Abstract This study aimed to investigate the antimicrobial activity of kefir fermented fruit by-products against strains of Alicyclobacillus spp., and determine their chemical characterization and antioxidant activity. According to the results, extracts fermented for a longer period (72 h) showed greater inhibition, and the extract of acerola by-product fermented for 72 h achieved the best results. For all strains, the Minimal Inhibitory Concentration (MIC) was 0.78%, except for A. acidocaldarius subsp. rittmannii, (1.56%). The same applies to the Minimal Bactericidal Concentration (MBC), in which 1.56% of the extract was capable of inactivating A. cycloheptanicus and A. acidocaldarius. In addition, damages to the structure of the microorganisms caused by the extracts were detected by Scanning Electron Microscopy. Metabolite identification through liquid chromatography (UHPLC-Qtof-MS) showed that the fermented extracts had a greater number of compounds compared to the non-fermented ones, such as glucuronic, succinic and glutaric acids. In conclusion, the extracts of fruit by-products showed to have bioactive properties, such as antibacterial potential and antioxidant activity against the Alicyclobacillus strains tested in this study, not to mention the value added by the use of a food by-product.

RESUMO: Protozoários do gênero Phytomonas são parasitas prejudiciais a várias culturas agrícolas de importância econômica. Devido a sua atividade biológica reconhecida, extratos brutos de folhas de Piper aduncum, P. crassinervium, P. hispidum e P. amalago, foram testadas pela técnica de microdiluição em placa para avaliar o seu potencial antiparasitário contra Phytomonas serpens. Os resultados mostraram que o extrato bruto etanólico de P. crassinervium e P. amalago apresentaram as melhores concentrações inibitórias para 50% das células (IC50), 16,5 µg mL-1 na fase clorofórmio e 18 µg mL-1 na fase aquosa, respectivamente, após 48 h de tratamento. Análises de citotoxicidade foram realizadas através do método colorimétrico da sulforodamina-B, em células de mamíferos LLCMK2. A fase clorofórmio de P. crassinervium foi submetida ao processo de fracionamento, no qual as frações acetato de etila e diclorometano obtiveram melhores valores de IC50. Imagens de microscopia eletrônica de varredura (MEV) mostraram alterações na membrana celular dos parasitas tratados com fase aquosa de P. amalago. Os dados obtidos indicam potencial efeito antiparasitário das espécies de Piper analisadas contra P. serpens, sendo consideradas candidatas promissoras para formulações de bioprodutos para controle do parasito.

ABSTRACT: Protozoa of the genus Phytomonas are harmful parasites to several agricultural crops of economic importance. Due to their recognized biological activity, crude extracts of Piper aduncum, P. crassinervium, P. hispidum, and P. amalago leaves, were tested using the microdilution plate technique to assess the antiparasitic potential against Phytomonas serpens. Results showed that the ethanolic crude extract of P. crassinervium and P. amalago presented the best inhibitory concentration for 50% of the cells (IC50), 16.5 µg mL-1 in chloroform phase, and 18 µg mL-1 in aqueous phase, respectively, after 48 h treatment. Cytotoxicity analyses were performed using the colorimetric method of sulforhodamine-B in LLCMK2 mammalian cells. The chloroform phase of P. crassinervium was subjected to the fractionation process, in which the ethyl acetate and dichloromethane fractions obtained better IC50 values. Scanning electron microscopy (SEM) images showed alterations in the cell membrane of the treated parasites. The data obtained indicate a potential antiparasitic effect of the Piper species analyzed against P. serpens, being considered promising candidates for formulations of bioproducts to control the parasite.

RESUMO: Os óleos essenciais são possíveis alternativas ao uso de praguicidas sintéticos no controle da contaminação por fungos. O óleo essencial de gengibre (Zingiber officinale - OEG) é conhecido por possuir propriedades antifúngicas e antiaflatoxigênicas, mas seu uso como fumigante in situ ainda não foi estudado. O objetivo deste estudo foi avaliar os efeitos do OEG sobre Aspergillus flavus como agente fumigante em grãos de milho armazenados. Os principais compostos encontrados no OEG foram α-zingibereno (23,85%) e geranial (14,16%), caracterizados por cromatografia gasosa acoplada a espectrometria de massas e ressonância magnética nuclear. O OEG foi utilizado como fumigante em grãos de milho irradiados em concentrações variando de 5 a 50 µg/g e os efeitos resultantes foram comparados a um agente antifúngico sintético (carbendazim e tiram), antifúngico tradicionalmente usado para o tratamento de sementes. A eficácia antifúngica do OEG contra A. flavus foi comprovada de maneira dependente da dose através do teste in situ (grãos de milho). O OEG inibiu a produção de aflatoxina nas concentrações 25 e 50 µg/g e controlou o crescimento de fungos. Portanto, o OEG pode ser usado como uma alternativa eficaz e não tóxica aos tratamentos convencionais em grãos de milho armazenados para o controle natural de A. flavus.

ABSTRACT: Essential oils are possible alternatives to the use of synthetic pesticides for control of fungal contamination. Ginger (Zingiber officinale) essential oil (GEO) is known for having antifungal and antiaflatoxigenic properties, but its use as a fumigant in situ has not been studied yet. The aim of this study was to evaluate GEO’s effects upon Aspergillus flavus as a fumigant agent in stored maize grains. The main compounds reported in GEO were α-zingiberene (23.85%) and geranial (14.16%), characterized by gas chromatography-mass spectrometry and nuclear magnetic resonance. The GEO was used as a fumigant in irradiated maize grains in concentrations ranging from 5 to 50 µg/g and the resulting effects were compared to a synthetic antifungal agent (carbendazim and thiram), an antifungal traditionally used for seed treatment. The antifungal efficacy of GEO against A. flavus has been proven in a dose-dependent manner through in situ (maize grains) test. The GEO inhibited aflatoxin production at concentrations 25 and 50 µg/g and controlled fungal growth. Therefore, GEO can be used as an effective and non-toxic alternative to conventional treatments in stored maize grains for the natural control of A. flavus.

RESUMO: O objetivo deste estudo foi avaliar o efeito da carga microbiana inicial, temperatura e tempo de contato na formação de biofilme de Alicyclobacillus acidoterrestris em aço inoxidável e borracha natural de qualidade alimentar utilizando suco de laranja como meio de cultura. A baixa carga inicial de A. acidoterrestris (2 log UFC/mL) levou à formação de biofilme na superfície do aço inoxidável após 48 h de contato a 28 ºC e após 24 h a 45 ºC, e na superfície natural de borracha de qualidade alimentar após 48 h de contato nas duas temperaturas. A alta carga microbiana inicial (5 log UFC/mL) levou à formação de biofilme em aço inoxidável após 4 h de contato a 28 °C e 45 °C, enquanto o biofilme foi formado em borracha natural de qualidade alimentar após 8 h de contato a 28 °C e 4 h a 45 °C. A carga microbiana também afetou a presença de esporos no biofilme, o que foi observado em ambas as superfícies apenas com altas cargas iniciais de A. acidoterrestris.

ABSTRACT: The objective of this study was to evaluate the effect of the initial microbial load, temperature and contact time on the biofilm formation of Alicyclobacillus acidoterrestris on stainless steel and natural food-grade rubber using orange juice as culture medium. The low initial load of A. acidoterrestris (2 log CFU/mL) led to biofilm formation on the stainless steel surface after 48 h of contact at 28 ºC and after 24 h at 45 ºC, and on natural food-grade rubber surface after 48 h of contact at both temperatures. The high initial microbial load (5 log CFU/mL) led to biofilm formation on stainless steel after 4 h of contact at 28 °C and 45 °C, while biofilm was formed on natural food-grade rubber after 8 h of contact at 28 °C and 4 h at 45 °C. The microbial load also affected the presence of spores in biofilm, which was observed on both surfaces only at high initial loads of A. acidoterrestris.

Resumo O principal objetivo do presente estudo foi sintetizar um nanocompósito usando grafeno e nanopartículas de ferrita de manganês (MnFe2O4-G) e avaliar sua atividade antibacteriana para aplicações em processos de tratamento de água. Suas características morfológicas foram avaliadas por técnicas instrumentais tais como microscopia eletrônica de varredura e microscopia eletrônica de transmissão. Os resultados de caracterização indicaram que o nanocompósito apresentou nanopartículas de aproximadamente 25 nm bem dispersas em nanofolhas de grafeno grandes (14 μm) e transparentes. A atividade antibacteriana foi avaliada em um experimento batelada usando uma concentração de 40 μg mL-1 de nanocompósito (MnFe2O4-G, nanopartículas de MnFe2O4 ou óxido de grafeno), 1x105 CFU mL-1 de Escherichia coli e 8 h de tempo de contato à temperatura ambiente. A maior capacidade antibacteriana foi observada para o nanocompósito híbrido (91,91%), decorrente do efeito sinérgico do grafeno e as nanopartículas de ferrita de manganês. Vários mecanismos foram propostos para explicar atividade antibacteriana efetiva do MnFe2O4-G, tais como aprisionamento, stress oxidativo, efeito de corte afiado, entre outros. Portanto, os resultados mostraram que MnFe2O4-G é uma alternativa em potencial para processes de tratamento de água como agente antibacteriano.

Abstract The main objective of this study was to synthesize a nanocomposite using graphene and manganese ferrite nanoparticles (MnFe2O4-G) and to evaluate its antibacterial activity for water treatment purposes. Its morphological characteristics were evaluated by instrumental techniques, such as scanning electron microscopy and transmission electron microscopy. The characterization results indicated that the nanocomposite presented nanoparticles of approximately 25 nm well dispersed in transparent and large (14 μm) graphene nanosheets. The antibacterial activity was evaluated in a batch experiment using a concentration of 40 μg mL-1 of nanocomposite (MnFe2O4-G, bare MnFe2O4 nanoparticles or graphene oxide), 1x105 CFU mL-1 of Escherichia coli, and 8 h of contact time at room temperature. The highest antibacterial capacity was observed for the hybrid nanocomposite (91.91%), due to the synergic effect of graphene and MnFe2O4 nanoparticles. Various mechanisms were proposed to explain the effective antibacterial activity of MnFe2O4-G, such as wrapping, oxidative stress, sharp-edge cutting effect, among others. The results showed that MnFe2O4-G is a potential alternative in water treatment processes as an antibacterial agent.

ABSTRACT The present study evaluated the antibacterial and antibiofilm activity of carvacrol against Salmonella Typhimurium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined and the time-kill curve and scanning electron microscopy (SEM) were performed to evaluate antibacterial activity. Antibiofilm activity was evaluated by quantifying total biomass using crystal violet assay, and metabolic activity was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The action of carvacrol against preformed biofilm on polypropylene and stainless steel was also evaluated by colony counting and SEM. The MIC and MBC was 312 µg mL-1. Carvacrol at MIC and 2 x MIC eliminated cells after 6 and 1 h of treatment, respectively, as exhibited in the time-kill curve. The greatest reduction in biofilm biomass and metabolic activity was 1,719 OD550 and 0,089 OD550 respectively, both at 4 x MIC of carvacrol. In carvacrol treated biofilms of S. Typhimurium on polypropylene, a reduction of 5.12 log was observed with 4 x MIC, while on stainless steel, carvacrol at 4 x MIC reduced bacterial counts by 5 log. The results showed that carvacrol exhibits antibacterial activity and can be used as an alternative for the control of S. Typhimurium biofilms.

The biosynthesis of amylase and collagenase, produced by A. acidocaldarius and A. sendaiensis respectively, were studied, and different matrices evaluated for the microorganisms immobilization and enzymes production optimization, such as loofa sponge, alginate-sponge and alginate, followed by concentration through ultrafiltration. Using a wheat bran substrate, the amylase enzyme displayed enzymatic activity of 0.45 U mL-1 and optimum temperature and pH conditions of 75 °C and pH 3.0, respectively. Thermal stability was in the range of 55 to 60 °C. The apparent Km and Vmax were 3.2 mg mL-1 and 0.5 U mL-1, respectively. The production of collagenase by A. sendaiensis was carried out with potato dextrose broth substrate and the activity obtained was 7.2 U mL-1. For amylase, the best results were obtained from immobilization in loofa sponge and the use of ultrafiltration (0.67 U mL-1) and for the collagenase extract, from the free biomass and ultrafiltration (13.6 U mL-1). The use of an ultrafiltration system enabled an average increase of 54% in the activity of both enzymes. Therefore, Alicyclobacillus are capable of producing enzymes of industrial interest, with the possibility of economically viable application of the substrate, and the use of immobilization and ultrafiltration produced positive results.

As bactérias do gênero Alicyiclobacillusspp.formam esporos e se desenvolvem em meios ácidos, podendo causar deterioração em sucos cítricos. O Brasil é o maior exportador de suco de laranja concentrado do mundo e, assim, este gênero vem sendo estudado por causar alterações de odor e sabor. Vários estudos relatam diferentes meios de culturas empregados para a detecção e enumeração de Alicyclobacillusspp.Este estudo teve como objetivo avaliar a recuperação de esporos de Alicyclobacillusspp.nos meios ALI, BAT, K ágar e YSG, utilizando a metodologia indicada pela ABECitrus. Cinco inóculos diferentes foram utilizados, sendo dois de linhagens-referência e os outros três isolados de suco concentrado de laranja pasteurizado. Também foi verificada a recuperação das células após o enriquecimento em suco de laranja reconstituído. Foi inoculada uma população inicial de 6 log UFC/mL. Os meios ALI, BAT e YSG conseguiram recuperar esta população nos diferentes inóculos, não existindo diferenças significativas entre os resultados. Contudo, devido à facilidade do preparo, os meios ALI e YSG mostraram-se mais vantajosos quando comparados ao meio BAT. O meio K ágar apresentou recuperação inferior aos outros meios para todos os inóculos, porém, houve diferença significativa apenas para Alicyclobacillus acidocaldarius 0298T (3,66 log UFC/mL) e Alicyclobacillus pomorum-like CBMAI 0278 (4,11 log UFC/mL).

Bacteria of the genus Alicyiclobacillus spp. form spores and develop in acid media, leading to the spoilage of citrus juices. Brazil is the largest exporter of orange juice concentrate, and yet, it has been extensively studied due to changes in taste and smell. Several investigations have reported different culture media used to detect and enumerate Alicyiclobacillus spp. The objective of the present study was to evaluate the recovery of Alicyiclobacillus spp. spores grown in ALI, BAT, K agar and YSG media using the methodology suggested by ABECitrus. Five inocula were used, two from reference strains and three from pasteurized concentrated orange juice. Cell recovery after the enrichment in reconstituted orange juice was also analyzed. An initial population of 6 log CFU/mL was inoculated. ALI, BAT and YSG media were able to recover the initial population of all different inocula, with no significant differences between the results. When compared to BAT, however, the preparation of ALI and YSG media was simpler and had more advantages. The recovery with K agar medium was lower than the other media for all the tested inocula, with significant differences found for Alicyclobacillus acidocaldarius0298T (3.66 log CFU/mL) and Alicyclobacillus pomorum-like CBMAI 0278 (4.11 log CFU/mL).

A porcentagem de amostras positivas para Aeromonas foi de 12.7% para água mineral, 8.3% para água de poço artesiano e 6.5% para água do sistema público de abastecimento. O isolamento de Aeromonas spp. foi significativamente maior em água mineral e água de poço artesiano do que em água do sistema público. A ocorrência de Aeromonas spp. não teve correlação significativa com os indicadores de contaminação tradicionalmente utilizados (coliformes totais) em amostras de água de poço artesiano. No entanto, esta correlação foi positiva e significativa em água mineral e água do sistema público. A presença ou ausência de correlação entre bactérias indicadoras e a presença de Aeromonas pode refletir o aparecimento ocasional de patógenos em águas para consumo humano e as diferentes taxas de sobrevivência e isolamento destes agentes comparados com os indicadores fecais de contaminação. A constatação de que 41.6%, 14.8% e 9.0% respectivamente amostras de água de poço, água mineral e água do abastecimento público utilizadas neste estudo apresentaram índices de coliformes maiores do que os aceitáveis pela legislação brasileira é um fato preocupante. Estes números mostram a necessidade de melhoria nos sistemas de monitoramento para a indústria de águas minerais e o sistema público de abastecimento. As cepas isoladas pertencentes ao gênero Aeromonas foram identificadas ao nível de espécie como A. hydrophila e A. jandaei. A significância do grande número de isolamentos de espécies de Aeromonas em saúde pública ainda não está clara. É necessário o estudo dos efeitos de cepas específicas utilizando modelos animais de infecção. Estes resultados podem contribuir para a reavaliação dos critérios empregados para a análise da qualidade microbiológica da água e a definição de limites de densidades para o gênero Aeromonas em águas destinadas ao consumo humano.

The aim of this work was to study the occurrence of Aeromonas sp in the bottled mineral water, well water and tap water from the municipal supplies. Positive samples were found for Aeromonas spp. 12.7% from the mineral water, 8.3% from the artesian water and 6.5% from the tap water. The recovery of Aeromonas spp. was significantly higher in the bottled mineral and artesian water than in the tap water from municipal supplies. The occurrence of the Aeromonas spp. did not correlate significantly with the contamination indicator bacteria (i.e. total coliforms) in the artesian water samples. However, a significant correlation was found between Aeromonas spp. and total coliforms in the both mineral water and tap water samples. The presence or absence of a correlation between the indicator bacteria and Aeromonas could reflect the occasional appearance of the pathogen in the drinking water and the different rates of survival and recovery of these agents compared with those fecal indicators. The finding that 41.6, 14.8 and 9.0 % of the artesian water, bottled mineral water and tap water, respectively, sampled in the current study failed to meet the Brazilian standard for total coliforms in the drinking water should therefore be of concern.

Ethanolic crude extracts from the roots of Chaptalia nutans, traditionally used in Brazilian folk medicine, were screened against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by using the disk diffusion test technique. S. aureus with 14 mm inhibition zone was considered susceptible. E. coli and P. aeruginosa without such a zone were considered resistant. As a result of this finding, the ethanolic crude extract was fractionated on silica gel column chromatography into five fractions. The ethyl acetate fraction was active against S. aureus and Bacillus subtilis. Further column chromatography separation of the ethyl acetate fraction afforded 30 fractions, which were assayed against S. aureus. Fractions 16 and 17 showed inhibition zones with S. aureus, indicating the presence of active compounds, and were subjected to purification by repeated preparative thin layer chromatography. The pure compound 7-O-beta-D-glucopyranosyl-nutanocoumarin inhibited B. subtilis and S. aureus at concentrations of 62.5 µg/ml and 125 µg/ml, respectively. The antibacterial property of C. nutans appears to have justified its use for the treatment of wounds, which are contaminated through bacterial infections.

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.