Autochthonous and Probiotic Lactic Acid Bacteria Employed for Production of "Advanced Traditional Cheeses".

Microbial characterization of two Italian traditional cheeses, Giuncata and Caciotta Leccese, was carried out, with the aim to isolate autochthonous bacterial strains to be used as starters to improve and standardize the quality of these cheeses. More than 400 bacterial isolates were found, using PCR-based identification, to belong to 12 species of the Streptococcus, Lactococcus, Lactobacillus, and Leuconostoc genera. The dominant strains were screened for antagonistic activity against pathogenic and spoilage bacteria and exopolysaccharide production, acidification, and proteolytic activity. Since Streptococcus macedonicus was found to be the most prevalent lactic acid bacteria species present in milk and in both types of cheese, the best performing strain of this species was successfully used, alone or in combination with a selected autochthonous Lactococcus lactis strain, in pilot-scale productions of Giuncata and Caciotta Leccese cheeses, respectively. The combined inoculums of selected autochthonous strains positively influenced the sensory characteristics of both Giuncata and Caciotta cheeses. Finally, the selected autochthonous cultures were enriched with a potentially probiotic Lactobacillus rhamnosus strain and successfully used in pilot-scale productions of these traditional cheeses. To the best of our knowledge, this is the first study reporting the use of an autochthonous S. macedonicus strain as a starter for the production of cheeses with added probiotics. In addition, the identification of the probiotic strain in the feces of healthy volunteers fed with the advanced traditional cheese proved its effectiveness as a carrier for the delivery of probiotics to the human body.

Improvement of Ayran quality by the selection of autochthonous microbial cultures.

Ayran is a traditional Turkish milk drink which is fermented and salted. Inadequate production and storage conditions contribute to its variable organoleptic quality and stability during shelf-life. A thorough physico-chemical, nutritional and microbial characterization of artisanal Ayran was carried out in order to standardize its overall quality without altering its original traits. Ayran microbial ecosystem was largely dominated by Streptococcus thermophilus (ST) and Lactobacillus delbrueckii subsp. bulgaricus (LDB). High counts of other lactic acid bacteria species, including Lactobacillus helveticus (LH), Lactobacillus fermentum (LF), and Lactobacillus paracasei (LP), were also found. Selected LDB, LP and LH strains grew well in milk displaying fast acidification and high proteolysis, differently from ST and LF strains that did not cause noticeable changes. A selected autochthonous three-strain culture (TSC), composed of one strain of LDB, LP and ST, was applied for the pilot-scale production of traditional Ayran. The Ayran produced with this TSC resulted in the most extensive shelf-life (one month) and in the best terms of its nutritional and sensory quality nevertheless altering its typical pleasant yogurt and cottage cheese notes. This TSC is at disposal of SMEs who need to standardize the overall quality of this traditional fermented milk, preserving its typical traits.

Eradication of high viable loads of Listeria monocytogenes contaminating food-contact surfaces.

This study demonstrates the efficacy of cold gaseous ozone treatments at low concentrations in the eradication of high Listeria monocytogenes viable cell loads from glass, polypropylene, stainless steel, and expanded polystyrene food-contact surfaces. Using a step by step approach, involving the selection of the most resistant strain-surface combinations, 11 Listeria sp. strains resulted inactivated by a continuous ozone flow at 1.07 mg m(-3) after 24 or 48 h of cold incubation, depending on both strain and surface evaluated. Increasing the inoculum level to 9 log CFU coupon(-1), the best inactivation rate was obtained after 48 h of treatment at 3.21 mg m(-3) ozone concentration when cells were deposited onto stainless steel and expanded polystyrene coupons, resulted the most resistant food-contact surfaces in the previous assays. The addition of naturally contaminated meat extract to a high load of L. monocytogenes LMG 23775 cells, the most resistant strain out of the 11 assayed Listeria sp. strains, led to its complete inactivation after 4 days of treatment. To the best of our knowledge, this is the first report describing the survival of L. monocytogenes and the effect of ozone treatment under cold storage conditions on expanded polystyrene, a commonly used material in food packaging. The results of this study could be useful for reducing pathogen cross-contamination phenomena during cold food storage.

Microbiological, physico-chemical, nutritional and sensory characterization of traditional Matsoni: selection and use of autochthonous multiple strain cultures to extend its shelf-life.

Matsoni, a traditional Georgian fermented milk, has variable quality and stability besides a short shelf-life (72-120 h at 6 °C) due to inadequate production and storage conditions. To individuate its typical traits as well as select and exploit autochthonous starter cultures to standardize its overall quality without altering its typicality, we carried out a thorough physico-chemical, sensorial and microbial characterization of traditional Matsoni. A polyphasic approach, including a culture-independent (PCR-DGGE) and two PCR culture-dependent methods, was employed to study the ecology of Matsoni. Overall, the microbial ecosystem of Matsoni resulted largely dominated by Streptococcus (S.) thermophilus and Lactobacillus (Lb.) delbrueckii subsp. bulgaricus. High loads of other lactic acid bacteria species, including Lb. helveticus, Lb. paracasei and Leuconostoc (Leuc.) lactis were found to occur as well. A selected autochthonous multiple strain culture (AMSC) composed of one Lb. bulgaricus, one Lb. paracasei and one S. thermophilus strain, applied for the pilot-scale production of traditional Matsoni, resulted the best in terms of enhanced shelf-life (one month), sensorial and nutritional quality without altering its overall typical quality. This AMSC is at disposal of SMEs who need to exploit and standardize the overall quality of this traditional fermented milk, preserving its typicality.

Antimicrobial efficacy of pepsin-digested bovine lactoferrin on spoilage bacteria contaminating traditional Mozzarella cheese.

The aim of this work was to check the efficacy of bovine lactoferrin (BLF) and its pepsin-digested hydrolysate (LFH) to control spoilage bacteria contaminating the governing liquid of high moisture (HM) Mozzarella cheese during cold storage. These natural substances resulted effective when tested in vitro against five potential spoilage bacteria contaminating cold-stored HM Mozzarella cheese. Among six LFH fractions, only the fraction containing lactoferricins, mainly represented by LfcinB17₋42, resulted effective against Escherichia coli K12 at the same extent of the whole pepsin-digested hydrolysate. LFH tested throughout seven days for its antimicrobial activity against the main bacterial groups growing in cold-stored commercial HM Mozzarella cheese samples delayed significantly the growth of pseudomonads and coliforms in comparison with the un-treated samples. This is the first report providing a direct evidence of the ability of LFH to inhibit the growth of cheese spoilage bacteria.

Occurrence of non-lactic acid bacteria populations involved in protein hydrolysis of cold-stored high moisture Mozzarella cheese.

The aim of this study was to analyse non-lactic acid bacteria populations (NLABPs) and evaluate their role in proteolysis of cold-stored high moisture (HM) Mozzarella cheese. NLABPs reached values close to 8 log cfu mL⁻¹ after seven days of cold storage. Sequencing of 16 rDNA and rpoB genes and molecular biotyping allowed to identify 66 bacterial strains belonging to 25 species from 15 genera, mainly represented by Pseudomonas, Acinetobacter, and Rahnella. Fifteen strains showed proteolytic activity values higher than 1000.00 µg Gly mL⁻¹ after 24 h of growth in skimmed milk. Moreover, as shown by Urea-PAGE, 11 proteolytic strains caused partial or total disappearance of at least one of the caseins. Their proteolytic behaviour was assessed even when they grew inside the governing liquid together with HM Mozzarella cheese at 4 °C for 12 days. This is the first report that throws light on the complexity of NLABPs in HM Mozzarella cheese, demonstrating that some strains caused the partial hydrolysis of α, ß, and γ caseins on its outer surface where a concomitant wrinkling and successive exfoliation became visible without significant changes in texture characteristics.

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks.

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.

Novel PCR-based identification of Weissella confusa using an AFLP-derived marker.

An extensive use of Weissella (W.) confusa is currently being made for the production of a variety of fermented foods and beverages although some strains of this species have emerged as opportunistic pathogens for humans and animals. Nevertheless, no rapid methods are available for the reliable identification of W. confusa. We developed a novel PCR using AFLP (Amplified Fragment Length Polymorphism)-derived primers for the rapid and unequivocal identification of W. confusa. Fluorescent AFLP of 30 strains of W. confusa, Leuconostoc citreum, Lactobacillus (Lb.) brevis, Lb. rossiae, Lb. plantarum and Lb. buchneri allowed us to detect, purify and sequence several W. confusa specific AFLP fragments. The homology search in BLAST of a 303 bp nucleotide sequence revealed a ≤ 77% identity of the purified fragment with the lepA gene of several lactic acid bacteria. A PCR assay targeting 225 bp of this fragment was developed and tested against the DNA of 109 strains, including 34 foodborne and clinical W. confusa and 75 strains of 47 phylogenetically closely and distantly related species, resulting in 100% specificity with a detection limit of 16 pg. Being the first species-specific PCR to date developed for the rapid and unambiguous identification of W. confusa, this novel assay could be a reliable and efficient tool for detecting W. confusa not only in food and beverages, but also in clinical specimens, thus contributing to clarify its real significance in human and animal infections.

Rapid and reliable identification of Staphylococcus aureus harbouring the enterotoxin gene cluster (egc) and quantitative detection in raw milk by real time PCR.

A TaqMan and a SYBR Green real time PCR (rt-PCR) were developed for the reliable identification and quantitative detection of Staphylococcus (S.) aureus strains harbouring the enterotoxin gene cluster (egc) regardless of its variants. Both approaches revealed 100% specificity against a panel of 70 reference strains, including 29 clinical and foodborne S. aureus strains harbouring all the egc variants to date known, 4 egc⁻S. aureus strains and 37 strains of phylogenetically closely and distantly related species. Standard curves made by 10 fold dilutions of either genomic DNA or cells from an egc(+)S. aureus log-phase broth culture showed a good linearity of response (R²≥0.993) for six orders of magnitude, with about 100% relative accuracy and a low inter-assay variability (CV≤3.02). The overall limit of quantification (LOQ) for both rt-PCR assays (about 100% PCR efficiency; running time 30 min) was 10 cfu or 10 genome equivalents per reaction mixture although 1 cfu or 1 genome equivalent was detected with a 33.33% probability. These performances were confirmed in raw milk artificially contaminated with log-phase broth cultures of either a single egc(+)S. aureus strain or a mixture of S. aureus strains harbouring all the egc variants to date known. Similar results were also obtained with a raw milk based standard curve of the S. aureus egc(+) mixture in the presence of 106 cfu/mL of egc⁻S. aureus strains harbouring some of the commonest enterotoxin genes associated to the staphylococcal food poisoning. Nonetheless, the TaqMan based approach resulted in a lower sensitivity (LOQ=100 cfu equivalents per reaction mixture) than the SYBR Green based assay (LOQ=10 cfu equivalents per reaction mixture). When applied to real milk samples, both PCR assays provided a good response with 100% diagnostic specificity and 96-107% relative accuracy, as compared to conventional culture-based PCR approaches. Due to the high specificity, the wide dynamic range of detection and the high sensitivity demonstrated even in a complex and potentially highly contaminated raw milk matrix, the SYBR Green rt-PCR assay is a useful diagnostic tool for quick, high throughput and reliable routine screening of egc(+)S. aureus isolates. Moreover, the SYBR Green based quantitative detection of these pathogens in raw milk could remarkably contribute to clarify their actual role in staphylococcal food poisoning and other clinical syndromes associated with the consumption of milk and milk-based products.

An Rhs-like genetic element is involved in bacteriocin production by Pseudomonas savastanoi pv. savastanoi.

The main aim of this work was the identification of genetic determinants involved in bacteriocin production by strain ITM317 of Pseudomonas savastanoi pv. savastanoi, besides bacteriocin characterization. The bacteriocin was observed to be a heat-sensitive, high molecular weight proteinaceous compound. We identified a transposon (Tn5)-induced mutant which had lost its ability to produce the bacteriocin. The Tn5 insertion's responsibility for the above mutated phenotype was demonstrated by marker-exchange mutagenesis. An EcoRI DNA fragment, corresponding to the EcoRI Tn5-containing fragment of the mutant, was also cloned from the wild-type strain, and its introduction into the mutant complemented the mutation. Moreover, that fragment enabled bacteriocin production by P. s. pv. savastanoi ITM302, a strain not previously capable of doing so. DNA sequence analysis revealed that Tn5 insertion occurred in the mutant within a large ORF encoding a protein which showed similarity with proteins from the Rhs family. The DNA region including that ORF showed features which have been considered typical of the Rhs genetic elements previously identified in other bacteria but whose function is as yet unclear. The results of this study for the first time identify an Rhs-like element in P. s. pv. savastanoi, and for the first time indicate that an Rhs element is involved in bacteriocin production, also suggesting this possible function for Rhs genetic elements previously characterized in other bacteria.

Molecular and physiological characterization of natural microbial communities isolated from a traditional Southern Italian processed sausage.

The development of the natural microbial populations during traditional processing of the "Salame di Senise", a typical little known sausage produced in the South of Italy, was investigated by using molecular and physiological techniques for taxonomic identification and technological characterization of strains. The application of RAPD-PCR over more than 90 colonies made it possible to isolate 18 bacterial and two yeast biotypes identified by partial rDNA sequencing as belonging mainly to three species of Bacillus, three species of Lactobacillus, three species of Staphylococcus and Debaryomyces hansenii. The physiological analyses revealed that the isolates belonging to Lactobacillus genus were the most acidifying, whereas Staphylococcus strains did not develop significant proteolytic and lipolytic activities. Interestingly, some Bacillus strains produced the highest values of proteolytic and lipolytic activities. The results for the technological properties of Bacillus strains isolated from this Southern Italian sausage, made without a selected starter, suggest that Bacillus strains, always present in meat curing, could play a role in the development of texture and organoleptic characteristics of the sausages.

Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages.

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.