Exploring hydrodynamic cavitation for citrus waste valorisation in Malta: from beverage enhancement to potato sprouting suppression and water remediation.

Introduction: The endorsement of circular economy, zero-waste, and sustainable development by the EU and UN has promoted non-thermal technologies in agro-food and health industries. While northern European countries rapidly integrate these technologies, their implementation in Mediterranean food-supply chains remains uncertain. Aims: We evaluated the usefulness of hydrodynamic cavitation (HC) for valorizing orange peel waste in the fresh orange juice supply chain of the Maltese Islands. Method: We assessed: a) the effectiveness of HC in extracting bioactive compounds from orange peels (Citrus sinensis) in water (35°C) and 70% (v/v) ethanol (-10°C) over time, compared to conventional maceration, and b) the potato sprouting-suppression and biosorbent potential of the processed peel for copper, nitrate, and nitrite binding. Results: Prolonged HC-assisted extractions in water (high cavitation numbers), damaged and/or oxidized bioactive compounds, with flavonoids and ascorbic acid being more sensitive, whereas cold ethanolic extractions preserved the compounds involved in radical scavenging. HC-processing adequately modified the peel, enabling its use as a potato suppressant and biosorbent for copper, nitrate, and nitrite. Conclusion: Coupling HC-assisted bioactive compound extractions with using leftover peel for potato-sprouting prevention and as biosorbent for water pollutant removal offers a straightforward approach to promoting circular economic practices and sustainable agriculture in Malta.

Insight to the diversity of Photobacterium spp. isolated from European plaice (Pleuronectes platessa) based on phylogenetic analysis, phenotypic characterisation and spoilage potential.

This study aimed to explore the diversity of fifty-four Photobacterium strains isolated from muscle tissue of European plaice (Pleuronectes platessa) caught at different fishing seasons and stored 14-days under various conditions. Single phylogenetic markers (16S rRNA, gapA, gyrB and recA) and multilocus sequence analysis (MLSA) were employed to classify isolates at species level. Furthermore, intra- and interspecies variability in the phenotypic traits, maximum specific growth rate (µmax) and spoilage potential of the Photobacterium isolates were investigated. The isolates were classified into the P. iliopiscarium (53.7 %), P. phosphoreum (40.7 %) and P. piscicola (5.6 %) clades using MLSA. Two housekeeping genes, gyrB and recA, exhibited a consistent phylogenetic relationship with MLSA, suggesting that they might be used as individual phylogenetic markers for the Photobacterium genus. Intra- and interspecies variability in the expression of phenotypic characteristics and the production of trimethylamine (TMA), inosine (HxR), and hypoxanthine (Hx) were observed. A growth optimum temperature for P. iliopiscarium was approximately 20 °C, while those for P. phosphoreum and P. piscicola were closer to 15 °C. All isolates exhibited the highest growth density at 1.5 % NaCl, followed by 0.5 %, 3 %, and 6 % NaCl. However, P. phosphoreum demonstrated a higher NaCl tolerance than the other two species. Although, the high CO2 atmosphere significantly inhibited the growth of all strains at 4 °C, P. phosphoreum and P. piscicola showed higher growth density at 15 °C than P. iliopiscarium. Notably, all strains demonstrated H2S production. The µmax varied considerably within each species, highlighting the significance of strain-level variability. This study demonstrates that P. iliopiscarium and P. piscicola, alongside P. phosphoreum, are efficient TMA-, HxR-, Hx-, and H2S-producers, suggesting their potential contribution to synergistic off-odour generation and spoilage. Moreover, the Photobacterium isolates seem to exhibit diverse adaptations to their environments, resulting in fluctuated growth and spoilage potential. Understanding intra- and interspecies variability will facilitate modelling seafood spoilage in microbial risk assessments and developing targeted hurdles to prolong products' shelf-life.

Quantitative tools in microbial and chemical risk assessment.

The EU-FORA programme 'Quantitative tools in microbial and chemical risk assessment' was dedicated to training on predictive microbiology fundamentals, implementation of different modelling strategies, design of experiments and software tools such as MATLAB, GInaFiT and DMFit. The fellow performed MATLAB training on maximum specific growth rate (µmax) determination according to the Ratkowsky model. GInaFiT training on different models for bacterial inactivation and DMFit training on growth parameters of Vibrio parahaemolyticus were also carried out. Optical density measurements of V. parahaemolyticus bacterial cultures were performed. The obtained kinetics of optical density measurements were used to estimate µmax. Hereafter, Minimum inhibitory concentrations and non-inhibitory concentrations of aminoglycoside antibiotics were estimated based on the quantification of the fractional areas of the optical density vs time. It can be concluded that the results of the quantitative characterisation of V. parahaemolyticus are reliable and can be used for exposure assessments. Also, the turbidimetric assay can be applied for successful estimation of minimum inhibitory concentrations and non-inhibitory concentrations.

Quantitative tools in microbial and chemical risk assessment.

The popularity of biological origin food protection substances is driven by demands from consumers for natural and clean label product, increasing various food-related safety and health concerns and sustainability issues. Lactic acid bacteria (LAB) are most promising because they are a large group of beneficial microorganisms commonly used in food protection due to their ability to inhibit the growth of pathogenic bacteria and enhance food safety. Extensive scientific research has been conducted to understand the mechanisms by which LAB exert their protective effects in various food systems. Even though LAB activity against various food pathogens and spoilers is distinguished, use of cell-free supernatant (CFS) is still under investigation. This report is dedicated to present how qualitative measures can elaborate in new bacteria-origin food additive investigation. As part of the EU-FORA programme, the fellow was involved in the risk assessment tasks and projects which include gaining basic knowledge in predicative microbiology fundamentals, including different types of modelling strategies; delivering essential understanding about experimental design, knowledge in three specific software tools (MATLAB, GInaFiT and DMFit) and gained overall understanding what are the main differences while modelling growth or inactivation models. Secondary activities were included as a way to expand competences beyond qualitative measures to overall all activities done regarding risk assessment and build a strong network of food safety experts and professionals to continue engaging in risk assessment beyond fellowship programme.

Escherichia coli K-12 Transcriptomics for Assessing the Mechanism of Action of High-Power Ultrasound.

An investigation into the mechanisms of action on bacteria involving exposure to stress factors was conducted in this study. The effects of ultrasound on Escherichia coli K-12 MG1655 and its isogenic mutant, ∆gadW, under high power ultrasound treatments (26 kHz) were screened and identified by analysing their transcriptome differences between primary and secondary sequential treatments using RNA-Seq. This also helped to assess any developed protection for cells between different generations. According to our results, 1825 genes of all tested conditions were expressed, playing different roles in the cell. The expression of these genes is associated with DNA damage, cell membrane integrity, and also metabolic effects. The studied strains also showed different differential expressed genes (DEGs), with some genes being directly responsible for defence mechanisms, while others play an indirect effect due to cell damage. A gradual decrease in the expression of the genes, as we moved from just one cycle of ultrasound treatment to sequential treatment, was evident from a heat map analysis of the results. Overall, E. coli K-12 builds a self-protection mechanism by increasing the expression of genes involved in the respiration for increased growth, and production of flagellum and pili. It can be concluded that high power ultrasound is a technology that triggers several different defence mechanisms which directly link to E. coli.

The role of temperature and carbon dioxide climatic stress factors on the growth kinetics of Escherichia coli.

AIMS: The global level of carbon dioxide and temperature in the atmosphere is expected to increase, which may affect the survival of the stress-adapted bacteria. In this study, the effect of temperature and dissolved carbon dioxide on the growth rate of Escherichia coli-eGFP tagged strain was studied, thus assessing its response to induced environmental stress factors. METHODS AND RESULTS: A kinetic assay has been performed using a microplate reader with a spectrofluorometer to determine the specific growth rates. Polynomial models were developed to correlate the environmental conditions of temperature and carbon dioxide with Escherichia coli BL21 (DE3) growth in culture media and dairy by-products. At a temperature of 42°C, as the dissolved CO2 increased, a decrease in µmax by 0.76 h-1 was observed. In contrast, at 27°C, this increase led to an increase in µmax by 0.99 h-1. Moreover, a correction factor was added when applying the model to dairy whey samples. CONCLUSIONS: The application of this developed model can be considered a useful tool for predicting the growth of Escherichia coli using climate projections.

Aqueous and gaseous plasma applications for the treatment of mung bean seeds.

Sprouts are particularly prone to microbial contamination due to their high nutrient content and the warm temperatures and humid conditions needed for their production. Therefore, disinfection is a crucial step in food processing as a means of preventing the transmission of bacterial, parasitic and viral pathogens. In this study, a dielectric coplanar surface barrier discharge (DCSBD) system was used for the application of cold atmospheric plasma (CAP), plasma activated water (PAW) and their combination on mung bean seeds. Germination assessments were performed in a test tube set-up filled with glass beads and the produced irrigation water. Overall, it was found that the combined seed treatment with direct air CAP (350 W) and air PAW had no negative impact on mung bean seed germination and growth, nor the concentration of secondary metabolites within the sprouts. These treatments also reduced the total microbial population in sprouts by 2.5 log CFU/g. This research reports for first time that aside from the stimulatory effect of plasma discharge on seed surface disinfection, sustained plasma treatment through irrigation of treated seeds with PAW can significantly enhance seedling growth. The positive outcome and further applications of different forms, of plasma i.e., gaseous and aqueous, in the agro-food industry is further supported by this research.

Principal component analysis of hyperspectral data for early detection of mould in cheeselets.

The application of non-destructive process analytical technologies in the area of food science got a lot of attention the past years. In this work we used hyperspectral imaging to detect mould on milk agar and cheese. Principal component analysis is applied to hyperspectral data to localise and visualise mycelia on the samples' surface. It is also shown that the PCA loadings obtained from a set of training samples can be applied to hyperspectral data from new test samples to detect the presence of mould on these. For both the agar and cheeselets, the first three principal components contained more than 99 % of the total variance. The spatial projection of the second principal component highlights the presence of mould on cheeselets. The proposed analysis methods can be adopted in industry to detect mould on cheeselets at an early stage and with further testing this application may also be extended to other food products.

Bacterial and fungal contaminants in caprine and ovine cheese: A meta-analysis assessment.

The dairy industry is of great importance to the European economy contributing towards € 8.7 billion of the total trade surplus. Caprine and ovine milk amount to 3.1% of the 152 million tonnes of milk produced in Europe, 95% of which is transformed into dairy products such as cheese. This cheese is mostly produced in small holdings from untreated milk, making it a high-risk dairy product for human consumption. A total of 49 foodborne disease outbreaks caused by dairy products were registered in 2017 in Europe. Therefore, these products remain a serious health risk. This meta-analysis examined 30 studies assessing bacterial or fungal contamination of caprine or ovine milk cheeses. The significantly contaminating microbes were found to be Acremonium spp. (19%), Aspergillus spp. (23%), Bacillus spp. (2%), Brucella spp. (34%), Enterobactericae spp. (36%), Enterococcus spp. (28%), Escherichia spp. (15%), Fusarium spp. (21%), Geotrichum spp. (22%), Listeria spp. (11%), Mucor spp. (15%), Penicillium spp. (25%), Phoma spp. (20%), Rhizopus spp. (15%), Salmonella spp. (3%), Scopulariopsis spp. (19%) and Staphylococcus spp. (25%) in caprine and ovine cheese, indicating a variety of food pathogens as well as spoilers. Raw milk is nutritious hence prone to contamination. However, since traditional cheese is often made from untreated milk, it is important to educate cheesemakers of key safety measures and good manufacturing practice allowing for the safe production of these food items.

Perspectives from CO+RE: How COVID-19 changed our food systems and food security paradigms.

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Characterization of Indigenous Lactic Acid Bacteria in Cow Milk of the Maltese Islands: A Geographical and Seasonal Assessment.

A geographical and seasonal assessment of indigenous lactic acid bacteria (LAB) in Maltese cow milk was conducted in this study. To investigate this, milk was collected from different regions of Malta during winter and summer seasons. Total viable counts (TVC) and LAB population were enumerated. Afterwards, LAB were isolated and identified by molecular methods. According to the results, similar TVC were enumerated on winter and summer samples, while highest LAB population was detected on summer samples. LAB isolates were grouped in seven different clusters which were assigned to Lactobacillus casei, Pediococcus pentosaceus, Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus rhamnosus, Lactococcus lactis, and Lactococcus garvieae. In addition, Enterococcus and Streptococcus species were also isolated. Season seemed to affect the genus / species of LAB since Lactobacillus were mainly isolated from winter samples, while Lactococcus and Enterococcus species were the main genera identified in summer samples. Regarding the geographical distribution, the majority of the Lactobacillus spp. were isolated from the South-eastern region in both seasons. In conclusion, through this study the diversity of indigenous LAB in the Maltese cow milk was monitored for the first time and highlighted that the microbial communities are affected by seasonality and geographical distribution of the farms.

Characterization of Fungal Surface Contaminants of the Small Maltese June Pear, Pyrus communis var. bambinella.

ABSTRACT: Fungal pathogens cause surface contamination and potential premature fruit spoilage of bambinella, a fruit endemic to the Maltese islands, leading to the loss of fruit during the postharvest phase. The objective of this study was to isolate, quantify, and characterize fungal contaminants of the small Maltese June Pear and describe their growth kinetics. In total, 284 fungicide-free fruits were collected over three consecutive summers (2014, 2015, 2016). The isolated fungi were identified by using forward and reverse colonial morphology. Species identification was determined using PCR-based methods. The number of CFU per square centimeter of bambinella outer skin was calculated. Mycelium diameter growth rate studies of the isolates were also carried out at seven different temperatures, ranging from 5 to 35°C. Fungi isolated from bambinella included Cladosporium ramotenellum, Alternaria arborescens, Penicillium lanosum, Penicillium expansum, and Aspergillus sydowii, listed from the most abundant to the least abundant. The Rosso model was fitted to the growth kinetic data and showed that the optimal temperatures for growth of all five fungi were in the range of 20 to 22°C, whereas growth was slower at temperatures below 10°C and above 30°C. As observed in the diameter studies, the order of highest to lowest germination rate was found to be P. expansum, A. sydowii, P. lanosum, C. ramotenellum, and A. arborescens. Germination studies showed that the highest germination rate was observed for P. lanosum, followed by A. arborescens, C. ramotenellum, P. expansum, and A. sydowii, in descending order. The highest germination lag time was observed for A. arborescens, followed by C. ramotenellum, P. expansum, P. lanosum, and A. sydowii, in ascending order.

Safety Control of Whole Berries by Cold Atmospheric Pressure Plasma Processing: A Review.

HIGHLIGHTS: CAPP technology has high application potential for decontamination of berries. Impacts of CAPP in aspects of food safety and security still need to be addressed. Optimized treatment parameters need to be investigated for each berry type.

Modelling the growth of pear postharvest fungal isolates at different temperatures.

The effect of temperature on the mycelium growth kinetics of four postharvest fungal isolates (i.e., Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer) was assessed. A cardinal model with inflection (CMI) was used to describe the effect of the temperature on the growth rate (µ) and the lag time (λ) of each isolate. Cardinal temperature values such as Tmin, Tmax and Topt were estimated and isolates were sorted according to their growth rate and lag time duration. Additionally, model validation was performed on a medium prepared from mashed pear pulp and on artificially wound-inoculated pear fruits. P. expansum was shown to be the most psychotrophic fungus with the lowest estimated Tmin = -8.78. Model validation on pear pulp agar showed growth rate over-prediction in the case of R. stolonifer and B. cinerea but a good correlation in the case of P. expansum and A. alternata. In vivo experiments on pear fruits showed discrepancies from the synthetic and the simulated counterparts for all the fungi with the only exception of P. expansum.

Assessing the efficacy of zinc oxide nanoparticles against Penicillium expansum by automated turbidimetric analysis.

Public concerns about food safety have triggered a worldwide implementation of new legislations aimed at banning many of the most popular food conventional antifungal treatments. There is therefore an urgent need to identify novel and safer solutions to prevent fungal contamination of food. The antifungal effect of zinc oxide nanoparticles (ZnO NPs) against the postharvest pathogenic fungus Penicillium expansum has been investigated in this study. An automated turbidimetric assay, with a standard 96-well microplate, has been developed and optimised regarding the selection of the inoculum size in order to collect sequential optical density measurements. Data were processed by the updated version of the Lambert Pearson model to estimate the minimum inhibitory concentration and the non-inhibitory concentration values which were found to be 9.8 and 1.8 mM (i.e. 798 and 147 ppm), respectively. The current results show that turbidimetry is a reliable technique for assessing the antifungal activity of metal nanoparticles and that zinc oxide (ZnO) is an effective fungicide which can be potentially used to control food safety.

Turbidimetric Assessment of the Growth of Filamentous Fungi and the Antifungal Activity of Zinc Oxide Nanoparticles.

Rapid assessment of fungal growth and screening antifungal compounds, such as nanoparticles (NPs), for effectiveness is a challenging procedure because no primary standards exist as they do for yeasts and bacteria. Because fungi do not grow as single cells, but as hyphal filaments, they cannot be quantified by the usual enumeration techniques used in bacteriology. The growth of three postharvest fungal isolates ( Alternaria alternata, Rhizopus stolonifer, and Botrytis cinerea) was investigated at different inoculum concentrations and in three nutrient media (Sabouraud dextrose agar, potato dextrose agar, and yeast extract dextrose agar [YED]) with a turbidimetric assay. Sequential measurements were performed to generate optical density versus time plots, whereas the growth responses were expressed quantitatively as the generated trapezoidal area. YED medium showed the lowest variation among replicated experiments; potato dextrose agar showed the next lowest, but there was no significant difference. The inoculum size had a minimal effect on the variation of the fungal dynamics. Microscopic assessment of the fungal growth confirmed that YED medium allowed the most homogeneous development of the studied fungi. Therefore, we developed a rapid and reliable technique to evaluate the efficacy of novel antifungal compounds such as zinc oxide NPs. Turbidimetric assessment showed that these NPs were able to inhibit the growth of all three isolates. A. alternata and B. cinerea did not show a significant difference in the level of inhibition at 15 mM, whereas R. stolonifer showed the highest inhibition at the same concentration.

Physiological effects and mode of action of ZnO nanoparticles against postharvest fungal contaminants.

Increasing concerns continue to be expressed about health hazards and environmental pollution resulting from the use of conventional fungicides for postharvest disease control. Nanoparticles represent an alternative solution for postharvest disease management. The objective of this work was to assess the physiological effects and the antifungal efficiency of ZnO nanoparticles (ZnO NPs) against a number of fungal contaminants. The efficacy of ZnO NPs was qualitatively and quantitatively assessed against: Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer. Mycelium growth diameters were measured onto Potato Dextrose Agar (PDA) plates loaded with different ZnO NPs concentrations (from 0mM to 15mM). Hereafter, the rate of the fungal diameter increase was quantified by linear regression modelling. Microscopic analysis was performed by scanning electron microscopy (SEM) images of agar plugs excised from plates with 0mM and 12mM ZnO. All the fungi were inhibited by ZnO NPs at concentrations higher than 6mM. SEM images showed clear morphological aberrations in the fungal structures of all the isolates grown in presence of ZnO. Additionally, knowing that the chelating agent EDTA sequesters metal ions, it was added to fungal inoculated PDA plates with ZnO to study the NPs' mode of action. Cultures where ZnO was mixed with EDTA showed a decrease in the antifungal effect of the nanoparticles. In conclusion, ZnO NPs are therefore a good candidate as an effective postharvest disease control antifungal agent.

Assessing the anti-fungal efficiency of filters coated with zinc oxide nanoparticles.

Air filters support fungal growth, leading to generation of conidia and volatile organic compounds, causing allergies, infections and food spoilage. Filters that inhibit fungi are therefore necessary. Zinc oxide (ZnO) nanoparticles have anti-fungal properties and therefore are good candidates for inhibiting growth. Two concentrations (0.012 M and 0.12 M) were used to coat two types of filters (melt-blown and needle-punched) for three different periods (0.5, 5 and 50 min). Rhizopus stolonifer and Penicillium expansum isolated from spoiled pears were used as test organisms. Conidial suspensions of 105 to 103 spores ml-1 were prepared in Sabouraud dextrose agar at 50°C, and a modified slide-culture technique was used to test the anti-fungal properties of the filters. Penicillium expansum was the more sensitive organism, with inhibition at 0.012 M at only 0.5 min coating time on the needle-punched filter. The longer the coating time, the more effective inhibition was for both organisms. Furthermore, it was also determined that the coating process had only a slight effect on the Young's Moduli of the needle-punched filters, while the Young's Moduli of the melt-blown filters is more susceptible to the coating method. This work contributes to the assessment of the efficacy of filter coating with ZnO nanopaticles aimed at inhibiting fungal growth.

Controlling Microbial Safety Challenges of Meat Using High Voltage Atmospheric Cold Plasma.

Atmospheric cold plasma (ACP) is a non-thermal technology, effective against a wide range of pathogenic microorganisms. Inactivation efficacy results from plasma generated reactive species. These may interact with any organic components in a test matrix including the target microorganism, thus food components may exert a protective effect against the antimicrobial mode of action. The effect of an in-package high voltage ACP process applied in conjunction with common meat processing MAP gas compositions as well as bacteria type and meat model media composition have been investigated to determine the applicability of this technology for decontamination of safety challenges associated with meat products. E. coli, L. monocytogenes, and S. aureus in PBS were undetectable after 60 s of treatment at 80 kVRMS in air, while ACP treatment of the contaminated meat model required post-treatment refrigeration to retain antimicrobial effect. The nutritive components in the meat model exerted a protective effect during treatment, where 300 s ACP exposure yielded a maximum reduction of 1.5 log using a high oxygen atmosphere, whilst using air and high nitrogen atmospheres yielded lower antimicrobial efficacy. Furthermore, an ROS assay was performed to understand the protective effects observed using the meat model. This revealed that nutritive components inhibited penetration of ROS into bacterial cells. This knowledge can assist the optimization of meat decontamination using ACP technology where interactions with all components of the food matrix require evaluation.