Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

Biofilm formation under food-relevant conditions and sanitizers' tolerance of a Pseudomonas fluorescens group strain.

AIMS: The aim of this study was to determine the biofilm-forming ability of a strain belonging to the Pseudomonas fluorescens group isolated from the dairy environment under food-relevant conditions. Moreover, the effects of commercial sanitizers against preformed biofilms were assessed both in terms of viability and structure. METHODS AND RESULTS: The biofilms were formed on polystyrene, stainless steel (SS), and polytetrafluoroethylene (PTFE) in a wide range of temperatures (4-25°C) and were subjected to the action of 10 different sanitizers. The strain under study showed to be a strong biofilm-former regardless of temperature, particularly on polystyrene. The biofilms were mostly sensitive to chlorine and peracetic acid-based sanitizers. For some sanitizers (e.g. amphoteric), a relationship was observed between the material and the tolerance, while the temperature was not statistically significant. The formation of long-term biofilms on SS was also structurally affected by the temperature, showing microcolonies more irregular in shape and with lower cellularity at 4°C compared to 15°C, where the biofilm was more compact and with a high presence of EPS. CONCLUSIONS: The strain belonging to the P. fluorescens group was shown to quickly adhere and form mature biofilm at temperatures and on materials relevant to the food sector; however, biofilms formed under different conditions were differently tolerant to disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study could provide a basis for developing targeted sanitation protocols in food plants.

Application of pre-adaptation strategies to improve the growth of probiotic lactobacilli under food-relevant stressful conditions.

While formulating a probiotic food, it is mandatory to make sure that the viability of probiotics is adequate at the point of consumption, which can be strongly compromised by stressful conditions due to low pH and high osmolarity. In this study, three probiotic lactobacilli were subjected to different pre-adaptation conditions, and the turbidimetric growth kinetics in challenging conditions (pH 4.0-6.5, NaCl 1-7%, sucrose 0.1-0.7 M) were evaluated. Different effects were observed for Lactobacillus acidophilus, Lacticaseibacillus casei, and Lactiplantibacillus plantarum. Indeed, pre-exposition to sub-optimal conditions in terms of pH and % NaCl significantly improved the ability of L. acidophilus and L. casei to overcome the osmotic stress due to salt or sucrose, and similar effects were observed for acidic stress. L. plantarum showed to be more tolerant to the challenging conditions applied in this study. Anyway, the pre-adaptation at conditions SUB_1 (pH 4.5 and NaCl 4%) and SUB_2 (pH 5 and NaCl 2%) speeded-up its growth kinetics by reducing the length of the lag phase under sucrose stress and enhancing the maximum growth rate at the highest pH tested. Moreover, an improvement in biomass amount was observed under sucrose stress. The whole data evidenced that the application of the appropriate pre-adaptation condition could contribute to making probiotics more robust towards challenging conditions due to food matrix, processing, and storage as well as gastrointestinal transit. Further studies will be necessary to gain insight into the proteomics and metabolomics responsible for increased tolerance to stressful conditions.

Raw milk preservation by hyperbaric storage: Effect on microbial counts, protein structure and technological functionality.

The possibility to apply hyperbaric storage (HS) at room temperature (20 °C) as a sustainable approach for preservation of raw skim milk was studied. Samples were stored at 200 and 150 MPa for up to 6 days. Optimal pressure for milk HS was found to be 150 MPa, since no clotting was detected for up to 6 days. 150 MPa-HS caused the irreversible inactivation of inoculated Escherichia coli (5.13 ± 0.33 logCFU mL-1) and Staphylococcus aureus (5.66 ± 0.93 logCFU mL-1) within 2 and 6 days, respectively. Inactivation of total and faecal coliforms (3.0 log reductions) below the detection limit was achieved after just 2 days, whereas lactic acid bacteria and coagulase-positive Staphylococci were inactivated after 6 days. Pressurized storage also caused an increase in proteose peptones and the release of submicelles from casein micelles. Micelles progressively aggregated with pressure-unfolded ß-Lactoglobulin. These phenomena led to milk presenting up to 4-fold better foaming capacity, probably due to ß-Lactoglobulin unfolding or higher proteose peptones content. This work demonstrated the capability of HS to guarantee milk preservation during storage, and brought attention on the opportunity to consider the technology for milk pasteurization and functionality improvement.

Turbidimetric definition of growth limits in probiotic Lactobacillus strains from the perspective of an adaptation strategy.

The application of an adaptation strategy for probiotics, which may improve their stress tolerance, requires the identification of the growth range for each parameter tested. In this study, 4 probiotics (Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus rhamnosus, and Lactiplantibacillus plantarum) were grown under different pH, NaCl, and sucrose concentrations at 25°C, 30°C, and 37°C. Turbidimetric growth curves were carried out and lag phase duration, maximum growth rate, and amplitude (i.e., the difference between initial and stationary phase optical density) were estimated. Moreover, cell morphology was observed, and cell length measured. The growth response, as well as the morphological changes, were quite different within the 4 species. The L. acidophilus was the most sensitive strain, whereas L. plantarum was shown to better tolerate a wide range of stressful conditions. Frequently, morphological changes occurred when the growth curve was delayed. Based on the results, ranges of environmental parameters are proposed that can be considered suboptimal for each strain, and therefore could be tested. The quantitative evaluation of the growth kinetics as well as the morphological observation of the cells can constitute useful support to the choice of the parameters to be used in an adaptation strategy, notwithstanding the need to verify the effect on viability both in model systems and in foods.

Imprinting Pentaphyrin on Conductive Electropolymerized Dipyrromethane Films: A New Strategy towards the Synthesis of Photokilling Materials.

We report herein the synthesis and photoinduced bactericidal activity of two new polymeric materials, obtained by imprinting the photosensitizer 20-(4-carboxyphenyl)-2,13-dimethyl-3,12-diethyl-[22]pentaphyrin (PCox, 1) into suitable electropolymerized dipyrromethane films. 5-Phenyl-dipyrromethane (5-ph-DP) and 5-(4-pyridyl)dipyrromethane (5-py-DP) have been selected as the monomers for the synthesis of the materials in order to assess the correlation between the substituent in C5 and the capability in Pcox uptake. Both films have been tested in their photokilling ability toward Staphylococcus aureus by using a multi-LED blue lamp at a fluence rate of 40 W/m2 . Poly-5-py-DP/PCox, with a PCox load of 10-8  mol/cm2 , achieved a 4-log reduction in microbial viability after 60 min of irradiation. The polymeric films proved to be stable over time and under oxidation conditions; in addition, no loss of photosensitizer was observed during the experiments, thus demonstrating that the bactericidal action was effectively brought by the ROS generated by PCox immobilized in the material. After use, the films were recharged with PCox, with almost complete recovery of their photodynamic efficiency.

Inactivation of Foodborne Bacteria Biofilms by Aqueous and Gaseous Ozone.

In this study, the efficacy of treatments with ozone in water and gaseous ozone against attached cells and microbial biofilms of three foodborne species, Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes, was investigated. Biofilms formed on AISI 304 stainless steel coupons from a mixture of three strains (one reference and two wild strains) of each microbial species were subjected to three types of treatment for increasing times: (i) ozonized water (0.5 ppm) by immersion in static condition, (ii) ozonized water under flow conditions, and (iii) gaseous ozone at different concentrations (0.1-20 ppm). The Excel add-in GinaFit tool allowed to estimate the survival curves of attached cells and microbial biofilms, highlighting that, regardless of the treatment, the antimicrobial effect occurred in the first minutes of treatment, while by increasing contact times probably the residual biofilm population acquired greater resistance to ozonation. Treatment with aqueous ozone under static conditions resulted in an estimated viability reduction of 1.61-2.14 Log CFU/cm2 after 20 min, while reduction values were higher (3.26-5.23 Log CFU/cm2) for biofilms treated in dynamic conditions. S. aureus was the most sensitive species to aqueous ozone under dynamic conditions. With regard to the use of gaseous ozone, at low concentrations (up to 0.2 ppm), estimated inactivations of 2.01-2.46 Log CFU/cm2 were obtained after 60 min, while at the highest concentrations a complete inactivation (<10 CFU/cm2) of the biofilms of L. monocytogenes and the reduction of 5.51 and 4.72 Log CFU/cm2 of P. fluorescens and S. aureus respectively after 60 and 20 min were achieved. Considering the results, ozone in water form might be used in daily sanitation protocols at the end of the day or during process downtime, while gaseous ozone might be used for the treatment of confined spaces for longer times (e.g., overnight) and in the absence of personnel, to allow an eco-friendly control of microbial biofilms and consequently reduce the risk of cross-contamination in the food industry.

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing.

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines.

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon.

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.

Identification of the Enterobacteriaceae in Montasio cheese and assessment of their amino acid decarboxylase activity.

The aim of the study was to identify the species of Enterobacteriaceae present in Montasio cheese and to assess their potential to produce biogenic amines. Plate count methods and an Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) approach, combined with 16S rDNA sequencing, were used to investigate the Enterobacteriaceae community present during the cheesemaking and ripening of 6 batches of Montasio cheese. Additionally, the potential decarboxylation abilities of selected bacterial isolates were qualitatively and quantitatively assessed against tyrosine, histidine, ornithine and lysine. The most predominant species detected during cheese manufacturing and ripening were Enterobacter cloacae, Escherichia coli and Hafnia alvei. The non-limiting physico-chemical conditions (pH, NaCl% and a(w)) during ripening were probably the cause of the presence of detectable levels of Enterobacteriaceae up to 120 d of ripening. The HPLC test showed that cadaverine and putrescine were the amines produced in higher amounts by almost all isolates, indicating that the presence of these amines in cheese can be linked to the presence of high counts of Enterobacteriaceae. 44 isolates produced low amounts of histamine (<300 ppm), and four isolates produced more than 1000 ppm of this amine. Only 9 isolates, belonging to the species Citrobacter freundii, Esch. coli and Raoultella ornithinolytica, appeared to produce tyramine. These data provided new information regarding the decarboxylase activity of some Enterobacteriaceae species, including Pantoea agglomerans, Esch. fergusonii and R. ornithinolytica.

Influence of hydroponic and soil cultivation on quality and shelf life of ready-to-eat lamb's lettuce (Valerianella locusta L. Laterr).

BACKGROUND: Nowadays, there is an increasing interest in the hydroponic floating system to cultivate leafy vegetables for ready-to-eat salads. It is reasonable that different growing systems could affect the quality and shelf life of these salads. RESULTS: The quality and shelf life of ready-to-eat lamb's lettuce grown in protected environment in soil plot or in soil-less system over hydroponic solution with or without the addition of 30 µmol L⁻¹ silicon were evaluated. Minimum effects were observed on colour, firmness and microbial counts. Hydroponic cultivation largely affected plant tissue hydration, leading to weight loss and structural modifications during refrigerated storage. The shelf life of lamb's lettuce was limited by the development of visually detectable unpleasant sensory properties. Shelf life, calculated by survival analysis of consumer acceptability data, resulted about 7 days for soil-cultivated salad and 2 days for the hydroponically grown ones. The addition of silicon to the hydroponic solution resulted in an interesting strategy to increase plant tissue yield and reduce nitrate accumulation. CONCLUSIONS: Although hydroponic cultivation may have critical consequences on product quality and shelf life, these disadvantages could be largely counterbalance by increased yield and a reduction of nitrate accumulation when cultivation is performed on nutritive solutions with supplemental addition of silicon.

Comparison of culture-dependent and -independent methods for bacterial community monitoring during Montasio cheese manufacturing.

The microbial community in milk is of great importance in the manufacture of traditional cheeses produced using raw milk and natural cultures. During milk curdling and cheese ripening, complex interactions occur in the microbial community, and accurate identification of the microorganisms involved provides essential information for understanding their role in these processes and in flavor production. Recent improvements in molecular biological methods have led to their application to food matrices, and thereby opened new perspectives for the study of microbial communities in fermented foods. In this study, a description of microbial community composition during the manufacture and ripening of Montasio cheese was provided. A combined approach using culture-dependent and -independent methods was applied. Culture-dependent identification was compared with 16S clone libraries sequencing data obtained from both DNA and reverse-transcribed RNA (cDNA) amplification and real-time quantitative PCR (qPCR) assays developed to detect and quantify specific bacterial species/genera (Streptococcus thermophilus, Lactobacillus casei, Pediococcus pentosaceus, Enterococcus spp., Pseudomonas spp.). S. thermophilus was the predominant LAB species throughout the entire ripening period of Montasio cheese. The culture-independent method demonstrates the relevant presence of Pseudomonas spp. and Lactococcus piscium at the beginning of ripening. The culture-dependent approach and the two culture-independent approaches produced complementary information, together generating a general view of cheese microbial ecology.

Microbiological characterization of artisanal Montasio cheese: analysis of its indigenous lactic acid bacteria.

The aim of this study was to investigate the dynamics of the microflora during Montasio cheese ripening, with specific reference to some characteristics of biotechnological interest. Nine batches of Montasio cheese produced in different plants were analyzed. Streptococcus thermophilus was the predominant species throughout the whole ripening period of Montasio cheese. Enterococci were also frequently present. This microbial group resulted probably from milk, and its proportion decreased rapidly during ripening. The most acidifying microbial species was S. thermophilus, while the most proteolytic strains belonged to the genera Enterococcus. A high degree of phenotypic diversity occurred within the microbial species.