Leonardo Caputo

Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding

Thirty-five isolates from pig feces were identified as Lactobacillus reuteri (12 strains), Lactobacillus mucosae (7), Lactobacillus plantarum (6), Lactobacillus kitasatonis (3), Lactobacillus rossiae (2), Lactobacillus ultunensis (2), Lactobacillus crispatus (2), and Lactobacillus intestinalis (1) by partial sequence analysis of the 16S rRNA. All isolates were detected at 8-9 log CFU g(-1). Preliminarily, strains were selected based on resistance to heat treatments (ca. 70 degrees C for 10 s). The decrease in viability for some L. reuteri, L. mucosae, L. plantarum, L. kitasatonis, and L. rossiae strains was lower than 1 log cycle. Selected strains were further characterized for acid and bile salt resistance, and antibacterial activity. Except for L. kitasatonis, tolerance to simulated gastric and intestinal conditions was enhanced for all strains by addition of reconstituted skimmed milk. Antibacterial activity was found against Gram-positive and -negative potential pathogens. L. reuteri 8.1, 3S7, 6.2, and 1.2, L. mucosae 1.1R, L. plantarum 4.1, and L. rossiae 4.4 were freeze-dried and mixed (1%, w/w) into pig feed before pelleting. After pelleting, pig feed contained 10-9 log CFU kg(-1) of lactobacilli. L. plantarum 4.1, and L. reuteri 3S7 were selected based on their bile salt resistance, pH tolerance, antimicrobial activity and heat resistance. The findings in this study provide a strong basis for exploring the potential of porcine lactobacilli isolates to be used in pelleted feeding as probiotic additives.

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.

Proteomic Analysis by Two-Dimensional Gel Electrophoresis and Starch Characterization of Triticum turgidum L. var. durum Cultivars for Pasta Making

Criteria for durum wheat quality are continuously evolving in response to market pressure and consumers preference. Specific attributes of durum wheat for different end products require more rapid and objective means to grade and classify wheat parcels based on processing potential. A total of 10 durum wheat cultivars were compared for compositional, protein, and starch characteristics. Mean values for the gross composition differed for total protein, gluten, and starch. Two-dimensional electrophoresis (2DE) analysis showed the proteome diversity among the cultivars. As shown by the principal component analysis (PCA) applied to 2DE data of gliadin and glutenin fractions, cultivars differed mainly from the number of proteins and levels of protein expression. As determined by the rapid viscoanalyzer (RVA), swelling power, starch damage, amylose content, and starch pasting properties of 10 cultivars differed significantly. 2DE fingerprinting and amylose content seemed to distinguish specific cultivars being useful tools for selecting suitable durum wheat cultivars for pasta making.

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.

Efficacy of lactoferricin B in controlling ready-to-eat vegetable spoilage caused by Pseudomonas spp.

The microbial content of plant tissues has been reported to cause the spoilage of ca. 30% of chlorine-disinfected fresh vegetables during cold storage. The aim of this work was to evaluate the efficacy of antimicrobial peptides in controlling microbial vegetable spoilage under cold storage conditions. A total of 48 bacterial isolates were collected from ready-to-eat (RTE) vegetables and identified as belonging to Acinetobacter calcoaceticus, Aeromonas media, Pseudomonas cichorii, Pseudomonas fluorescens, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas putida, Pseudomonas simiae and Pseudomonas viridiflava species. Reddish or brownish pigmentation was found when Pseudomonas strains were inoculated in wounds on leaves of Iceberg and Trocadero lettuce and escarole chicory throughout cold storage. Bovine lactoferrin (BLF) and its hydrolysates (LFHs) produced by pepsin, papain and rennin, were assayed in vitro against four Pseudomonas spp. strains selected for their heavy spoiling ability. As the pepsin-LFH showed the strongest antimicrobial effect, subsequent experiments were carried out using the peptide lactoferricin B (LfcinB), well known to be responsible for its antimicrobial activity. LfcinB significantly reduced (P ≤ 0.05) spoilage by a mean of 36% caused by three out of four inoculated spoiler pseudomonads on RTE lettuce leaves after six days of cold storage. The reduction in the extent of spoilage was unrelated to viable cell density in the inoculated wounds. This is the first paper providing direct evidence regarding the application of an antimicrobial peptide to control microbial spoilage affecting RTE leafy vegetables during cold storage.

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.

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.

Changes in prolamin and high resistant starch composition during the production process of Boza, a traditional cereal-based beverage

The aim of this work was to analyze changes occurring in cereal protein and starch composition throughout the production process of Boza for its potential exploitation as functional beverage. A sharp decrease in the extractability of albumins, globulins, glutenins and prolamins was found in the processed samples after the cooking step. This phenomenon was confirmed analyzing the extracted cereal proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which also revealed an apparent decrease in the number of wheat albumin/globulin, glutenin and prolamin bands in the gruel samples in comparison with that shown by the related flours. The application of an improved extraction procedure and reverse-phase high-performance liquid chromatography analysis confirmed that the prolamin content in Boza decreased markedly in comparison with that of the flour samples. Under the experimental conditions applied, the resistant starch content of Boza resulted significantly higher than that of flours; thus, this beverage can be regarded as a “high fiber” food in accordance with the recent European regulations. This is the first report that throws light on changes occurring in protein and starch components throughout the production process of Boza and provides direct evidence on the low prolamin and high resistant starch contents of this traditional cereal-based beverage.

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.