Publications by authors named "Antonio F Logrieco"

80 Publications

Mycotoxin Profile and Phylogeny of Pathogenic Species Isolated from Symptomatic Tomato Plants in Lebanon.

Toxins (Basel) 2021 Jul 22;13(8). Epub 2021 Jul 22.

Institute of Science of Food Production-ISPA, Research National Council-CNR, Via Amendola, 122/O, 70126 Bari, Italy.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including species, can infect tomato plants during the whole growing cycle. infections cause severe production and economic losses in field and during storage. In addition, species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several species were detected on tomatoes, among which the most important are , , and . A set of 49 strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic section and grouped in three well-separated sub-clades, namely (24 out of 49), (12 out of 49), and morpho-species (12 out of 49). One strain showed high genetic similarity with an reference strain. Chemical analyses showed that most of the strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although and strains were slightly more aggressive than morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of species on tomatoes.
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http://dx.doi.org/10.3390/toxins13080513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402471PMC
July 2021

Genetic structure of Fusarium verticillioides populations from maize in Iran.

Fungal Genet Biol 2021 Jul 29:103613. Epub 2021 Jul 29.

Institute of Sciences of Food Production, National Research Council (ISPA-CNR), via Amendola 122/O, 70126 Bari, Italy. Electronic address:

Fusarium verticillioides is one of the most important fungal pathogens of maize since it causes severe yield losses and produces the mycotoxins fumonisins that represent a major concern for human and animal health. Information about genetic diversity and population structure of fungal pathogens is essential for developing disease management strategies. The aim of this research was to investigate the genetic structure of F. verticillioides isolated from different provinces of Iran through determination of mating type idiomorphs, phylogenetic analyses based on translation elongation factor-1 alpha (EF-1α), RNA Polymerase II Subunit (RPB2), beta-tubulin (tub2) and Calmodulin (cmdA) genes and genetic diversity analyses based on 6 simple-sequence repeats (SSRs). Both mating types were detected in Iranian populations of F. verticillioides, particularly in Qazvin and Khuzestan, with equal frequency, which highlighted that sexual reproduction is favorable under field conditions. However, the linkage disequilibrium indices did not support the hypothesis of random mating in Khuzestan and Fars. Although assessment of nucleotide diversity based on housekeeping genes showed low level of variation among strains, genotype diversity based on SSRs revealed a high level of genetic diversity within Iranian populations. AMOVA analysis highlighted that the genetic variation of F. verticillioides in Iran was mainly distributed within population of a single area (97%), while a small proportion of genetic variation (3%) resided among populations. These patterns of variation are likely explained by the continuous gene flow among populations isolated from different areas. On the other hand, principal coordinate analysis indicated that the distribution of genetic variation among populations could be explained by the geographical distances. Consequently, to reduce pathogen gene flow among regions, the quarantine processes in Iran should be intensified.
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http://dx.doi.org/10.1016/j.fgb.2021.103613DOI Listing
July 2021

Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes.

Molecules 2021 Apr 8;26(8). Epub 2021 Apr 8.

National Research Council, Institute of Sciences of Food Production, (CNR-ISPA, Lecce), Via Prov.le Lecce-Monteroni, 73100 Lecce, Italy.

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.
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http://dx.doi.org/10.3390/molecules26082153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068360PMC
April 2021

A PCR method to identify ochratoxin A-producing Aspergillus westerdijkiae strains on dried and aged foods.

Int J Food Microbiol 2021 Apr 23;344:109113. Epub 2021 Feb 23.

United States Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, IL, USA.

Ochratoxins are a group of mycotoxins that frequently occur as contaminants in agricultural commodities and foods, including dry-cured meats and cheeses. The fungus Aspergillus westerdijkiae is frequently isolated from aged foods and can produce ochratoxin A (OTA). However, individual strains of the fungus can have one of two OTA production phenotypes (chemotypes): OTA production and OTA nonproduction. Monitoring and early detection of OTA-producing fungi in food are the most effective strategies to manage OTA contamination. Therefore, we examined genome sequence data from five A. westerdijkiae strains isolated from the surface of cheese from southern Italy to identify genetic markers indicative of the twoOTA chemotypes. This analysis revealed a naturally occurring deletion of the OTA regulatory gene, otaR, in an OTA-nonproducing isolate.We used this information to design a polymerase chain reaction (PCR) method that could identify A. westerdijkiae and distinguish between the two OTA chemotypes. In this method, the PCR primers were complementary to conserved sequences flanking otaR and yielded different-sized amplicons from strains with the different chemotypes. The primers did not yield ota-region-specific amplicons from other OTA-producing species. Because the method is specific to A. westerdijkiae and can distinguish between the two OTA chemotypes, it has potential to significantly improve OTA monitoring programs.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2021.109113DOI Listing
April 2021

Microbial quality and safety of milk and milk products in the 21st century.

Compr Rev Food Sci Food Saf 2020 07 23;19(4):2013-2049. Epub 2020 May 23.

Department of Microbiology and Biotechnology, Max-Rubner Institut, Kiel, Germany.

Milk and milk products have been utilized by humans for many thousands of years. With the advent of metagenomic studies, our knowledge on the microbiota of milk and milk products, especially as affected by the environment, production, and storage parameters, has increased. Milk quality depends on chemical parameters (fat and protein content and absence of inhibitory substances), as well as microbial and somatic cells counts, and affects the price of milk. The effects of hygiene and effective cooling on the spoilage microbiota have shown that proteolytic and lipolytic bacteria such as Pseudomonas or Acinetobacter spp. predominate the spoilage bacterial populations. These bacteria can produce heat-stable proteases and lipases, which remain active after pasteurization and thus can spoil the milk during prolonged storage. Additionally, milk can become contaminated after pasteurization and therefore there is still a high demand on developing better cleaning and sanitation regimes and equipment, as well as test systems to (quantitatively) detect relevant pathogenic or spoilage microorganisms. Raw milk and raw milk cheese consumption is also increasing worldwide with the growing demand of minimally processed, sustainable, healthy, and local foods. In this context, emerging and re-emerging pathogens once again represent a major food safety challenge. As a result of global warming, it is conceivable that not only microbiological risks but also chemical risks relating to presence of mycotoxins or plant toxins in milk will increase. Herein, we provide an overview of the major microbial hazards occurring in the 21st century.
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http://dx.doi.org/10.1111/1541-4337.12568DOI Listing
July 2020

An In-Silico Pipeline for Rapid Screening of DNA Aptamers against Mycotoxins: The Case-Study of Fumonisin B1, Aflatoxin B1 and Ochratoxin A.

Polymers (Basel) 2020 Dec 14;12(12). Epub 2020 Dec 14.

Institute of Sciences of Food Production (ISPA), CNR-National Research Council of Italy, Via G. Amendola 122/O, 70126 Bari, Italy.

Aptamers are single-stranded oligonucleotides selected by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) able to discriminate target molecules with high affinity and specificity, even in the case of very closely related structures. Aptamers have been produced for several targets including small molecules like mycotoxins; however, the high affinity for their respective target molecules is a critical requirement. In the last decade, the screening through computational methods of aptamers for their affinity against specific targets has greatly increased and is becoming a commonly used procedure due to its convenience and low costs. This paper describes an in-silico approach for rapid screening of ten ssDNA aptamer sequences against fumonisin B1 (FB1, = 3), aflatoxin B1 (AFB1, = 2) and ochratoxin A (OTA, = 5). Theoretical results were compared with those obtained by testing the same aptamers by fluorescent microscale thermophoresis and by magnetic beads assay for their binding affinity () revealing a good agreement.
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http://dx.doi.org/10.3390/polym12122983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764985PMC
December 2020

Editorial: -Derived Mycotoxins in the Feed and Food Chain.

Front Microbiol 2020 11;11:606108. Epub 2020 Nov 11.

Institute of Sciences of Food Production, Italian National Research Council, Bari, Italy.

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http://dx.doi.org/10.3389/fmicb.2020.606108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693537PMC
November 2020

Studies on the efficacy of electrolysed oxidising water to control Aspergillus carbonarius and ochratoxin A contamination on grape.

Int J Food Microbiol 2021 Jan 27;338:108996. Epub 2020 Nov 27.

National Research Council, Institute of Sciences of Food Production, Bari, Italy. Electronic address:

Ochratoxin A (OTA) occurrence in grapes is caused by black Aspergilli (Aspergillus carbonarius followed by A. niger) vineyards contamination. It depends on climatic conditions, geographical regions, damage by insects, and grape varieties. Good agricultural practices, pesticides, and fungicides seem adequate to manage the problem during low OTA risk vintages, but the development of new strategies is always encouraged, especially when an extremely favourable condition occurs in the vineyard. Electrolysed oxidising water (EOW) has become an interesting alternative to chemicals in agriculture, mainly during the post-harvest phase. This study tested the fungicidal efficacy of EOW generated by potassium chloride, in vitro, on black Aspergilli conidia, and detached grape berries infected by A. carbonarius. Then, during field trials on Primitivo cv vineyard treated with EOW, A. carbonarius contamination, and OTA levels were compared with Switch® fungicide treatment (0.8 g/l). Black Aspergilli conidia were killed on plate assay after 2 min of treatment by EOW containing >0.4 g/l of active chlorine. EOW (0.6 g/l active chlorine) treatment reduced the rate of A. carbonarius infections in vitro of about 87-92% on detached berries and, more than half in the field trials, although Switch® showed better performance. A significant reduction in the OTA concentration was observed for the EOW and Switch® treatments in vitro (92% and 96%, respectively), while in the field trials, although the average decrease in OTA was recorded in the treated grapes, it was not statistically significant. These results highlighted that EOW could be considered effective, as a substitute for fungicides, to reduce the contamination of A. carbonarius and OTA on grapes.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2020.108996DOI Listing
January 2021

FvmnSOD is involved in oxidative stress defence, mitochondrial stability and apoptosis prevention in Fusarium verticillioides.

J Basic Microbiol 2020 Nov 23;60(11-12):994-1003. Epub 2020 Nov 23.

Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.

Superoxide dismutases are key enzymes in elimination of the superoxide anion radical (O ) generated intracellularly or by exogenous oxidative stress eliciting agents, like menadione. In this study, we investigated the physiological role of the manganese superoxide dismutase-encoding gene in Fusarium verticillioides via the construction of a gene deletion mutant, ΔFvmnSOD and comparing its phenotype with that of the wild-type parental strain and a ΔFvmnSOD' C strain, complemented with the functional manganese superoxide dismutase gene. Deletion of FvmnSOD had no effect on the relative intracellular superoxide ratio but increased the sensitivity of the fungus to menadione sodium bisulphite on Czapek-Dox stress agar plates. The lack of FvmnSOD caused changes in mitochondrial morphology and physiology: The volumetric ratio of these cell organelles in the second hyphal segment, as well as the total, the KCN-sensitive cytochrome c-dependent and the KCN+SHAM (salicylhidroxamic acid)-resistant residual respiration rates, were higher in the mutant as compared to the wild-type and the complemented strains. Nevertheless, changes in the respiration rates were attributable to the higher volumetric ratio of mitochondria found in the gene deletion mutant. Changes in the mitochondrial functions also brought about higher sensitivity to apoptotic cell death elicited by the Penicillium chrysogenum antifungal protein. The gene deletion mutant developed significantly thinner hyphae in comparison to the wild-type strain. Deletion of FvmnSOD had no effect on fumonisin B and B production of the fungus grown in Myro medium as a static culture.
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http://dx.doi.org/10.1002/jobm.202000560DOI Listing
November 2020

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic that Includes the Species Complex.

Phytopathology 2021 Sep 9:PHYTO08200330LE. Epub 2021 Sep 9.

Departamento de Genética, Campus de Excelencia Internacional Agroalimentario, Universidad de Córdoba, Córdoba, Spain.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the community voiced near unanimous support for a concept of that represented a clade comprising all agriculturally and clinically important species, including the species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus into seven genera, including the FSSC described as members of the genus , with subsequent justification in 2018 based on claims that the 2013 concept of is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of including the FSSC. We reassert the practical and scientific argument in support of a genus that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus were recombined in genus , and nine others were renamed Here the global community voices strong support for the inclusion of the FSSC in , as it remains the best scientific, nomenclatural, and practical taxonomic option available.
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http://dx.doi.org/10.1094/PHYTO-08-20-0330-LEDOI Listing
September 2021

Unravelling the Antifungal Effect of Red Thyme Oil ( L.) Compounds in Vapor Phase.

Molecules 2020 Oct 16;25(20). Epub 2020 Oct 16.

Institute of Sciences of Food Production, National Research Council of Italy, Via G. Amendola 122/O, 70126 Bari, Italy.

The aim of this work was to evaluate the antifungal activity in vapor phase of thymol, p-cymene, and γ-terpinene, the red thyme essential oil compounds (RTOCs). The Minimum Inhibitory Concentration (MIC) of RTOCs was determined against postharvest spoilage fungi of the genera , , , and , by measuring the reduction of the fungal biomass after exposure for 72 h at 25 °C. Thymol showed the lowest MIC (7.0 µg/L), followed by γ-terpinene (28.4 µg/L) and p-cymene (40.0 µg/L). In the case of ITEM 9569, resistant to commercial RTO, a better evaluation of interactions among RTOCs was performed using the checkerboard assay and the calculation of the Fractional Inhibitory Concentration Index (FICI). During incubation, changes in the RTOCs concentration were measured by GC-MS analysis. A synergistic effect between thymol (0.013 ± 0.003 L/L) and γ-terpinene (0.990 ± 0.030 L/L) (FICI = 0.50) in binary combinations, and between p-cymene (0.700 ± 0.010 L/L) and γ-terpinene (0.290 ± 0.010 L/L) in presence of thymol (0.008 ± 0.001 L/L) (FICI = 0.19), in ternary combinations was found. The synergistic effect against the strain ITEM 9569 suggests that different combinations among RTOCs could be defined to control fungal strains causing different food spoilage phenomena.
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http://dx.doi.org/10.3390/molecules25204761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587587PMC
October 2020

New insight into microbial degradation of mycotoxins during anaerobic digestion.

Waste Manag 2021 Jan 15;119:215-225. Epub 2020 Oct 15.

Institute of Sciences of Food Production, National Research Council, Bari, Italy.

Anaerobic digestion represents an interesting approach to produce biogas from organic waste materials contaminated by mycotoxins. In this study a shotgun metagenomic analysis of lab-scale bioreactors fed with mycotoxin-contaminated silage has been carried out to characterize the evolution of microbial community under the operating conditions and the key enzymatic activities responsible for mycotoxin degradation. The study was conducted at two different level of contamination for fumonisins and aflatoxin B. After 15 days biogas production was not influenced by the presence of mycotoxins. Metagenomic analysis revealed that a high contamination rate of mycotoxins interfere with microbial diversity. Degradation of mycotoxins accounted in about 54% for aflatoxin B and 60% for fumonisins. The degradation activity of fumonisins resulted in the presence of partially hydrolyzed forms in both tested contamination levels. Accordingly, metagenomic functional analysis revealed the presence of two new carboxylesterase genes belonging to D. bacterium and P. bacterium putatively involved in fumonisin degradation.
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http://dx.doi.org/10.1016/j.wasman.2020.09.048DOI Listing
January 2021

FvatfA regulates growth, stress tolerance as well as mycotoxin and pigment productions in Fusarium verticillioides.

Appl Microbiol Biotechnol 2020 Sep 27;104(18):7879-7899. Epub 2020 Jul 27.

Department of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.

FvatfA from the maize pathogen Fusarium verticillioides putatively encodes the Aspergillus nidulans AtfA and Schizasaccharomyces pombe Atf1 orthologous bZIP-type transcription factor, FvAtfA. In this study, a ΔFvatfA deletion mutant was constructed and then genetically complemented with the fully functional FvatfA gene. Comparing phenotypic features of the wild-type parental, the deletion mutant and the restored strains shed light on the versatile regulatory functions played by FvAtfA in (i) the maintenance of vegetative growth on Czapek-Dox and Potato Dextrose agars and invasive growth on unwounded tomato fruits, (ii) the preservation of conidiospore yield and size, (iii) the orchestration of oxidative (HO, menadione sodium bisulphite) and cell wall integrity (Congo Red) stress defences and (iv) the regulation of mycotoxin (fumonisins) and pigment (bikaverin, carotenoid) productions. Expression of selected biosynthetic genes both in the fumonisin (fum1, fum8) and the carotenoid (carRA, carB) pathways were down-regulated in the ΔFvatfA strain resulting in defected fumonisin production and considerably decreased carotenoid yields. The expression of bik1, encoding the polyketide synthase needed in bikaverin biosynthesis, was not up-regulated by the deletion of FvatfA meanwhile the ΔFvatfA strain produced approximately ten times more bikaverin than the wild-type or the genetically complemented strains. The abolishment of fumonisin production of the ΔFvatfA strain may lead to the development of new-type, biology-based mycotoxin control strategies. The novel information gained on the regulation of pigment production by this fungus can be interesting for experts working on new, Fusarium-based biomass and pigment production technologies. Key points • FvatfA regulates vegetative and invasive growths of F. verticillioides. • FvatfA also orchestrates oxidative and cell wall integrity stress defenses. • The ΔFvatfA mutant was deficient in fumonisin production. • FvatfA deletion resulted in decreased carotenoid and increased bikaverin yields.
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http://dx.doi.org/10.1007/s00253-020-10717-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447684PMC
September 2020

Application of an Integrated and Open Source Workflow for LC-HRMS Plant Metabolomics Studies. Case-Control Study: Metabolic Changes of Maize in Response to Infection.

Front Plant Sci 2020 5;11:664. Epub 2020 Jun 5.

Institute of Sciences of Food Production, National Research Council, Bari, Italy.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform to investigate biological systems. Reproducible and standardized workflows allow obtaining a meaningful biological interpretation. The purpose of this study was to set up and apply an open-source workflow for LC-HRMS plant metabolomics studies. Key steps of the proposed workflow were as follows: (1) experimental design, (2) sample preparation, (3) LC-HRMS analysis, (4) data processing, (5) custom database search, (6) statistical analysis, (7) compound identification, and (8) biochemical interpretation. Its applicability was evaluated through the study of metabolomics changes of two maize recombinant inbred lines with contrasting phenotypes with respect to disease severity after infection of seedlings. Analysis of data from the case-control study revealed abundance change in metabolites belonging to different metabolic pathways, including two amino acids (L-tryptophan and tyrosine), five flavonoids, and three -hydroxynnamic acid amides.
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http://dx.doi.org/10.3389/fpls.2020.00664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290002PMC
June 2020

Aflatoxin Reduction in Maize by Industrial-Scale Cleaning Solutions.

Toxins (Basel) 2020 05 17;12(5). Epub 2020 May 17.

Bühler AG, 9240 Uzwil, Switzerland.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water-slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize.
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http://dx.doi.org/10.3390/toxins12050331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290569PMC
May 2020

A Novel and Potentially MultifacetedDehydroascorbate Reductase Increasing theAntioxidant Systems Is Induced by Beauvericinin Tomato.

Antioxidants (Basel) 2020 May 16;9(5). Epub 2020 May 16.

Department of Biology, University of Bari "Aldo Moro", Via E. Orabona 4, 70125 Bari, Italy.

Dehydroascorbate reductases (DHARs) are important enzymes that reconvert the dehydroascorbic acid (DHA) into ascorbic acid (ASC). They are involved in the plant response to oxidative stress, such as that induced by the mycotoxin beauvericin (BEA). Tomato plants were treated with 50 µM of BEA; the main antioxidant compounds and enzymes were evaluated. DHARs were analyzed in the presence of different electron donors by native and denaturing electrophoresis as well as by western blot and mass spectrometry to identify a novel induced protein with DHAR activity. Kinetic parameters for dehydroascorbate (DHA) and glutathione (GSH) were also determined. The novel DHAR was induced after BEA treatment. It was GSH-dependent and possessed lower affinity to DHA and GSH than the classical DHARs. Interestingly, the mass spectrometry analysis of the main band appearing on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a chloroplast sedoheptulose 1,7-bisphosphatase, a key enzyme of the Calvin cycle, and a chloroplast mRNA-binding protein, suggesting that the DHA reducing capacity could be a side activity or the novel DHAR could be part of a protein complex. These results shed new light on the ascorbate-glutathione regulation network under oxidative stress and may represent a new way to increase the plant antioxidant defense system, plant nutraceutical value, and the health benefits of plant consumption.
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http://dx.doi.org/10.3390/antiox9050435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278833PMC
May 2020

Characterization of Micronutrients, Bioaccessibility and Antioxidant Activity of Prickly Pear Cladodes as Functional Ingredient.

Molecules 2020 May 6;25(9). Epub 2020 May 6.

Institute of Sciences of Food Production (ISPA), National Council of Research (CNR), 70126 Bari, Italy.

The (L.) (OFI) is used as a nutritional and pharmaceutical agent in various dietary and value added products. This study underlines the possible use of native prickly pear cladode powder as a functional ingredient for health-promoting food production. To summarise, chemical characterization of polyphenols, minerals and soluble dietary fibre was performed; furthermore, the antioxidant activity and bioaccessibility of polyphenols and minerals were assessed. Eleven compounds between phenolic acids and flavonoids were identified, with piscidic acid and isorhamnetin derivatives being the most abundant. dietary fibre was mainly constituted of mucilage and pectin, and was composed of arabinose, galactose, glucose, mannose, rhamnose, and xylose sugars. The polyphenols' bioaccessibility was very high: piscidic acid at 200%, eucomic and ferulic acids >110% and flavonoids from 89% to 100%. The prickly pear cladode powder is also a source of minerals, as cations (calcium, sodium, potassium and magnesium) and anions (sulphate and chloride), with high magnesium bioaccessibilty (93%). OFI powder showed good capacity of radical scavenging measured by DPPH and ABTS methods, with 740 and 775 μmol Trolox/100 g OFI, respectively. Finally, the presented results allow the consideration of this natural product as a source of several essential nutrients, with a possible use in the food industry as a functional ingredient.
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http://dx.doi.org/10.3390/molecules25092176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248772PMC
May 2020

A loop-mediated isothermal amplification (LAMP) assay for rapid detection of fumonisin producing Aspergillus species.

Food Microbiol 2020 Sep 28;90:103469. Epub 2020 Feb 28.

Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Bari, Italy.

Fumonisins contamination of food commodities is a worldwide problem, especially for maize. The ability to produce fumonisinsis a trait of several species of Fusarium, mainly F. verticillioides and F. proliferatum on maize, and some Aspergillus species. A. niger and its sister species A. welwitschiae, can contribute to fumonisin B (FB) accumulation in maize kernels, although to a lesser extent than fumonisin-producing Fusarium species. Fumonisins risk monitoring represents an effective strategy in the integrated approach for mycotoxin risk management and reduction. The availability of a user-friendlymolecular assay for the detection oftoxigenic fungal species represents a valuable tool in understanding and managing upcoming mycotoxin contamination. In this study, we developed a LAMP assay, based on the detection of fum10, for a rapid and specific molecular detection of FB-producing A. niger and A. welwistchiae, potentially useful to perform monitoring directly "on site" in maize chain. Results showed that very low amounts of conidia are suitable to detect the presence of the target gene, thus providing information about the presence of FB-producing Aspergillus species and the possible upcoming fumonisins contamination in maize. The assay was combined with a suitable protocol for "in field" crude DNA extraction and a colorimetric method for easy naked-eye evaluationof results, offering a reliable and user-friendly tool to support effective reduction strategies of mycotoxin contamination in crop management programs.
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http://dx.doi.org/10.1016/j.fm.2020.103469DOI Listing
September 2020

Critical Comparison of Analytical Performances of Two Immunoassay Methods for Rapid Detection of Aflatoxin M in Milk.

Toxins (Basel) 2020 04 22;12(4). Epub 2020 Apr 22.

Institute of Sciences of Food Production, National Research Council of Italy, via Amendola 122/O, 70126 Bari, Italy.

Aflatoxin B (AFB) is a secondary metabolite produced by some . fungi affecting many crops and feed materials. Aflatoxin M (AFM), the 4-hydroxylated metabolite of AFB is the main AFB-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a "group 1 human carcinogen". The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM levels of 25, 50, 75 ng/kg and blank samples (AFM < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSD) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM at levels within "not detected" (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.
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http://dx.doi.org/10.3390/toxins12040270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232151PMC
April 2020

Plant Bioactive Compounds in Pre- and Postharvest Management for Aflatoxins Reduction.

Front Microbiol 2020 12;11:243. Epub 2020 Mar 12.

Institute of Sciences of Food Production, Italian National Research Council, Bari, Italy.

Aflatoxins (AFs) are secondary metabolites produced by spp., known for their hepatotoxic, carcinogenic, and mutagenic activity in humans and animals. AF contamination of staple food commodities is a global concern due to their toxicity and the economic losses they cause. Different strategies have been applied to reduce fungal contamination and AF production. Among them, the use of natural, plant-derived compounds is emerging as a promising strategy to be applied to control both spoilage and AF contamination in food and feed commodities in an integrated pre- and postharvest management. In particular, phenols, aldehydes, and terpenes extracted from medicinal plants, spices, or fruits have been studied in depth. They can be easily extracted, they are generally recognized as safe (GRAS), and they are food-grade and act through a wide variety of mechanisms. This review investigated the main compounds with antifungal and anti-aflatoxigenic activity, also elucidating their physiological role and the different modes of action and synergies. Plant bioactive compounds are shown to be effective in modulating spp. contamination and AF production both and . Therefore, their application in pre- and postharvest management could represent an important tool to control aflatoxigenic fungi and to reduce AF contamination.
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http://dx.doi.org/10.3389/fmicb.2020.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080658PMC
March 2020

Enzymatic transformation of aflatoxin B by Rh_DypB peroxidase and characterization of the reaction products.

Chemosphere 2020 Jul 25;250:126296. Epub 2020 Feb 25.

Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR), Via G. Amendola 122/O, 70126 Bari, Italy. Electronic address:

In some environments, a number of crops, notably maize and nuts can be contaminated by aflatoxin B and related compounds resulting from the growth of aflatoxin-producing Aspergilli. Fungal peroxidases have been shown to degrade a number of mycotoxins, including aflatoxin B (AFB). Therefore, the purpose of this study was to investigate the in vitro enzymatic degradation AFB by a recombinant type B dye decolorizing peroxidase (Rh_DypB). Analysis of the reaction products by HPLC-MS analysis showed that under optimized conditions AFB was efficiently transformed by Rh_DypB, reaching a maximum of 96% conversion after 4 days of reaction at 25 °C. Based on high resolution mass spectrometry analysis, AFB was demonstrated to be quantitatively converted to AFQ, a compound with a significantly lower toxicity. A number of low molecular mass compounds were also present in the final reaction mixture in small quantities. The results presented in this study are promising for a possible application of the enzyme Rh_DypB for aflatoxin reduction in feed.
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http://dx.doi.org/10.1016/j.chemosphere.2020.126296DOI Listing
July 2020

Quality evaluation of table grapes during storage by using H NMR, LC-HRMS, MS-eNose and multivariate statistical analysis.

Food Chem 2020 Jun 21;315:126247. Epub 2020 Jan 21.

Consiglio Nazionale delle Ricerche (CNR), Istituto Scienze delle Produzioni Alimentari (ISPA), Via Amendola 122/O, 70126 Bari, Italy.

Three non-targeted methods, i.e. H NMR, LC-HRMS, and HS-SPME/MS-eNose, combined with chemometrics, were used to classify two table grape cultivars (Italia and Victoria) based on five quality levels (5, 4, 3, 2, 1). Grapes at marketable quality levels (5, 4, 3) were also discriminated from non-marketable quality levels (2 and 1). PCA-LDA and PLS-DA were applied, and results showed that, the MS-eNose provided the best results. Specifically, with the Italia table grapes, mean prediction abilities ranging from 87% to 88% and from 98% to 99% were obtained for discrimination amongst the five quality levels and of marketability/non-marketability, respectively. For the cultivar Victoria, mean predictive abilities higher than 99% were achieved for both classifications. Good models were also obtained for both cultivars using NMR and HRMS data, but only for classification by marketability. Satisfying models were further validated by MCCV. Finally, the compounds that contributed the most to the discriminations were identified.
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http://dx.doi.org/10.1016/j.foodchem.2020.126247DOI Listing
June 2020

Degradation of Aflatoxin B by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of .

Toxins (Basel) 2020 01 14;12(1). Epub 2020 Jan 14.

Institute of Sciences of Food Production, National Research Council (CNR), Via G. Amendola 122/O, 70126 Bari, Italy.

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U gdry matter) and low MnP activity (0.4 U gdry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1 whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable.
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http://dx.doi.org/10.3390/toxins12010049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020430PMC
January 2020

A CRISPR-Cas9 System for Genome Editing of Fusarium proliferatum.

Sci Rep 2019 12 27;9(1):19836. Epub 2019 Dec 27.

Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy.

Fusarium proliferatum causes diverse diseases of many economically important plants. The fungus produces several mycotoxins of which the fumonisins are the most toxic. Currently, deletion of key genes for mycotoxin biosynthesis is a laborious and time-consuming procedure. We developed a novel CRISPR/Cas9-based genome-editing tool for the direct delivery of preassembled Cas9 ribonucleoproteins into protoplasts of F. proliferatum. Our CRISPR-Cas9 system couples a site-specific double-strand DNA break mediated by two Cas9 ribonucleoproteins with microhomology recombination requiring only 50-bp regions flanking the target gene. This system reduces the risk of off-target mutations and minimizes the risk of altering any gene adjacent to the target region. We used this tool to delete a polyketide synthase gene (FUM1) required for fumonisin biosynthesis. The mutants generated are no longer able to produce fumonisins, confirming the key role of FUM1 in fumonisin biosynthesis. Our CRISPR-Cas9 system is an important new tool for genetic studies of Fusarium.
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http://dx.doi.org/10.1038/s41598-019-56270-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934662PMC
December 2019

Preservation, Characterization and Exploitation of Microbial Biodiversity: The Perspective of the Italian Network of Culture Collections.

Microorganisms 2019 Dec 12;7(12). Epub 2019 Dec 12.

Department of Life Sciences and Systems Biology, University of Turin, viale Mattioli, 25, 10125 Torino, Italy.

Microorganisms represent most of the biodiversity of living organisms in every ecological habitat. They have profound effects on the functioning of any ecosystem, and therefore on the health of our planet and of human beings. Moreover, microorganisms are the main protagonists in food, medical and biotech industries, and have several environmental applications. Accordingly, the characterization and preservation of microbial biodiversity are essential not only for the maintenance of natural ecosystems but also for research purposes and biotechnological exploitation. In this context, culture collections (CCs) and microbial biological resource centres (mBRCs) are crucial for the safeguarding and circulation of biological resources, as well as for the progress of life sciences. This review deals with the expertise and services of CCs, in particular concerning preservation and characterization of microbial resources, by pointing to the advanced approaches applied to investigate a huge reservoir of microorganisms. Data sharing and web services as well as the tight interconnection between CCs and the biotechnological industry are highlighted. In addition, guidelines and regulations related to quality management systems (QMSs), biosafety and biosecurity issues are discussed according to the perspectives of CCs and mBRCs.
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http://dx.doi.org/10.3390/microorganisms7120685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956255PMC
December 2019

Effects of temperature and water activity change on ecophysiology of ochratoxigenic Aspergillus carbonarius in field-simulating conditions.

Int J Food Microbiol 2020 Feb 5;315:108420. Epub 2019 Nov 5.

Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Bari, Italy.

Ochratoxin A (OTA) is the primary mycotoxin threat in wine and dried vine fruits. Its presence in grape and wine is strongly related to climatic conditions and the expected climate change could represent a risk of increasing fungal colonization and OTA contamination in grapes. In this regard, the interacting effect of i) different conditions of water availability (0.93 and 0.99a) and ii) different 10 h/14 h dark/light alternating temperature conditions simulating a nowadays (18/31 °C) and climate change scenario (20/37 °C) in high OTA risk areas of Apulia region, were studied. Lag phases prior to growth, mycelial growth rate, the expression of biosynthesis, transcription factors and regulatory genes of OTA cluster and OTA production were analysed in Aspergillus carbonarius ITEM 5010 under the combined effect of different climatic factors. At 18/31 °C and under water stress conditions (0.93 a) the growth rate was slower than at 0.99 a; on the contrary, at 20/37 °C a higher growth rate was observed at 0.93 a. An over-expression of OTA genes and genes belonging to the global regulator Velvet complex was observed at 18/31 °C and 0.99 a, with the specific OTA pathway transcription factor bZIP showing the highest expression level. The up-regulated transcription profile of the genes positively correlated with OTA production higher at 18/31 °C than at 20/37 °C and 0.99 a; while no OTA production was detected at 0.93 a at each of the temperature conditions tested. These findings provide preliminary evidence that the possible increase of the temperature, likely to happen in some areas of the Apulia region, may results in a reduction of both A. carbonarius spoilage and OTA production in grapes.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2019.108420DOI Listing
February 2020

The life and times of yeasts in traditional food fermentations.

Crit Rev Food Sci Nutr 2020 26;60(18):3103-3132. Epub 2019 Oct 26.

Department of Microbiology and Biotechnology, Max Rubner-Institut, Kiel, Germany.

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations.
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http://dx.doi.org/10.1080/10408398.2019.1677553DOI Listing
November 2020

Fluorescence Polarization Immunoassay for the Determination of T-2 and HT-2 Toxins and Their Glucosides in Wheat.

Toxins (Basel) 2019 07 1;11(7). Epub 2019 Jul 1.

Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), 70126 Bari, Italy.

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside) may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA) was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides, expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC = 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). The FPIA has been used to develop two rapid and easy-to-use methods using two different extraction protocols, based on the use of organic (methanol/water, 90:10, /) and non-organic (water) solvents, for the determination of these toxins in wheat. The two proposed methods showed analytical performances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relative standard deviations ≤13%), fulfilling the criteria for acceptability of an analytical method for the quantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore, the methods were then validated in accordance with the harmonized guidelines for the validation of screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analytical performances, in terms of intermediate precision (≤25%), cut-off level (80 and 96 µg/kg for the two methods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methods as screening method for assessing the content of these toxins in wheat at the EU indicative levels reported for T-2 and HT-2 toxins.
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http://dx.doi.org/10.3390/toxins11070380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669535PMC
July 2019

Occurrence of Multiple Mycotoxins in Various Types of Rice and Barley Samples in Thailand.

J Food Prot 2019 Jun;82(6):1007-1015

1 Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.

Highlights: Contamination with multiple mycotoxins was found in rice and barley. BEA, DAS, ZEA, and aflatoxins were the mycotoxins most frequently found in samples. The assessed mycotoxin exposure does not represent a health risk for consumers.
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http://dx.doi.org/10.4315/0362-028X.JFP-18-456DOI Listing
June 2019

Equilibrium Isotherm Approach to Measure the Capability of Yeast Cell Wall to Adsorb .

Foodborne Pathog Dis 2019 09 17;16(9):630-637. Epub 2019 May 17.

National Research Council, Institute of Sciences of Food Production (CNR-ISPA), Bari, Italy.

Yeast cell wall (YCW) products are currently used as substitutes to antibiotic growth promoters, to improve animal performances, and to reduce the incidence of infectious diseases in livestock. They are claimed to bind enteropathogens, thus interfering with their colonization in the intestinal mucosa. Although the anti-infectious activity of YCW products on Gram-positive pathogens like has been reported , evidences on the adsorption of by YCW fractions are not yet available. Preliminary results showed that purified YCW products exert antimicrobial activity toward . Using the adsorption isotherm approach, we measured the ability of YCW products in adsorbing , thus reducing its viability. Dosages of YCW products >1 mg/mL adsorbed 4 Log colony-forming unit (CFU)/mL of in buffered solution. The maximum adsorption of the bacterium was reached in 3 h, whereas only one product of four YCW products retained the adsorption up to 6 h. The analysis of equilibrium isotherms and adsorption kinetics revealed that all products adsorb in a dose- and time-dependent manner, with high affinity and capacity, sequestering up to 4 Log CFU/mg of product. The determination of adsorption parameters allows to differentiate among adsorbents and select the most efficient product. This approach discriminated among YCW products more efficiently than the antimicrobial assay. In conclusion, this study suggests that the ability of YCW products in reducing viability can be the result of an adsorption mechanism.
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http://dx.doi.org/10.1089/fpd.2018.2606DOI Listing
September 2019
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