The food industry by-products in bread making: single and combined effect of carob pod flour, sugar beet fibers and molasses on dough rheology, quality and food safety.

Obtaining high-quality value-added bread with extended shelf-life by utilizing food industry by-products that would have minimal negative effect on gluten structure while avoiding the usage of synthetic preservatives, was the challenge of this study. For this purpose the influence of the combination of carob pod flour (C, 0-15%), sugar beet fibers (F, 0-10%) and sugar beet molasses (M, 0-6%) on dough rheology and bread quality was investigated. Selected materials were of good microbiological quality, while carob flour and sugar beet fibers were rich in dietary fibers (43.6% and 67.0%, respectively). The presence of high share of dietary fibers (combination of C and F) increased dough resistance to extension up to 2.5 times, while dough extensibility was reduced by 50%. The addition of molasses had less pronounced single effect on dough properties however its impact in combination with fiber-rich materials outweighed the individual effect (an increase of dough resistance at about 55%). Molasses alone and in combination with other ingredients had positive effect on bread quality. Sample M6F5 attained crumb quality score (6.0 of maximum 7.0) higher than in control sample (5.6). High share of C and F reduced loaf volume and crumb quality of bread up to 56% and 50%, respectively and increased hardness 7.5 times. However, carob flour (7.5%) increased polyphenols content and antioxidant capacity 3 and 4 times, respectively. Moreover, carob flour and molasses and their combination showed inhibitory effect on mold mycelia growth and spores formation. Regarding all of the above, C7.5M3F5 was chosen as optimal sample.

Phenolic Profile, Antioxidant Capacity and Antimicrobial Activity of Nettle Leaves Extracts Obtained by Advanced Extraction Techniques.

Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS2 and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative Pseudomonas fragi ATCC 4973 and Campylobacter jejuni NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.

Development of Biodegradable Whey-Based Laminate Functionalised by Chitosan-Natural Extract Formulations.

In this research, antimicrobial polysaccharide chitosan and natural extracts were used as surface coating of a plastic laminate with an integrated whey layer on the inside. The aim was to establish the biodegradable and active concept of packaging laminates. For this purpose, chitosan nanoparticles (CSNPs) with embedded rosemary or cinnamon extracts were synthesised and characterised. Additionally, a whey-based laminate was functionalised: i) chitosan macromolecular solution was applied as first layer and ii) cinnamon or rosemary extracts encapsulated in CSNPs were applied as upper layer (layer wise deposition). Such functionalised whey-based laminate was physicochemically characterized in terms of elemental surface composition, wettability, morphology and oxygen permeability. The antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli, Aspergillus flavus and Penicillium verrucosum. The antioxidant properties were determined using the ABTS assay. It could be shown that after functionalization of the films with the above-mentioned strategy, the wettability was improved. Furthermore, such whey-based laminates still show excellent barrier properties, good antimicrobial activity and a remarkable antioxidative activity. In addition to the improved biodegradability, this type of lamination could also have a positive effect on the shelf-life of products packaged in such structured films.

Bioactive Characterization of Packaging Foils Coated by Chitosan and Polyphenol Colloidal Formulations.

Polypropylene (PP) and polyethylene (PE) foils, previously activated by ultraviolet (UV)/ozone, were functionalized using chitosan-extract nanoparticle dispersions. A solution of macromolecular chitosan was applied onto foils as a first layer, followed by the deposition of various extracts encapsulated into chitosan nanoparticles, which were attached as an upper layer. Functionalized foils were analyzed from a bioactive point of view, i.e., regarding antimicrobial and antioxidant activity. Desorption kinetics were also studied. Moreover, barrier properties were examined, as the most important parameter influencing antimicrobial and antioxidant activity. Finally, all these properties were correlated with different surface parameters, determined previously, in order to understand if there is any direct correlation between surface elemental composition, surface charge, contact angle, or morphology and a specific bioactive property. It was shown that great bioactive properties were introduced due to the additive effect of antimicrobial chitosan and antioxidative plant extracts. Moreover, oxygen permeability decreased significantly, and the migration of polyphenols and chitosan from the foil surface was below the OML (overall migration limit), which is very important for food industry applications. Furthermore, surface properties of foils influence to some extent the desired bioactivity.

Combination of rosemary extract and buffered vinegar inhibits Pseudomonas and Shewanella growth in common carp (Cyprinus carpio).

BACKGROUND: Aquaculture is the fastest growing food-production sector, and common carp (Cyprinus carpio) is one of the most cultivated fish species in the world. Due to its intrinsic characteristics, fish meat is highly susceptible to microbiological spoilage. Pseudomonas and Shewanella are the primary and secondary occurring microbiota during storage of fish meat, with significant contribution to spoilage with the formation of hydrolytic enzymes (lipases and proteases). RESULTS: With in vitro testing, we show that rosemary extract (Inolens4), buffered vinegar and their combination (SyneROX) exhibit antimicrobial effects against P. fragi, P. psychrophila, S. putrefaciens and S. xiaemensis at concentrations of 3.13 and 1.56 mg mL-1 . The combination was the most effective in inhibiting growth of selected bacteria in food model, and production of lipases and proteases during 9 days at 5 °C. In situ testing of antimicrobial dip treatment of carp meat determined that aerobic mesophilic, total psychrotrophic, Pseudomonas and hydrogen sulfide producer counts were reduced in all treatments, with the most prominent influence being shown by the combination and buffered vinegar. CONCLUSIONS: Our study highlights the importance of a multilevel assessment of the antimicrobial potential of biopreservatives under conditions comparable to those of the selected food. Investigation with bacteria and food model provided coherent and consistent data for the evaluation of the antimicrobial potential for carp meat. Combination of buffered vinegar (as active antimicrobial) and rosemary extract, with well-known and researched antioxidant properties but low in situ antimicrobial activity, represents a good potential for combined effect in preservation of fish meat. © 2020 Society of Chemical Industry.

Spoilage Pseudomonas biofilm with Escherichia coli protection in fish meat at 5 °C.

BACKGROUND: Pseudomonas are part of the indigenous microbiota of different foods, where they gradually cause spoilage. In fish meat, Pseudomonas fragi and Pseudomonas psychrophila have been identified as important spoilers. The initial aim of this study was to investigate the physiological characteristics, adhesion, and biofilm of P. fragi and P. psychrophila under temperatures related to the fish-processing industry. The further aim was to define the problem of increased growth of pathogenic bacteria in the presence of spoilage bacteria in vitro and in fish meat. RESULTS: Temperature dependence on physiological characteristics, adhesion, and biofilm was observed. Hydrophobicity and autoaggregation were most prominent at 15 °C, and at this temperature floating biofilm was also formed. The adhesion of these Pseudomonas was up to 2 log CFU cm-1 more pronounced on stainless steel than polystyrene, with up to five times greater biofilm biomass production at 5 °C on polystyrene. This paralleled at least a 0.5 log CFU g-1 increase in the pathogenic bacterium Escherichia coli in fish meat. CONCLUSION: Pseudomonas fragi and P. psychrophila adhesion and biofilm depend on the temperature, and are stimulated by temperatures that can occur during the processing and storage of fish meat. Strong Pseudomonas biofilm formation under refrigeration conditions is protective for E. coli, potentially by providing more favorable conditions by ensuring a higher concentration of nutrients. Interactions between spoilage Pseudomonas and pathogenic bacteria can occur through different mechanisms, and an understanding of these is of particular importance to ensure the overall quality and safety of fish meat and other proteinaceous foods. © 2019 Society of Chemical Industry.

Bleeding of Common Carp (Cyprinus carpio) Improves Sensory Quality of Fillets and Slows Oxidative and Microbiological Changes During Refrigerated Aerobic Storage.

Common carp (Cyprinus carpio) aquaculture is one of the most important and rapidly growing productions around the world. However, for consumers, carp is often not acceptable due to its distinctive colour and odour. In this study, we investigated the effects of bleeding of common carp on fillet quality. The obtained results show that carp bleeding by cutting the gill arches is an effective way of reducing the total haem content, which here decreased from (9.6±1.6) in unbled carp to (2.34±0.8) µmol/kg of haemoglobin in bled carp. Furthermore, fillets from bled carp showed reduced formation of primary and secondary lipid oxidation products and growth of microorganisms during 12 days of refrigerated aerobic storage. On the last day of storage, the amount of lipid hydroperoxides decreased from (88.9±4.2) in unbled to (62.1±2.9) µmol/kg of cumene hydroperoxide in bled carp, TBARS decreased from (4.2±0.5) in unbled to (2.6±0.4) µmol/kg of malondialdehyde in bled carp, mesophilic and psychrotrophic bacteria count decreased from (6.4±0.1) and (6.2±0.3) log CFU/g in unbled to (4.0±0.2) and (4.2±0.2) log CFU/g in bled carp, respectively. These raw bled fillets showed increased lightness L*, and reduced redness a* and yellowness b* compared to unbled fillets. Sensory analysis showed improved colour, odour and overall acceptability of bled raw fillets. Overall, bleeding improves the quality of carp fillets. Thus, inclusion of bleeding into processing of carp fillets has the potential to improve their acceptance by consumers and prolong their shelf-life.

Aqueous Extracts of Wild Mushrooms Show Antimicrobial and Antiadhesion Activities against Bacteria and Fungi.

Mushrooms represent promising sources of novel bioactive compounds and can be applied as innovative strategies to control microbial contamination and infection via the food chain. We characterized aqueous extracts from 21 wild basidiomycete mushrooms and the cultivated oyster mushroom, Pleurotus ostreatus, as putative sources of antimicrobial and antiadhesive compounds. Broth microdilutions and adhesion to a polystyrene surface were evaluated on Gram-positive and Gram-negative bacteria and on fungi. The aqueous extracts tested showed antimicrobial and antiadhesive activities against these microorganisms. Biochemical analyses of the P. ostreatus extract indicated the involvement of several compounds with different molecular masses. Copyright © 2017 John Wiley & Sons, Ltd.

Beyond Birth: Pioneering Insights into Colostrum Quality Variation among Bitches with Different Types of Parturition.

This study deals with the immunological quality of canine colostrum in relation to the different parturition modes in bitches. It included four groups of bitches, who underwent vaginal parturition (VP), emergency cesarean section (EM-CS), elective cesarean section (EL-CS), or elective cesarean section with aglepristone injection 24 h prior to surgery (EL-A). Colostrum samples from 40 bitches of 18 breeds were taken immediately after the birth of the first puppy or directly after surgery, and four hours later. The concentrations of immunoglobulin G (IgG) and IgA were measured using ELISA tests. The initial IgG concentration was 18.3 ± 10.2 g/L, and the IgA concentration was 13.7 ± 5.8 g/L, respectively. Significantly lower IgG and IgA concentrations were observed in the EL-CS group compared to other groups. The administration of aglepristone led to an increase in IgG and IgA concentrations. Despite the lower immunoglobulin levels with EL-CS, the survival rate of the puppies was not affected. The study shows that immunoglobulin concentrations in colostrum vary between bitches, with the type of birth significantly influencing the levels. Progesterone withdrawal before birth could play a decisive role in increasing IgG and IgA concentrations in the mammary glands of the bitches.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Pseudomonas fragi biofilm on stainless steel (at low temperatures) affects the survival of Campylobacter jejuni and Listeria monocytogenes and their control by a polymer molybdenum oxide nanocomposite coating.

Pseudomonas spp. are widely distributed bacteria on surfaces in the food production and processing environment, where they form extracellular polymeric substance rich biofilms that interact with other bacteria. In this study, the influence of biofilm of Pseudomonas fragi ATCC 4973 on Listeria monocytogenes ATCC 19115 and Campylobacter jejuni NCTC 11168 was investigated at 5 °C and 15 °C on stainless steel in broth and food homogenates (fish or chicken meat). Stainless steel was then coated with PVDF-HFP/PVP/MoO3 nanocomposite and examined for surface changes (scanning electron microscope, static contact angle, Vickers hardness and elastic modulus). The effect of the prepared nanocomposite coating on P. fragi and on L. monocytogenes and C. jejuni was evaluated in mono- and co-culture. P. fragi produced more biofilm at 15 °C than at 5 °C, especially when food homogenates were used as growth media. Co-cultivation with pathogens did not affect biofilm production by P. fragi, but significant changes were observed in L. monocytogenes and C. jejuni, resulting in a decrease and increase, respectively, in the determined number of culturable biofilm cells. The first change was probably due to competition for the surface, and the second to the oxygen gradient. Stainless steel was then coated with a PVDF-HFP/PVP/MoO3 nanocomposite, which was characterised by lower roughness and higher wettability, but lower hardness compared to uncoated stainless steel. The prepared nanocoating showed bactericidal activity when tested in phosphate buffered saline. When used in food homogenates, a reduction of over 95 % in bacterial counts was observed. An abundant biofilm of P. fragi proved protective to L. monocytogenes and C. jejuni against the functionalised nanocomposite surface when tested in food homogenates. The control of spoilage Pseudomonas spp., which are common in the food production and processing environment, is important for reducing the contamination of food with spoilage bacteria and with pathogens such as L. monocytogenes and C. jejuni, which may be present in the same environment. The PVDF-HFP/PVP/MoO3 nanocomposite showed good potential for use as a coating for food contact surfaces, but possible migration of nanoparticles from the nanocomposite coating to food should be evaluated before its commercial use.

SIMBA Method-Simultaneous Detection of Antimicrobial and Anti-biofilm Activity of New Compounds Using Salmonella Infantis.

The development of antimicrobial resistance and the formation of Salmonella biofilms are serious public health problems. For this reason, new natural compounds with antimicrobial and anti-biofilm activity are being sought, and wild fungi represent an untapped potential. Various extraction agents, including organic solvents and aqueous buffers, can be used to obtain bioactive compounds from natural sources. To evaluate their bioactivity, extensive screening studies are required to determine antimicrobial and anti-biofilm activity using methods such as broth microdilution or crystal violet assay, respectively, but none of these methods allow simultaneous evaluation of both activities against bacteria. Cold water extraction from wild fungi offers the advantage of extracting water-soluble compounds. The SIMultaneous detection of antiMicrobial and anti-Biofilm Activity (SIMBA) method combines the testing of both types of activity against bacteria with the evaluation of the 20 h growth curve of the Salmonella Infantis ZM9 strain determined with absorbance measurements at 600 nm in a 96-well plate. SIMBA method thus shortens the time to determine the bioactivity of extracts, reduces material consumption, and eliminates the need for additional reagents. SIMBA enables rapid selection of bioactive extracts for their fractionation and shortens the time to determine new natural products with antimicrobial and anti-biofilm activity. Graphical overview.

A novel approach using growth curve analysis to distinguish between antimicrobial and anti-biofilm activities against Salmonella.

Salmonella spp. are a commonly identified cause of outbreaks of food-borne diseases. Despite much research, there remains the need to find new antimicrobial and anti-biofilm agents against Salmonella. For this, it is necessary to distinguish between these two aspects. Agents that influence biofilm formation should not affect bacterial growth, to thus avoid further promotion of the development of resistance. In this study, we present the use of growth curves of Salmonella Infantis to simultaneously determine antimicrobial and anti-biofilm activities, for the screening for anti-Salmonella activities of 42 aqueous fungal extracts. The extract from Pseudohydnum gelatinosum showed good antimicrobial activity, and that from Pleurotus ostreatus showed good anti-biofilm activity. In extracts from Infundibulicybe geotropa and Infundibulicybe gibba, both activities were determined after fractionation. The antimicrobial activity was associated with protein-rich fractions and mediated by l-amino acid oxidase activity. The fractionation did not allow determination of the anti-biofilm active fraction, so further studies are needed to define these compounds. Growth curve analysis of S. Infantis is shown here to provide a fast and simple approach to distinguish between antimicrobial and anti-biofilm activities in a high-throughput setting, such that it can be easily implemented in screening and further bioassay-based purification of novel alternatives to antibiotics.

Listeria innocua Biofilm Assay Using NanoLuc Luciferase.

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution. Graphic abstract: Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

Characterisation of a new cell wall teichoic acid produced by Listeria innocua ZM39 and analysis of its biosynthesis genes.

Listeria innocua is genetically closely related to the foodborne human pathogen Listeria monocytogenes. However, as most L. innocua strains are non-pathogenic, it has been proposed as a surrogate organism for determining the efficacy of antimicrobial strategies against L. monocytogenes. Teichoic acids are one of the three major cell wall components of Listeria, along with the peptidoglycan backbone and cell wall-associated proteins. The polymeric teichoic acids make up the majority of cell wall carbohydrates; the type of teichoic acids directly attached to the peptidoglycan are termed wall teichoic acids (WTAs). WTAs play vital physiological roles, are important virulence factors, antigenic determinants, and phage-binding ligands. The structures of the various WTAs of L. monocytogenes are well known, whereas those of L. innocua are not. In the present study, the WTA structure of L. innocua ZM39 was determined mainly by 1D and 2D NMR spectroscopy and it was found to be the following: [→4)-[α-D-GlcpNAc-(1→3)]-ß-D-GlcpNAc-(1→4)-D-Rbo-(1P→]n This structure is new with respect to all currently known Listeria WTAs and it shares structural similarities with type II WTA serovar 6a. In addition, the genome of strain L. innocua ZM39 was sequenced and the majority of putative WTA synthesis genes were identified.

Seasonal Changes in Chemical Profile and Antioxidant Activity of Padina pavonica Extracts and Their Application in the Development of Bioactive Chitosan/PLA Bilayer Film.

Seaweeds are a potentially sustainable source of natural antioxidants that can be used in the food industry and possibly for the development of new sustainable packaging materials with the ability to extend the shelf-life of foods and reduce oxidation. With this in mind, the seasonal variations in the chemical composition and antioxidant activity of brown seaweed (Padina pavonica) extracts were investigated. The highest total phenolic content (TPC) and antioxidant activity (measured by ferric reducing/antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and oxygen radical absorbance capacity (ORAC)) were found for P. pavonica June extract. The TPC of 26.69 ± 1.86 mg gallic acid equivalent/g, FRAP of 352.82 ± 15.41 µmole Trolox equivalent (TE)/L, DPPH of 52.51 ± 2.81% inhibition, and ORAC of 76.45 ± 1.47 µmole TE/L were detected. Therefore, this extract was chosen for the development of bioactive PLA bilayer film, along with chitosan. Primary or quaternary chitosan was used as the first layer on polylactic acid (PLA) films. A suspension of chitosan particles with entrapped P. pavonica extract was used as the second layer. X-ray photoelectron spectroscopy confirmed the presence of layers on the material surface. The highest recorded antioxidant activity of the newly developed films was 63.82% inhibition. The developed functional films exhibited antifogging and antioxidant properties, showing the potential for application in the food industry.

Biomass-Derived Plant Extracts in Macromolecular Chitosan Matrices as a Green Coating for PLA Films.

Due to the growing problem of food and packaging waste, environmental awareness, and customer requirements for food safety, there is a great need for the development of innovative and functional packaging. Among these developments, the concept of active packaging is at the forefront. The shortcoming in this area is that there is still a lack of multifunctional concepts, as well as green approaches. Therefore, this work focuses on the development of active chemical substances of natural origin applied as a coating on polylactic acid (PLA) films. Biopolymer chitosan and plant extracts rich in phenolic compounds (blackberry leaves-Rubus fruticosus, needles of prickly juniper-Juniperus oxycedrus) obtained from plant biomass from Southeastern Europe were selected in this work. In order to increase the effectiveness of individual substances and to introduce multifunctionality, they were combined in the form of different colloidal structural formulations. The plant extracts were embedded in chitosan biopolymer particles and dispersed in a macromolecular chitosan solution. In addition, a two-layer coating, the first of a macromolecular chitosan solution, and the second of a dispersion of the embedded extracts in chitosan particles, was applied to the PLA films as a novel approach. The success of the coatings was monitored by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and the wettability was evaluated by contact angle measurements. Scanning electron microscopy SEM tracked the morphology and homogeneity of the coating. Antioxidation was studied by DPPH and ABTS spectrophotometric tests, and microbiological analysis of the films was performed according to the ISO 22196 Standard. Desorption of the coating from the PLA was monitored by reducing the elemental composition of the films themselves. The successful functionalization of PLA was demonstrated, while the XPS and ATR-FTIR analyses clearly showed the peaks of elemental composition of the extracts and chitosan on the PLA surface. Moreover, in all cases, the contact angle of the bilayer coatings decreased by more than 35-60% and contributed to the anti-fogging properties. The desorption experiments, due to decrease in the concentration of the specific typical element (nitrogen), indicated some migration of substances from the PLA's surface. The newly developed films also exhibited antioxidant properties, with antioxidant ABTS efficiencies ranging from 83.5 to 100% and a quite high inhibition of Gram-positive Staphylococcus aureus bacteria, averaging over 95%. The current functionalization of PLA simultaneously confers antifogging, antioxidant, and antimicrobial properties and drives the development of a biodegradable and environmentally friendly composite material using green chemistry principles.

Antioxidant and Antimicrobial Activity of Hydroethanolic Leaf Extracts from Six Mediterranean Olive Cultivars.

Phenolic profiles, antioxidant, and antimicrobial activities of hydroethanolic olive leaf extracts from six Mediterranean olive cultivars (Croatian: Lastovka, Levantinka, Oblica; Italian: Moraiolo, Frantoio, Nostrana di Brisighella) were investigated. As expected, various distributions of phenolic levels were observed for each cultivar and the total phenolic content showed high variability (ranging from 4 to 22 mg GAE/g of dry extract), with the highest amount of phenolics found in the Oblica sample, which also provided the highest antiradical (ORAC) and reducing activity (FRAP). The screening of individual compounds was performed by HPLC-PDA-ESI-QTOF-MS and the main detected compounds were oleuropein, hydroxytyrosol, oleoside/secologanoside, verbascoside, rutin, luteolin glucoside, hydroxyoleuropein, and ligstroside. While the antioxidant activity of the samples was relatively high, they showed no bactericidal and bacteriostatic activity against E. coli and S. Typhimurium; weak activity against Staphylococcus aureus, Bacillus cereus, and Listeria innocua; and inhibitory effects against Campylobacter jejuni at 0.5 mg dry extract/mL. The obtained results support the fact that olive leaf extracts, and especially those from the Oblica cultivar, could potentially be applied in various industries as natural preservatives and effective and inexpensive sources of valuable antioxidants.

The role of the Listeria monocytogenes surfactome in biofilm formation.

Listeria monocytogenes is a highly pathogenic foodborne bacterium that is ubiquitous in the natural environment and capable of forming persistent biofilms in food processing environments. This species has a rich repertoire of surface structures that enable it to survive, adapt and persist in various environments and promote biofilm formation. We review current understanding and advances on how L. monocytogenes organizes its surface for biofilm formation on surfaces associated with food processing settings, because they may be an important target for development of novel antibiofilm compounds. A synthesis of the current knowledge on the role of Listeria surfactome, comprising peptidoglycan, teichoic acids and cell wall proteins, during biofilm formation on abiotic surfaces is provided. We consider indications gained from genome-wide studies and discuss surfactome structures with established mechanistic aspects in biofilm formation. Additionally, we look at the analogies to the species L. innocua, which is closely related to L. monocytogenes and often used as its model (surrogate) organism.

Expression of NanoLuc Luciferase in Listeria innocua for Development of Biofilm Assay.

Studies of biofilm formation by bacteria are crucial for understanding bacterial resistance and for development of novel antibacterial strategies. We have developed a new bioluminescence biofilm assay for Listeria innocua, which is considered a non-pathogenic surrogate for Listeria monocytogenes. L. innocua was transformed with a plasmid for inducible expression of NanoLuc luciferase (Nluc). Concentration-dependent bioluminescence signals were obtained over a concentration range of more than three log units. This biofilm assay enables absolute quantification of bacterial cells, with the necessary validation. For biofilm detection and quantification, this "Nluc bioluminescence" method has sensitivity of 1.0 × 104 and 3.0 × 104 colony forming units (CFU)/mL, respectively, with a dynamic range of 1.0 × 104 to 5.0 × 107 CFU/mL. These are accompanied by good precision (coefficient of variation, <8%) and acceptable accuracy (relative error for most samples, <15%). This novel method was applied to assess temporal biofilm formation of L. innocua as a function of concentration of inoculant, in comparison with conventional plating and CFU counting, the crystal violet assay, and the resazurin fluorescence assay. Good correlation (r = 0.9684) of this Nluc bioluminescence assay was obtained with CFU counting. The limitations of this Nluc bioluminescence assay include genetic engineering of bacteria and relatively high cost, while the advantages include direct detection, absolute cell quantification, broad dynamic range, low time requirement, and high sensitivity. Nluc-based detection of L. innocua should therefore be considered as a viable alternative or a complement to existing methods.