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Item Antimicrobial activity of autoclaved and non autoclaved copaiba oil on Listeria monocytogenes(Ciência Rural, 2010-08) Pieri, Fábio Alessandro; José, Raphaela Mansur; Galvão, Newton Nascentes; Nero, Luis Augusto; Moreira, Maria Aparecida ScatamburloThe aim of this study was to evaluate the antimicrobial effect of different copaiba oil concentrations against the growth of Listeria monocytogenes, and analyze differences in inhibition of microorganisms with autoclaved and non autoclaved oil. This study provided an agar diffusion test with six isolates of bacteria and six different concentrations of autoclaved or non autoclaved copaiba oil and a negative control. The results showed sensitivity of five L. monocytogenes isolates related to the 10% autoclaved solution of copaiba oil. Four strains also showed sensitivity to the 5% autoclaved solution and one to 2.5% autoclaved solution. The 10% non autoclaved oil solution showed growth inhibition only for two strains. These results had pointed the 10% autoclaved solution of copaiba oil with higher inhibition as all other solutions and concentrations tested (P<0.05). For the other concentrations of both solutions, the 5 and 2.5% autoclaved and 10% non autoclaved solutions had presented statistically equal. All other concentrations of both copaiba solutions and the negative control did not presented any bacteria inhibition. In conclusion, the results of this study suggest that the autoclaved copaiba oil may be a potential new agent source for infection control or for food preservation, inhibiting the growth of food-borne bacteria such as L. monocytogenes.Item Evaluation of the microbiological safety and sensory quality of a sliced cured-smoked pork product with protective cultures addition and modified atmosphere packaging(Food Science and Technology International, 2019) Todorov, Svetoslav; Casquete, Rocı́o; Fonseca, Susana C.; Pinto, Ricardo; Castro, Sónia M.; Teixeira, Paula; Vaz-Velho, ManuelaThe aim of this study was to evaluate the effect of two protective lactic acid bacteria cultures combined with modified atmosphere packaging on the survival/growth of Listeria innocua 2030c (as a surrogate for Listeria monocytogenes) and on sensory attributes of ready-to-eat ‘lombo’ over storage time. Sliced ‘lombo’, a trad- itional cured-smoked pork loin, was inoculated with L. innocua 2030c, Lactobacillus sakei ST153 (isolated from ‘salpicão’) and BLC35 culture (with Lactobacillus curvatus, Staphylococcus xylosus and Pediococcus acidilactici; CHR Hansen) as protective cultures. Samples were packed in two modified atmosphere packa- ging conditions (20% CO 2 /80% N 2 and 40% CO 2 /60% N 2 ) and stored at 5 C for 124 days. Both cultures led to a reduction of 1–2 log CFU/g of L. innocua 2030c after 12 h; however, at the end of storage only Lb. sakei ST153 maintained this antilisterial effect, which was more evident at 40% CO 2 /60% N 2 . The influence of cultures addition and modified atmosphere packaging conditions on the sensory characteristics of the prod- uct were not significant. Thus, Lb. sakei ST153 combined with modified atmosphere packaging is a strong candidate to be used in a biopreservation strategy maintaining the traditional sensory quality of cured- smoked pork products and increasing their safety with respect to Listeria spp.Item Potential Control of Listeria monocytogenes by Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC Strains Isolated From Artisanal Cheese(Probiotics and Antimicrobial Proteins, 2019-03) Cavicchioli, Valéria Quintana; Camargo, Anderson Carlos; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoBacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC strains, previously isolated from artisanal cheese, were evaluated for their safety with the aim to determine whether they could be used as beneficial strains, especially in the control of Listeria monocytogenes. Both isolates survived simulated gastrointestinal conditions and showed high levels of auto- and co-aggregation with L. monocytogenes, although the hydrophobicity of cells varied. Using the agar-spot test with 33 commercial drugs from different groups, only anti-inflammatory drugs and drugs containing loratadine and propranolol hydrochloride were able to affect the growth of the tested strains. Both strains were resistant to 3 out of 11 antibiotics tested by the disc diffusion method, and low frequencies of antibiotic resistance-encoding genes were observed by PCR analysis. Tested strains neither presented biogenic amine-related genes nor produced these substances. Aside from some antibiotic resistance characteristics, the tested strains were considered safe as they lack other virulence-related genes. E. hirae ST57ACC and P. pentosaceus ST65ACC both presented beneficial properties, particularly their ability to survive gastrointestinal conditions and to aggregate with L. monocytogenes, which can facilitate the elimination of this pathogen. Further studies should be conducted to better understand these interactions.Item Lactobacillus curvatus UFV-NPAC1 and other lactic acid bacteria isolated from calabresa, a fermented meat product, present high bacteriocinogenic activity against Listeria monocytogenes(BMC Microbiology, 2019) Castilho, Nathália Parma Augusto; Colombo, Monique; Oliveira, Leandro Licursi de; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoBacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry.Three LAB strains (firstly named as Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles (rep-PCR, RAPD, bacteriocin-related genes) and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins.LAB isolates obtained from calabresa presented high inhibitory activity. Among these isolates, bacteriocins produced by L. curvatus 12, now named as L. curvatus UFV-NPAC1, presented the highest inhibitory performance and the purification procedures revealed four peptides with sequences not described for bacteriocins to date.Item Antibiotic resistance of Listeria monocytogenes isolated from meat-processing environments, beef products, and clinical cases in Brazil(Microbial Drug Resistance, 2015) Camargo, Anderson Carlos; Castilho, Natalia Parma Augusto de; Silva, Danilo Augusto Lopes da; Vallim, Deyse Christina; Hofer, Ernesto; Nero, Luís AugustoThe present study aimed to assess the antimicrobial resistance and the presence of virulence markers in 137 Listeria monocytogenes isolates obtained from meat-processing environments, beef products, and clinical cases. All isolates were subject to molecular serogrouping and their antibiotic resistance profiles were assessed against 12 antimicrobials. In addition, isolates were subjected to detection of virulence marker genes (inlA, inlC, inlJ). The isolates were classified into serogroups 4b, 4d, 4a, or 4c (46%), 1/2c or 3c (27%), 1/2a or 3a (13.9%), and 1/2b or 3b (13.1%). All tested isolates presented sensitivity to the majority of the tested antimicrobials, but most of them presented resistance or intermediate resistance to clindamycin (88.3%) and oxacillin (73.7%). Virulence markers were detected in all isolates, demanding further analysis to better characterize their pathogenic potential.Item Lactic acid bacteria (LAB) and their bacteriocins as alternative biotechnological tools to control Listeria monocytogenes biofilms in food processing facilities(Molecular Biotechnology, 2018-09) Camargo, Anderson C.; Todorov, Svetoslav D.; Chihib, N. E.; Drider, D.; Nero, Luís A.Bacteriocins are antimicrobial peptides produced by bacteria Gram-negative and Gram-positive, including lactic acid bacteria (LAB), organisms that are traditionally used in food preservation practices. Bacteriocins have been shown to have an aptitude as biofilm controlling agents in Listeria monocytogenes biofilms, a major risk for consumers and the food industry. Biofilms protect pathogens from sanitization procedures, allowing them to survive and persist in processing facilities, resulting in the cross-contamination of the end products. Studies have been undertaken on bacteriocinogenic LAB, their bacteriocins, and bioengineered bacteriocin derivatives for controlling L. monocytogenes biofilms on different surfaces through inhibition, competition, exclusion, and displacement. These alternative strategies can be considered promising in preventing the development of resistance to conventional sanitizers and disinfectants. Bacteriocins are “friendly” antimicrobial agents, and with high prevalence in nature, they do not have any known associated public health risk. Most trials have been carried out in vitro, on food contact materials such as polystyrene and stainless steel, while there have been few studies performed in situ to consolidate the results observed in vitro. There are strategies that can be employed for prevention and eradication of L. monocytogenes biofilms (such as the establishment of standard cleaning procedures using the available agents at proper concentrations). However, commercial cocktails using alternatives compounds recognized as safe and environmental friendly can be an alternative approach to be applied by the industries in the future.Item Characterization of a novel bacteriocin produced by Lactobacillus plantarum ST8SH and some aspects of its mode of action(Annals of Microbiology, 2015-11-17) Todorov, Svetoslav Dimitrov; Holzapfel, Wilhelm; Nero, Luis AugustoBacteriocins produced by lactic acid bacteria (LAB) are ribosomally synthesized antimicrobial peptides, with a diverse mode of bactericidal activity. This study focused on characterization of the bactericidal activity of bacteriocin ST8SH, with special attention to control of Listeria and Enteroccus species. Lactobacillus plantarum ST8SH produces a bacteriocin of the pediocin PA-1 family (sharing 96 % similarity on genetic level) with activity against several LAB, Enterococcus spp., Klebsiella pneumoniae, Listeria spp., Streptococcus spp. and some other human and foodborne pathogens. Addition of bacteriocin ST8SH to exponential or stationary phase cultures of L. monocytogenes ScottA and E. faecalis ATCC 19433 inhibited growth for 12 h. The effects of bacteriocin ST8SH on L. monocytogenes ScottA and E. faecalis ATCC 19433 were recorded indirectly based on enzyme, protein and nucleotide material leakage. Considering the antimicrobial activity of bacteriocin ST8SH against the tested microorganisms, and the physiological characteristics of Lb. plantarum ST8SH, either the bacteriocin or the strain may be used as tools for biopreservation.Item Lactobacillus plantarum isolated from cheese: production and partial characterization of bacteriocin B391(Annals of Microbiology, 2017-05-17) Nero, Luis Augusto; Fernandes, Paulo; Loureiro, Daniela; Monteiro, Vitor; Ramos, Carla; Todorov, Svetoslav Dimitrov; Guerreiro, Joana SantosLactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.Item Expression of genes associated with stress conditions by Listeria monocytogenes in interaction with nisin producer Lactococcus lactis(Food Research International, 2017-12-13) Miranda, Rodrigo Otávio; Campos-Galvão, Maria Emilene Martino; Nero, Luís AugustoThe use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2 log in BHI and 1 log in milk after 24 h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24 h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods.Item Genetic diversity and some aspects of antimicrobial activity of lactic acid bacteria isolated from goat milk(Applied Biochemistry and Biotechnology, 2015-01-31) Cavicchioli, Valéria Quintana; Dornellas, Wesley dos Santos; Perin, Luana Martins; Pieri, Fábio Alessandro; Franco, Bernadette Dora Gombossy de Melo; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoLactic acid bacteria (LAB, n = 57) were previously obtained from raw goat milk, identified as Lactococcus spp. (n = 24) and Enterococcus spp. (n = 33), and characterized as bacteriocinogenic. Fingerprinting by pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity, and 30 strains were selected and exhibited strong antimicrobial activity against 46 target strains (LAB, spoilage, and foodborne pathogens). Six strains (Lactococcus lactis: GLc03 and GLc05; and Enterococcus durans: GEn09, GEn12, GEn14, and GEn17) were selected to characterize their bacteriocinogenic features, using Listeria monocytogenes ATCC 7644 as the target. The six strains produced bacteriocins at higher titer when incubated in MRS at 37 °C up to 12 h, when compared to growth at 25 and 30 °C. The produced bacteriocins kept their antimicrobial activity after exposure to 100 °C for 2 h and 121 °C for 20 min; the antimicrobial activity was also observed after treatment at pH 2.0 to 10.0, except for GLc03. L. monocytogenes populations were reduced approximately two logs after treatment with cell-free supernatants from the selected strains. These data show that goat milk can contain a diverse microbiota able to inhibit L. monocytogenes, a common pathogen found in dairy products, and can be potentially employed in biopreservation of food produced under different processing conditions.