Federico Baruzzi

Genotyping and Toxigenic Potential of Bacillus subtilis and Bacillus pumilus Strains Occurring in Industrial and Artisanal Cured Sausages

ABSTRACT 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.

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, β, and γ caseins on its outer surface where a concomitant wrinkling and successive exfoliation became visible without significant changes in texture characteristics.

Pepsin-digested bovine lactoferrin prevents Mozzarella cheese blue discoloration caused by Pseudomonas fluorescens

The aim of this work was to check the efficacy of bovine lactoferrin hydrolyzed by pepsin (LFH) to prevent blue discoloration of Mozzarella cheese delaying the growth of the related spoilage bacteria. Among 64 Pseudomonas fluorescens strains, isolated from 105 Mozzarella samples, only ten developed blue discoloration in cold-stored Mozzarella cheese slices. When Mozzarella cheese samples from dairy were treated with LFH and inoculated with a selected P. fluorescens strain, no pigmentation and changes in casein profiles were found up to 14 days of cold storage. In addition, starting from day 5, the count of P. fluorescens spoiling strain was steadily ca. one log cycle lower than that of LFH-free samples. ESI-Orbitrap-based mass spectrometry analyses allowed to reveal the pigment leucoindigoidine only in the blue LFH-free cheese samples indicating that this compound could be considered a chemical marker of this alteration. For the first time, an innovative mild approach, based on the antimicrobial activity of milk protein hydrolysates, for counteracting blue Mozzarella event and controlling psychrotrophic pigmenting pseudomonads, is here reported.

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.

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.

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.

Gallium-modified chitosan/poly(acrylic acid) bilayer coatings for improved titanium implant performances

A gallium-modified chitosan/poly(acrylic acid) bilayer was obtained by electrochemical techniques on titanium to reduce orthopaedic and/or dental implants failure. The bilayer in vitro antibacterial properties and biocompatibility were evaluated against Escherichia coli and Pseudomonas aeruginosa and on MG63 osteoblast-like cells, respectively. Gallium loading into the bilayer was carefully tuned by the electrochemical deposition time to ensure the best balance between antibacterial activity and cytocompatibility. The 30min deposition time was able to reduce in vitro the viable cell counts of E. coli and P. aeruginosa of 2 and 3 log cfu/sheet, respectively. Our results evidenced that the developed antibacterial coating did not considerably alter the mechanical flexural properties of titanium substrates and, in addition, influenced positively MG63 adhesion and proliferation. Therefore, the gallium-modified chitosan/poly(acrylic acid) bilayer can be exploited as a promising titanium coating to limit bacterial adhesion and proliferation, while maintaining osseointegrative potential.

Reduction of whey protein concentrate antigenicity by using a combined enzymatic digestion and ultrafiltration approach

The global interest in saving food resources is leading to recycle wasted-food materials to extract useful nutrients. In dairy industry, the recycling of whey proteins determines their utilization in the healthy-addressed foods, which, however, can cause immunological responses in allergic subjects. In this work, a whey protein concentrate (WPC) was alternatively hydrolyzed with pepsin, papain, trypsin and rennin in order to attenuate or abolish the β-lactoglobulin (BLG) antigenicity. The electrophoretic profiles of both pepsin and papain WPC hydrolysates proved the disappearance of the BLG band, even though a slight antigenicity was still found by ELISA. Pepsin hydrolysates, filtered through a 10-kDa cut-off membrane, did not produce immunological response. A deeper investigation carried out on pepsin digested and ultrafiltered samples by LC–MS/MS showed the disappearance of the immunoreactive BLG-fragment IVTQMKGLDIQKVAGTW. The remaining peptides, partially overlapped to major IgE binding epitopes, were not able to give immunoreactivity response. The combined WPC pepsin digestion with ultrafiltration confirmed to be a user-friendly strategy to reduce markedly the WPC antigenicity. The improvement of this two-steps process could be used to produce novel hypoallergenic infant food formulas.

Efficacy of Combined Sous Vide-Microwave Cooking for Foodborne Pathogen Inactivation in Ready-to-Eat Chicory Stems

There is a variety of different food processing methods, which can be used to prepare ready-to-eat foods. However, the need to preserve the freshness and nutritional qualities leads to the application of mild technologies which may be insufficient to inactivate microbial pathogens. In this work, fresh chicory stems were packed under a vacuum in films, which were transparent to microwaves. These were then exposed to microwaves for different periods of time. The application of sous vide microwave cooking (SV-MW, 900 W, 2450 MHz), controlled naturally occurring mesophilic aerobic bacteria, yeasts and molds for up to 30 d when vacuum-packed vegetables were stored at 4 °C. In addition, the process lethality of the SV-MW 90 s cooking was experimentally validated. This treatment led to 6.07 ± 0.7 and 4.92 ± 0.65 log cfu/g reduction of Escherichia coli and Listeria monocytogenes inoculated over the chicory stems (100 g), respectively. With an initial load of 9 log cfu/g for both pathogens, less than 10 cfu/g of surviving cells were found after 90 s cooking. This shows that short-time microwave cooking can be used to effectively pasteurize vacuum-packed chicory stems, achieving >5 log cfu/g reduction of E. coli and L. monocytogenes.

Rapid profiling of antimicrobial compounds characterising B. subtilis TR50 cell-free filtrate by high-performance liquid chromatography coupled to high-resolution Orbitrap™ mass spectrometry.

RATIONALE Several Bacillus strains, typically isolated from different food sources, represent renowned producers of a multitude of low and high molecular weight compounds, including lipopeptides and macrolactones, with an importance for their antimicrobial activity. The high homology shared by many of these compounds also occurring as closely related isoforms poses a challenge in their prompt detection. METHODS Identification and structural elucidation is generally achieved by matrix-assisted laser desorption/ionization (MALDI) or liquid chromatography (LC) coupled to mass spectrometry (MS) after a pre-fractionation and/or purification step of the extract. In this paper we report the application of a method based on LC separation and high-resolution Orbitrap™-based MS for the rapid screening of raw filtrate of the strain Bacillus subtilis TR50 endowed with antimicrobial activity, without requiring any sample pre-treatment. RESULTS Upon direct analysis of the cell-free filtrate of Bacillus subtilis TR50 by high-resolution mass spectrometry (HRMS), different compounds families, that proved to exert a remarked antimicrobial activity against several foodborne pathogens, can be readily displayed along the chromatographic run. Among them, three different classes were identified and characterized belonging to the iturin, fengycin and surfactin groups. The high resolving power and accurate mass accuracy provided by the HRMS system in use ensured an enhanced selectivity compared to other mass spectrometers. In addition, after activation of the HCD cell, the HR-MS/MS spectra can provide insights in the structural elucidation of several compounds. CONCLUSIONS The acquisition of HRMS spectra of raw filtrates of subtilis strains allows untargeted analysis of the major classes of compounds produced to be performed, thus facilitating identification of other unknown bioactive molecules after retrospective analysis. These features make this approach a fast tool applicable to the rapid screening and further identification of antimicrobial compounds released by Bacillus strains in raw filtrates.