Alessandra Pino

Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses

In the present study 177 Lactobacillus spp. strains, isolated from Ragusano and Pecorino Siciliano cheeses, were inxa0vitro screened for probiotic traits, and their characteristics were compared to those of Lactobacillus rhamnosus GG, commercial strain. Based on acidic and bile salt resistance, thirteen Lactobacillus strains were selected. The multiplex-PCR application revealed that nine strains belonged to L. rhamnosus species and four to Lactobacillus paracasei species. All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal tract (GI), for adhesion capacity to human intestinal cell lines, for hydrophobicity, for co-aggregation and auto-aggregation and for antimicrobial activities. Moreover, antibiotic resistance, hemolytic and bile salt hydrolase activities were investigated for safety assessment. Viable counts after simulated gastric and duodenal transit revealed that overall the selected lactobacilli tolerated better pancreatic juice and bile salts than acidic juice. In particular, three L.xa0rhamnosus strains (FS10, FS2, and PS11) and one L.xa0paracasei strain (PM8) increased their cell density after the simulated GI transit. The same strains showed also high percentage of auto-aggregation and co-aggregation with Escherichia coli. All strains were effective against both Staphylococcus aureus and E.xa0coli and variability was achieved versus Listeria monocytogenes and Enterococcus faecalis used as pathogenic indicator strains. Different behavior was revealed by strains for adhesion ability and hydrophobicity, which are not always linked each other and are strongly strain-dependent. From the safety point of view, no isolate showed hemolytic and bile salt hydrolase activities, except one, and most of the strains were sensitive to a broad range of clinical antibiotics. This work showed that the L.xa0rhamnosus FS10 and the L.xa0paracasei PM8 are good promising probiotic candidates for further inxa0vivo investigations.

A large factory-scale application of selected autochthonous lactic acid bacteria for PDO Pecorino Siciliano cheese production.

The main hypothesis of this study was that the autochthonous lactic acid bacteria (LAB) selected for their dairy traits are able to stabilize the production of PDO (Protected Denomination of Origin) Pecorino Siciliano cheese, preserving its typicality. The experimental plan included the application of a multi-strain lactic acid bacteria (LAB) culture, composed of starter (Lactococcus lactis subsp. lactis CAG4 and CAG37) and non starter (Enterococcus faecalis PSL71, Lactococcus garviae PSL67 and Streptococcus macedonicus PSL72) strains, during the traditional production of cheese at large scale level in six factories located in different areas of Sicily. The cheese making processes were followed from milk to ripened cheeses and the effects of the added LAB were evaluated on the microbiological, chemico-physical and sensorial characteristics of the final products. Results highlighted a high variability for all investigated parameters and the dominance of LAB cocci in bulk milk samples. The experimental curds showed a faster pH drop than control curds and the levels of LAB estimated in 5-month ripened experimental cheeses (7.59 and 7.27 Log CFU/g for rods and cocci, respectively) were higher than those of control cheeses (7.02 and 6.61 Log CFU/g for rods and cocci, respectively). The comparison of the bacterial isolates by randomly amplified polymorphic DNA (RAPD)-PCR evidenced the dominance of the added starter lactococci over native milk and vat LAB, while the added non starter LAB were found at almost the same levels of the indigenous strains. The sensory evaluation showed that the mixed LAB culture did not influence the majority of the sensory attributes of the cheeses and that each factory produced cheeses with unique characteristics. Finally, the multivariate statistical analysis based on all parameters evaluated on the ripened cheeses showed the dissimilarities and the relationships among cheeses. Thus, the main hypothesis of the work was accepted since the quality parameters of the final cheeses were stabilized, but all cheeses maintained their local typicality.

Microbiota and metabolome during controlled and spontaneous fermentation of Nocellara Etnea table olives

This study is aimed to investigate bacterial community and its dynamics during the fermentation of Nocellara Etnea table olives and to study its effect on metabolome formation. Six different combination of bacterial cultures (BC1-BC6) were used as starters for table olive fermentation and one additional process, conducted without addition of any starters, was used as control (C). The processes were conducted in triplicate and, overall, 21 vessels were performed at industrial scale. The fermentation was monitored for 120 days through culture-dependent and -independent approaches. Microbial counts of the main microbial groups revealed slight differences among brine samples, with the exception of LAB counts and Enterobacteriaceae, which were higher and lower, respectively, in most of the inoculated samples than the control ones. In addition, results demonstrated that the use of bacterial cultures (except the BC1), singly or in different combinations, clearly influenced the fermentation process reducing the final pH value below 4.50. When microbiota was investigated through sequencing analysis, data revealed the presence of halophilic bacteria and, among lactobacilli, the dominance of Lactobacillus plantarum group at the initial stage of fermentation, in all brine samples, except in the BC5 in which dominated Lactobacillus casei group. At 60 and 120 days of fermentation, an overturned bacterial ecology and an increase of biodiversity was observed in all samples, with the occurrence of Lactobacillus paracollinoides, Lactobacillus acidipiscis and Pediococcus parvulus. Correlation between bacterial OTU and volatile organic compounds (VOCs) revealed that, aldehydes and alcohol compounds exhibited a positive correlation with Proteobacteria, while several esters with LAB and Hafnia. In particular, esters, associated with fruity and floral notes, were positively correlated to L.xa0paracollinoides, L. acidipiscis, and P.xa0parvulus species. Although the VOCs amounts were sample-specific, overall aldehydes were mostly produced at the beginning of the fermentation, while acids, alcohols and esters at the end of the process.

Survival of potential probiotic lactobacilli used as adjunct cultures on Pecorino Siciliano cheese ripening and passage through the gastrointestinal tract of healthy volunteers

In the present study, two lactobacilli strains, Lactobacillus rhamnosus H25 and Lactobacillus paracasei N24, used as adjunct cultures, were evaluated for their heat resistance both with and without prior heat adaptation and for their survival, at industrial scale, during the production and ripening of the Pecorino Siciliano cheese. In addition, the viability and persistence of the lactobacilli strains after passage through the gastrointestinal tract of healthy volunteers were evaluated by using rep-PCR analysis of viable cells. Both strains exhibited good heat resistance and survival throughout cheese production and ripening, and positively influenced the physico-chemical, the microbiological and the sensorial characteristics of the final product. In addition, the molecular typing of the lactobacilli isolates, retrieved from fecal samples of healthy volunteers during and after 15 days of the experimental cheese administration, revealed a high survival of the strains, highlighting their persistence during passage into the GI tract. In conclusion, this study proposes the two adjunct cultures as potential probiotic candidate deliverable by cheese.

Bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial

ABSTRACT Background: Bacterial vaginosis is the most frequent condition associated to the vaginal microbiota imbalance, affecting about the 40–50% of women in the world. Even if antibiotics are effcetive for bacterial vaginosis treatment a long-term recurrence rates, higher than 70%, is recorded. Lactoferrin is an iron-binding glycoprotein with bacteriostatic and bactericidal properties. It owns the ability to protect the host against infection, by binding and regulating the iron needed for the bacterial proliferation. Objective: The present study was an open prospective randomized trial (registration no. SHI-EVE-2014.01) aimed at characterizing the bacterial biota of women affected by bacterial vaginosis (BV) and assessing the effects of two different lactoferrin concentrations (100 mg and 200 mg vaginal pessaries) on the composition and dynamics of the vaginal bacterial biota. Design: Sixty women with BV were recruited and randomized into two groups to receive lactoferrin pessaries for 10 days. Clinical evaluation was based on Amsel criteria and Nugent scores. Culture-dependent methods and Ion Torrent PGM sequencing of the 16S rRNA gene were applied to study in depth the overall structure of the vaginal bacterial biota and its dynamics during the treatment. Results: Vaginal lactoferrin administration modified the vaginal microbiota composition in patients with BV. During treatment, both 100 mg and 200 mg lactoferrin vaginal pessaries significantly decreased the occurrence of bacteria associated with BV, such as Gardnerella, Prevotella, and Lachnospira, and increased the occurrence of Lactobacillus species. The bacterial biota balance was maintained up to 2 weeks after treatment only in women treated with 200 mg lactoferrin pessaries. Conclusions: This study indicates that lactoferrin could be proposed as an alternative therapeutic approach for BV. Our data showed, for the first time, the dominance of Lactobacillus helveticus species during and after vaginal lactoferrin treatment.

Influence of PDO Ragusano cheese biofilm microbiota on flavour compounds formation

The objectives of the present study were to characterize the biofilm microbiota of 11 different farms (from A to K), producing PDO Ragusano cheese, and to investigate on its ability to generate volatile organic compounds (VOCs) in milk samples inoculated with biofilm and incubated under Ragusano cheese making conditions. The biofilms were subjected to plate counting and PCR/T/DGGE analysis and the VOCs generated in incubated milk samples were evaluated through SmartNose, GC/O, and GC/MS. Streptococcus thermophilus was the dominant species both in biofilms and in incubated milks. Lactobacillus, Lactococcus, Enterococcus and Leuconostoc were also identified. Low levels of Pseudomonas spp. and yeasts counts were detected, whereas coliforms, Listeria monocytogenes and Salmonella spp., were never found. SmartNose and GC/O analyses were able to differentiate incubated milk samples on the basis of the odour compounds, highlighting that samples E and F overlapped and sample C was clearly separated from the others. These results complied with those acquired by GC/MS analysis, that detected in total 20 VOCs. Principal component analysis showed positive correlations (rxa0>xa00.6; Pxa0<xa00.05) between some lactic acid bacteria (LAB) and VOCs: such as Enterococcus hirae with alcohols, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus delbrueckii with aldehydes, and Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus hilgardii with ketones. This work demonstrates that biofilm represents an excellent source of LAB biodiversity, which contribute to generate VOCs during the production of PDO Ragusano cheese.

Fermentation of Nocellara Etnea table olives by functional starter cultures at different low salt concentrations

Nocellara Etnea is one of the main Sicilian cultivars traditionally used to produce both olive oil and naturally fermented table olives. In the present study, the effect of different salt concentrations on physico-chemical, microbiological, sensorial, and volatile organic compounds (VOCs) formation was evaluated in order to obtain functional Nocellara Etnea table olives. The experimental design consisted of 8 treatments as follow: fermentations at 4, 5, 6, and 8% of salt with (E1-E4 samples) and without (C1-C4 samples) the addition of starters. All the trials were carried out at room temperature (18 ± 2°C) and monitored for an overall period of 120 d. In addition, the persistence of the potential probiotic Lactobacillus paracasei N24 at the end of the process was investigated. Microbiological data revealed the dominance of lactic acid bacteria (LAB), starting from the 7th d of fermentation, and the reduction of yeasts and enterobacteria in the final product inoculated with starters. VOCs profile highlighted a high amount of aldehydes at the beginning of fermentation, which significantly decreased through the process and a concomitant increase of alcohols, acids, esters, and phenols. In particular, esters showed an occurrence percentage higher in experimental samples rather than in control ones, contributing to more pleasant flavors. Moreover, acetic acid, ethanol, and phenols, which often generate off-flavors, were negatively correlated with mesophilic bacteria and LAB. It is interesting to note that salt content did not affect the performances of starter cultures and slightly influenced the metabolome of table olives. Sensory data demonstrated significant differences among samples registering the highest overall acceptability in the experimental sample at 5% of NaCl. The persistence of the L. paracasei N24 strain in experimental samples, at the end of the process, revealed its promising perspectives as starter culture for the production of functional table olives with reduced salt content.

Polyphasic approach to study physico-chemical, microbiological and sensorial characteristics of artisanal Nicastrese goat's cheese

Nicastrese goats cheese is produced in the South of Italy under traditional procedures, from raw goat milk without any starter cultures addition. Samples from milk to ripened cheese provided by 4 different farms were subjected to a polyphasic approach to study their physico-chemical, microbiological and sensorial characteristics. In addition, volatile organic compounds formation in the final products was studied. Overall, gross composition and microbiological data revealed a significant variability among samples, which was confirmed by both the volatile organic compounds generated in the final products and by the sensorial data. Conventional technique allowed us to identify 720 isolates, mainly belonging to Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Leuconostoc mesenteroides, and Enterococcus faecalis. Culture-independent methods revealed shifts in the microbial community structure, with an increase in biodiversity of metabolically active bacterial species, from milk to cheese samples. Analysis of volatile organic compounds (VOCs) allowed the identification of 36 compounds; free fatty acids and ketones represented the main detected, followed by alcohols and esters. Moreover, statistical analysis was performed in order to correlate VOCs to bacterial species. Data showed that ester compounds as well as alcohol and aldehydes were positively correlated to NSLAB, indicating that the occurrence of L.xa0casei, L. plantarum and L.xa0brevis species is relevant for the VOCs formation in the final product.

Effects of selected bacterial cultures on safety and sensory traits of Nocellara Etnea olives produced at large factory scale

This work investigates the effects of different combinations of selected lactic acid bacteria strains on Lactobacillus species occurrence, on safety and on sensory traits of natural green table olives, produced at large factory scale. Olives belonging to Nocellara Etnea cv were processed in a 6% NaCl brine and inoculated with six different bacterial cultures, using selected strains belonging to Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus pentosus species. The fermentation process was strongly influenced by the added starters and the identification of lactic acid bacteria isolated throughout the process confirms that L. pentosus dominated all fermentations, followed by L. plantarum, whereas L. casei was never detected. Pathogens were never found, while histamine and tyrosine were detected in control and in two experimental samples. The samples with the lowest final pH values showed a safer profile and the most appreciated sensory traits. The present study highlights that selected starters promote prevalence of L. pentosus over the autochthonous microbiota throughout the whole process of Nocellara Etnea olives.

Enterococcus spp. in Ragusano PDO and Pecorino Siciliano cheese types: A snapshot of their antibiotic resistance distribution

In the present study, 110 enterococci were isolated from two Sicilian cheese types, Ragusano PDO and Pecorino Siciliano. Isolates, firstly identified by MALDI-TOF/MS and a multiplex PCR assay, were tested for susceptibility to the most relevant clinical antibiotics. Clonal relationships among isolates were evaluated by pulsed-field-gel electrophoresis (PFGE) analysis and the presence of vanA and vanB genes, in vancomycin resistant enterococci (VRE), was investigated. Overall, E. faecalis, E. durans (35% for each species) and E. faecium (28%) were the major identified species. Different occurrence between cheese types was revealed. Most isolates from Ragusano PDO cheese were identified as E. durans (46%) and/or E. faecalis (43%), while E. faecium (605) was mainly detected in Pecorino Siciliano cheese. High incidence of resistance (97% of total strains) was detected for rifampicin, erythromycin and ampicillin. Moreover, 83 isolates (75%) exhibited multidrug-resistant phenotypes and the one VRE (vanB) isolate was identified as E. durans. PFGE analysis clustered isolates into 22 genotypes and the presence of the same PFGE types, for both E. durans and E. faecalis, in the two cheese types, suggest the link between enterococci and geographical area of production. Results of present study raise concerns about possible role of dairy enterococci as reservoirs of antibiotic resistance.