Veterinária
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11842
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Resultados da Pesquisa
Item Beneficial properties of lactic acid bacteria naturally present in dairy production(BMC Microbiology, 2018-12) Colombo, Monique; Castilho, Nathália P. A.; Todorov, Svetoslav D.; Nero, Luís AugustoConsumers are increasingly demanding for natural and beneficial foods, in order to improve their health and well-being. Probiotics play an important role in such demand, and dairy foods are commonly used as vehicles for such bacteria, represented predominantly by lactic acid bacteria. Due to consumers demand, food industry is constantly looking for novel bacterial strains, leading to studies that aims the isolation and characterization of their beneficial features. This study aimed to characterize the naturally occurring lactic acid bacteria obtained from a dairy environment, in order to assess their potential use as probiotics.Preliminary screening and PCR analysis, based on 16S rRNA sequencing, were applied to select and identify 15 LAB strains from the genera Lactobacillus (n = 11), Pediococcus (n = 2) and Weissella (n = 2). All strains showed resistance to low pH and the evaluated bile salt concentrations in vitro. The API ZYM test characterized the enzymatic activity of the strains, and a high β-galactosidase activity was observed in 13 strains. All strains presented resistance to simulated gastric (3 h) and intestinal (4 h) conditions in vitro, the ability to auto- and co-aggregate with indicator microorganisms and a high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. All strains exhibited strong deconjugation of bile salts in vitro and all assimilated lactose.The phenotypes exhibited in vitro and the presence of beneficial genes revealed the beneficial potential of the studied strains, demanding further analyses in a food matrix and in vivo to allow the development of a functional product, with health-related properties.Item Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese(Journal of Dairy Science, 2010-07) Ortolani, M. B. T.; Moraes, P. M.; Perin, L. M.; Viçosa, G. N.; Carvalho, K. G.; Silva Júnior, A.; Nero, L. A.Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.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 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 Protocols for the isolation and detection of lactic acid bacteria with bacteriocinogenic potential(LWT - Food Science and Technology, 2010-11) Moraes, Paula Mendonça; Perin, Luana Martins Perin; Ortolani, Maria Beatriz Tassinari; Yamazi, Anderson Keizo; Viçosa, Gabriela Nogueira; Nero, Luís AugustoThe objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 °C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI + C) and M17 (both at 35 °C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 °C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 °C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI + C) in comparison to KFS (24 by M17 and 15 by BHI + C). The spot-on-the-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.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 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.Item Functional properties of Lactobacillus mucosa e strains isolated from brazilian goat milk(Probiotics and Antimicrobial Proteins, 2016-12-10) Nero, Luís Augusto; Todorov, Svetoslav Dimitrov; Moraes, Georgia Maciel Dias de; Abreu, Louricélia Rodrigues de; Egito, Antônio Silvio do; Salle, Hévila Oliveira; Silva, Liana Maria Ferreira da; 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 Bacterial ecology of artisanal Minas cheeses assessed by culture-dependent and -independent methods(Food Microbiology, 2017-02-12) Nero, Luís Augusto; Perin, Luana Martins; Sardaro, Maria Luisa Savo; Neviani, Erasmo; Gatti, MonicaArtisanal Minas cheese is produced in Minas Gerais state, Brazil and its varieties are named according to their geographical origin (Serro, Canastra, Serra do Salitre, Araxá and Campo das Vertentes). The cheese is produced with raw cow's milk and the whey from the previous cheese production (“pingo”). The high economic and cultural importance of artisanal cheese in Brazil justifies the efforts to ensure its safety, quality and provenance. This study aimed to characterize the microbial diversity composition, and geographical distribution of artisanal Minas cheese, focusing on the characterization of its autochthonous lactic acid bacteria (LAB) microbiota. Artisanal Minas cheese varieties from Serro, Canastra, Serra do Salitre, Araxá and Campo das Vertentes were analyzed by culture-dependent (culturing and LAB sequencing) and -independent (repetitive extragenic palindromic-PCR (rep-PCR) and length heterogeneity-PCR, LH-PCR) methods to characterize the microbiota. The microbial counts were variable between cheese samples, and some samples presented high number of coagulase positive bacteria and coliforms that may be associated with hygienic issues. In all samples was observed a prevalence of LAB. 16S rRNA sequencing and rep-PCR of the LAB strains identified four genus (Lactobacillus, Lactococcus, Enterococcus and Weissella), ten species and more than one strain per species. Lactobacillus was the most prevalent genera in all the cheeses. LH-PCR revealed a further six genera and ten species that were not identified by culturing, highlighting the importance of combining both culture-dependent and -independent methods to fully characterize microbiota diversity. Principal component analysis of the LH-PCR data and cluster analysis of rep-PCR data revealed that the artisanal Minas cheese microbiota was influenced not only by their geographical origin but also by the cheese farm. The lack of standardization in the milking and cheese manufacturing procedures between artisanal cheese farms could explain the microbial diversity.