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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 Enhanced bacteriocin production by Pediococcus pentosaceus 147 in co-culture with Lactobacillus plantarum LE27 on Cheese Whey Broth(Frontiers in Microbiology, 2018-12) Nero, Luis Augusto; Gutiérrez-Cortés, Carolina; Suarez, Héctor; Buitrago, Gustavo; Todorov, Svetoslav DimitrovThe production of bacteriocins by lactic acid bacteria (LAB) has been of wide interest in the food industry due to their potential application in biopreservation. The production of bacteriocins is usually low in single strain fermentation, but can improve when the bacteriocinogenic strain is cultured in association with another bacteria. The present work aims to evaluate the growth and production of bacteriocins by Pediococcus pentosaceus 147 (bacteriocinogenic strain) in co-culture with Lactobacillus plantarum LE27 (inducer strain) using a culture medium based on cheese whey (CW). Strains were inoculated in co-culture in a CW broth at 7.24 Log CFU/mL of initial concentration of P. pentosaceus 147 and incubated at 37◦ C. Bacteriocin production was measured after 24 h by the critical dilution method, biomass was measured by plating on MRS agar (1% aniline blue), and a mono-culture was used as a control. The titers of bacteriocins produced by P. pentosaceus 147 in mono-culture were 19,200 AU/mL lower than those obtained in co-culture with Lb. plantarum LE27 at 51,200 AU/mL. The effect of adding the inducer strain at different times of incubation (3, 6, 9, and 12 h) was evaluated, with the addition of the induction factor at the beginning of the incubation of P. pentosaceus 147 generating the highest bacteriocin activity. This study shows the potential of inducing bacteriocinogenesis using co-cultures of strains of the genera Pediococcus and Lactobacillus and using alternative substrates such as cheese whey.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 Bacteriocinogenic LAB from cheeses – Application in biopreservation?(Trends in Food Science & Technology, 2015-01) Penna, Ana Lúcia Barretto; Todorov, Svetoslav Dimitrov; Favaro, LorenzoOver the last decade, there has been an explosion of basic and applied research on lactic acid bacteria bacteriocins, because of their potential as biopreservatives and inhibition of the growth of spoilage bacteria. Although bacteriocins can be produced during cheese production, their titers are much lower than those achieved in vitro fermentations under optimal physical and chemical conditions. Safety and technological traits of the bacteriocinogenic lactic acid bacteria (LAB) have to be considered before their wide-spread applications. This review described the perspectives and hurdles to be solved in order to definitively disclose the potential of bacteriocins in the production of safe and healthy cheese commodities.Item In vitro assessment of safety and probiotic potential characteristics of Lactobacillus strains isolated from water buffalo mozzarella cheese(Annals of Microbiology, 2017-02-28) Todorov, Svetoslav Dimitrov; Casarotti, Sabrina Neves; Carneiro, Bruno Moreira; Nero, Luis Augusto; Rahal, Paula; Penna, Ana Lúcia BarrettoThe aim of this study was to evaluate the safety and probiotic potential characteristics of ten Lactobacillus spp. strains (Lactobacillus fermentum SJRP30, Lactobacillus casei SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus SJRP50, SJRP76, and SJRP149) that had previously been isolated from water buffalo mozzarella cheese. The safety of the strains was analyzed based on mucin degradation, hemolytic activity, resistance to antibiotics and the presence of genes encoding virulence factors. The in vitro tests concerning probiotic potential included survival under simulated gastrointestinal (GI) tract conditions, intestinal epithelial cell adhesion, the presence of genes encoding adhesion, aggregation and colonization factors, antimicrobial activity, and the production of the β-galactosidase enzyme. Although all strains presented resistance to several antibiotics, the resistance was limited to antibiotics to which the strains had intrinsic resistance. Furthermore, the strains presented a limited spread of genes encoding virulence factors and resistance to antibiotics, and none of the strains presented hemolytic or mucin degradation activity. The L. delbrueckii subsp. bulgaricus strains showed the lowest survival rate after exposure to simulated GI tract conditions, whereas all of the L. casei and L. fermentum strains showed good survivability. None of the tested lactobacilli strains presented bile salt hydrolase (BSH) activity, and only L. casei SJRP145 did not produce the β-galactosidase enzyme. The strains showed varied levels of adhesion to Caco-2 cells. None of the cell-free supernatants inhibited the growth of pathogenic target microorganisms. Overall, L. fermentum SJRP30 and L. casei SJRP145 and SJRP146 were revealed to be safe and to possess similar or superior probiotic characteristics compared to the reference strain L. rhamnosus GG (ATCC 53103).Item Functional properties of Lactobacillus mucosae strains isolated from brazilian goat milk(Probiotics and Antimicrobial Proteins, 2016-12-10) Nero, Luís Augusto; Moraes, Georgia Maciel Dias de; Abreu, Louricélia Rodrigues de; Egito, Antônio Silvio do; Salles, Hévila Oliveira; Silva, Liana Maria Ferreira da; Todorov, Svetoslav Dimitrov; Santos, Karina Maria Olbrich dosThe search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.Item Characterization of bacteriocins produced by strains of Pediococcus pentosaceus isolated from Minas cheese(Annals of Microbiology, 2018-05-18) Nero, Luis Augusto; Gutiérrez-Cortés, Carolina; Suarez, Héctor; Buitrago, Gustavo; Todorov, Svetoslav DimitrovInterest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.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 Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk(International Journal of Food Microbiology, 2014-06-12) Perin, Luana Martins; Miranda, Rodrigo Otávio; Todorov, Svetoslav Dimitrov; Franco, Bernadette Dora Gombossy de Melo; Nero, Luís AugustoThe present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest®). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems.