fermentation, was half of the value determined on the wine added with the SO2. Seventy-three LAB cultures isolated by different phases of both thesis were identified and characterized through molecular approach. Identification revealed the prevalence of Lactobacillus (Lb.) plantarum (about 60%) followed by the species Lb. mali, Lb. hilgardii, Lb. casei/paracasei/zeae and Leuconostoc mesenteroides subsp. mesenteroides. By evidences emerged from minimal inhibition concentration (MIC) to lysozyme, lactobacilli resistance appeared to be strain’s related; however, strains could be grouped into three classes according to their resistance behavior. Representative strains of these three classes, (Lactobacillus plantarum B30, Leuconostoc mesenteroides subsp. mesenteroides B1 and Lactobacillus mali B4p) were employed in a winemaking trial by using synthetic must. In detail, each strain was inoculated in must added of lysozyme (20 mg ml-1) singly or in combination with a commercial strain of Saccharomyces cerevisiae. Inoculated must was used as control. Lactic microflora was monitored for twenty days of fermentation, confirming evidences emerged by MIC determination. In all cases following to yeast addiction microbial loads appeared strongly reduced. In conclusion, results obtained in this study confirmed the relative lack of antimicrobial activity of lysozyme on Lactobacillus strains already recorded during preceding surveys (Delfini et al., 2004). Nevertheless, in the chosen challenging experimental conditions, lysozyme activity proved to be a potential suitable tool to control lactic bacteria populations and to manage the start of the malolactic fermentation only if combined with a highly fermentative yeast, as already pointed out by several authors (Ribéreau-Gayon et al., 1998). Since lysozyme is a protein that partially remains in the white treated wine, further investigations are needed to highlight possible secondary effects and understand how to properly use it in this Sicilian wine production.

Una lavorazione tipo “Sivigliano” di olive della varietà “Nocellara del Belice”, provenienti da coltura in irriguo e non, è stata sottoposta a monitoraggio chimico-fisico e microbiologico periodico sino al 150° giorno di fermentazione. La tecnologia adottata su tre repliche da 500 Kg ha previsto l’aggiunta di soda a 2,2°Bè a scopo di deamarizzare le olive in bagno alcalino e sei lavaggi sequenziali allo scopo di neutralizzare l’alcalinità, al termine dei quali si è provveduto alla colmatura con salamoia concentrata a 10°Bé. Il monitoraggio del pH e della concentrazione di zuccheri riduttori ha evidenziato il buon andamento del processo fermentativo per entrambe le tesi considerate, sebbene la temperatura si sia costantemente mantenuta, a partire dal 20 giorno, al di sotto di 18°C, che è convenzionalmente considerato il livello soglia minimo per un efficiente sviluppo microbico nelle salamoie di olive in fermentazione. Il risultato dell’evoluzione delle microflore spontanee, contaminanti e protecnologiche, valutata su 8 diversi substrati selettivi, ha evidenziato che le Enterobacteriaceae risultavano inferiori alle 10 UFC mL-1, già dopo 11 giorni di fermentazione; mentre i batteri sporigeni si sono costantemente mantenuti al di sotto delle 10 UFC mL-1 per l’intera durata del periodo di monitoraggio. Le sole differenze significative tra le due tesi considerate, ovvero in irriguo e non, riguardano l’evoluzione della microflora blastomicetica e degli stafilococchi, che in entrambi i casi registrano livelli di popolazione più elevati, finanche di tre decadi, nella tesi non irrigata. Per tutti gli altri parametri microbiologici considerati gli andamenti delle popolazioni risultano perfettamente sovrapponibili. Novanta colture di batteri lattici e 90 colture blastomicetiche, proveniente da diverse fasi delle olive in fermentazione sono state identificate e biotipizzate a mezzo di tecniche di biologia molecolare, nonché sottoposte a caratterizzazione fenotipica mirata all’individuazione di attività di specifico interesse protecnologico. Un ceppo afferente alla specie Lactobacillus plantarum è stato selezionato quale potenziale microrganismo starter ed adoperato per inoculare in diverse condizioni tecnologiche di produzione lotti da 25 Kg di olive delle medesima varietà “Nocellara del Belice”. Lavoro effettuato con il contributo dell’Istituto Regionale dell’Olio e dell’Olivo (IROO), Regione Sicilia.

A set of degenerate PCR primers was designed and used to amplify and sequence about 75% of the catalase (kat) gene from each of 49 staphylococcal strains. In some strains of Staphylococcus xylosus, S. saprophyticus, and S. equorum, two catalase genes, katA and katB, were found. A phylogenetic tree was generated and showed diversities among 66 partial (about 900-bp) staphylococcal kat nucleotide sequences (including 17 sequences found in GenBank) representing 26 different species. The topology of this tree showed a distribution of staphylococcal species similar, but not identical, to those reported previously based on 16S rRNA, hsp60, sodA, rpoB, tuf, and gap genes. The kat gene sequences were less conserved than those of 16S rRNA, rpoB, hsp60, and tuf genes and slightly more conserved than those of the gap gene. Therefore, kat gene sequence analysis may provide an additional marker for inferring phylogenetic relationships of staphylococci. Moreover, the discrete nucleotide polymorphism revealed in this gene could be exploited for rapid, low-cost identification of staphylococcal species through PCR-restriction fragment length polymorphism (RFLP) analysis. In this study, a PCR-RFLP assay performed by using only the TaqI restriction enzyme was successfully developed for rapid unequivocal identification/differentiation, at species and subspecies levels, of coagulase-positive staphylococci (CPS). The assay was validated by testing the DNA from 100 staphylococcal strains, including reference and wild CPS strains isolated from different environments. This reliable, rapid, and low-cost approach (requiring about 6 h from DNA isolation to the achievement of results and

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent Study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.