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      Survival, growth, and biogenic amine production of Enterococcus faecium FC12 in response to extracts and essential oils of Rubus fruticosus and Juniperus oxycedrus

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          Abstract

          Introduction

          Enterococci are lactic acid bacteria (LAB) usually found as food contaminants in fermented products such as cheeses and fermented sausages. Due to their antibiotic resistance, the presence of virulence factors, and the ability to produce biogenic amines (BAs), the determination of these bacteria is crucial to assure food quality and safety. BAs production and consequent accumulation in foods can cause toxicological effects on human health. Plant phenolic compounds are promising alternatives to chemical preservatives and reflect consumers' demand for “green” solutions. In this study, the antimicrobial effect of blackberry ( Rubus fruticosus) leaves and prickly juniper ( Juniperus oxycedrus) needles, both as phenolic extracts (PE) and essential oils (EO), were evaluated against Enterococcus faecium FC12, a known tyramine-producing strain.

          Methods

          The growth kinetics in the presence of sub-lethal concentrations of such plant derivatives were modeled (Gompertz equation) and BA production was monitored over time by HPLC. Moreover, flow cytometry (FCM) was used to study the effects of EOs and PEs on cell viability.

          Results

          The EOs showed a higher antimicrobial effect (especially R. fruticosus added at 0.75 mg/ml), determining an initial decrease of culturable cells followed by a recovery, even if with lower growth rates and final cell loads. Different rates of BA formation were observed, with tyramine concentrations ranging from 120 to 160 mg/l after 96 h of incubation, and 2-phenylethylamine was produced in lower amounts, usually after reaching the peak of tyramine. FCM confirmed the higher efficacy of R. fruticosus EO that induced cell membrane injury in 93% of the total population. However, complete recovery occurred in the following incubation, demonstrating transient damage.

          Discussion

          Although further research is required to better investigate this recovery and to assess the suitability of this approach in a real food system, the present study showed the potential antimicrobial activity of plant derivatives, especially R. fruticosus EO, against the tyramine-producing E. faecium FC12.

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          Most cited references57

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          Antimicrobial activity of flavonoids

          Flavonoids are ubiquitous in photosynthesising cells and are commonly found in fruit, vegetables, nuts, seeds, stems, flowers, tea, wine, propolis and honey. For centuries, preparations containing these compounds as the principal physiologically active constituents have been used to treat human diseases. Increasingly, this class of natural products is becoming the subject of anti-infective research, and many groups have isolated and identified the structures of flavonoids possessing antifungal, antiviral and antibacterial activity. Moreover, several groups have demonstrated synergy between active flavonoids as well as between flavonoids and existing chemotherapeutics. Reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably owing to inter- and intra-assay variation in susceptibility testing. However, several high-quality investigations have examined the relationship between flavonoid structure and antibacterial activity and these are in close agreement. In addition, numerous research groups have sought to elucidate the antibacterial mechanisms of action of selected flavonoids. The activity of quercetin, for example, has been at least partially attributed to inhibition of DNA gyrase. It has also been proposed that sophoraflavone G and (−)-epigallocatechin gallate inhibit cytoplasmic membrane function, and that licochalcones A and C inhibit energy metabolism. Other flavonoids whose mechanisms of action have been investigated include robinetin, myricetin, apigenin, rutin, galangin, 2,4,2′-trihydroxy-5′-methylchalcone and lonchocarpol A. These compounds represent novel leads, and future studies may allow the development of a pharmacologically acceptable antimicrobial agent or class of agents.
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            Modeling of the Bacterial Growth Curve

            Several sigmoidal functions (logistic, Gompertz, Richards, Schnute, and Stannard) were compared to describe a bacterial growth curve. They were compared statistically by using the model of Schnute, which is a comprehensive model, encompassing all other models. The t test and the F test were used. With the t test, confidence intervals for parameters can be calculated and can be used to distinguish between models. In the F test, the lack of fit of the models is compared with the measuring error. Moreover, the models were compared with respect to their ease of use. All sigmoidal functions were modified so that they contained biologically relevant parameters. The models of Richards, Schnute, and Stannard appeared to be basically the same equation. In the cases tested, the modified Gompertz equation was statistically sufficient to describe the growth data of Lactobacillus plantarum and was easy to use.
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              Enterococci as probiotics and their implications in food safety.

              Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis. Copyright © 2011 Elsevier B.V. All rights reserved.
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                Author and article information

                Contributors
                Journal
                Front Nutr
                Front Nutr
                Front. Nutr.
                Frontiers in Nutrition
                Frontiers Media S.A.
                2296-861X
                13 January 2023
                2022
                : 9
                : 1092172
                Affiliations
                [1] 1Department of Agricultural and Food Sciences, University of Bologna , Cesena, Italy
                [2] 2Interdepartmental Center for Industrial Agri-food Research, University of Bologna , Cesena, Italy
                [3] 3University Department of Marine Studies, University of Split , Split, Croatia
                [4] 4Department of Seafood Processing Technology, Faculty of Fisheries, Cukurova University , Adana, Turkey
                [5] 5Department of Agricultural and Food Sciences, University of Bologna , Bologna, Italy
                Author notes

                Edited by: Oriol Comas-Basté, University of Barcelona, Spain

                Reviewed by: Raffaele Coppola, University of Molise, Italy; Teresa Veciana-Nogues, University of Barcelona, Spain; Veronica Ortiz Alvarenga, Federal University of Minas Gerais, Brazil

                *Correspondence: Giulia Tabanelli ✉ giulia.tabanelli2@ 123456unibo.it

                This article was submitted to Nutrition and Food Science Technology, a section of the journal Frontiers in Nutrition

                Article
                10.3389/fnut.2022.1092172
                9880475
                36712524
                3dbeaeff-a24f-4c7d-bc08-80a49c4e2841
                Copyright © 2023 Montanari, Barbieri, Lorenzini, Gottardi, Šimat, Özogul, Gardini and Tabanelli.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 07 November 2022
                : 23 December 2022
                Page count
                Figures: 4, Tables: 1, Equations: 2, References: 58, Pages: 9, Words: 7004
                Funding
                Funded by: Horizon 2020 Framework Programme, doi 10.13039/100010661;
                This research was supported by the PRIMA program under BioProMedFood (Project ID 1467; CUP: J34I19004820005). The PRIMA program was supported by the European Union H2020 program.
                Categories
                Nutrition
                Original Research

                enterococcus faecium,juniperus oxycedrus,rubus fruticosus,antimicrobial activity,growth kinetics,flow cytometry,tyramine production

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