RESUMO
BACKGROUND: The use of boar back fat for processing of fermented sausages may cause the presence of abnormal odours. In dry-cured products, ripening time is essential to develop the sensory characteristics. Yeast has been proposed as an alternative to mask boar taint odour through its metabolic activity but it is necessary to elucidate which mechanisms are involved. The aim is to study the effect of Debaryomyces hansenii inoculation on the lipolysis process and generation of aroma compounds in fermented sausages manufactured with boar back fat at two different ripening times. RESULTS: D. hansenii inoculated sausages had a higher degree of lipolysis as demonstrated by higher content of free fatty acids, ester compounds and branched aldehydes which contribute the fruity odour. The increase in lipolysis produced by D. hansenii inoculation was not followed by an increase in oxidation during processing possibly due to the metabolic activity of yeast. The effect of back fat type was scarcely appreciated whereas ripening time had a stronger effect on sausage. Boar sausages were characterised by a lower polyunsaturated fatty acid profile and lesser lipolysis than gilt sausages. CONCLUSION: Yeast inoculation with D. hansenii and long ripening time were appropriate strategies to limit the perception of boar taint in dry fermented sausages. © 2017 Society of Chemical Industry.
Assuntos
Debaryomyces/metabolismo , Aromatizantes/metabolismo , Produtos da Carne/microbiologia , Animais , Fermentação , Aromatizantes/análise , Microbiologia de Alimentos , Lipólise , Masculino , Produtos da Carne/análise , Odorantes/análise , Sódio/análise , Sódio/metabolismo , Sus scrofa , SuínosRESUMO
We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.
Assuntos
Pressão Osmótica , Estresse Oxidativo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/fisiologia , Sais/toxicidade , Estresse Fisiológico , Divisão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Citoplasma/química , Humanos , Fluidez de Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potássio/análise , Espécies Reativas de Oxigênio/análise , Saccharomycetales/química , Saccharomycetales/crescimento & desenvolvimento , Sódio/análiseRESUMO
Debaryomyces hansenii showed an increased growth in the presence of either 1 M, KCl or 1 M NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86Rb+ and 22Na+. 86Rb+ transport was saturable, with K(m) and Vmax values higher for cells grown in 1 M NaCl. 22Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1 M KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.
Assuntos
Cátions Monovalentes/metabolismo , Cloreto de Potássio/metabolismo , Saccharomycetales/fisiologia , Cloreto de Sódio/metabolismo , Trifosfato de Adenosina/análise , Cátions Monovalentes/farmacologia , Etanol/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana , Consumo de Oxigênio , Potássio/análise , Cloreto de Potássio/farmacologia , Saccharomycetales/crescimento & desenvolvimento , Sódio/análise , Cloreto de Sódio/farmacologiaRESUMO
The successful establishment of a drug screening system for intracellular cultivable and noncultivable mycobacteria based on the mass spectrometric determination of bacterial viability is described. To compare drug efficacies on intra- and extracellular mycobacteria, the mycobacteria were subjected to drug treatment either after phagocytosis by the mouse macrophage cell line RAW 264.7 or in cell-free medium. After reisolation, their viability was monitored by analyzing the intrabacterial sodium-to-potassium ratios for a limited number of individual organisms. This approach offers a reliable and quick tool for monitoring the influence of intracellular growth and of additional permeation barriers on intracellular drug efficacy and will thus provide useful information for the rational development and testing of optimized antimycobacterial drugs. In particular, the methodology is applicable to the noncultivable species Mycobacterium leprae, because the mass spectrometric analysis of the intrabacterial sodium-to-potassium ratio allows the determination of bacterial viability independent from their ability to multiply in vitro. Because of the improved metabolic activity of intracellularly growing M. leprae compared with that of extracellularly growing M. leprae, the spectrum of antileprosy drugs that can be tested in vitro could even be extended to those interfering with DNA replication and cell division.
Assuntos
Macrófagos/microbiologia , Mycobacterium/efeitos dos fármacos , Mycobacterium/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Antituberculosos/farmacologia , Tatus/microbiologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hansenostáticos/farmacologia , Macrófagos/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Mycobacterium/crescimento & desenvolvimento , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fagocitose/efeitos dos fármacos , Potássio/análise , Sódio/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TempoRESUMO
In a clinical trial including 17 multibacillary leprosy patients the in vivo effectiveness of ofloxacin on Mycobacterium leprae was tested via mass spectrometric determination of intrabacterial ratios of the concentrations of the sodium and potassium ions of individual organisms and of the ATP content per 10(6) bacteria isolated from skin biopsies. After 3 months of treatment, the in vivo drug effect could be determined with at least one of the two methods in 14 cases. Both methods revealed that in two cases the bacteria definitely did not respond to a 3-month ofloxacin monotherapy (200 mg twice daily). In three further cases a nonresponse of the M. leprae organisms was suspected from the mass spectrometric measurements. In the responder cases, the M. leprae were severely impaired. From the intrabacterial cation ratios the percentage of viable organisms averaged over all untreated biopsies was determined to be 58% and the percentage-killing during the first 3 months of treatment was 72%.
Assuntos
Trifosfato de Adenosina/análise , Eletrólitos/análise , Hanseníase/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Ofloxacino/farmacologia , Adolescente , Adulto , Idoso , Biópsia , Feminino , Humanos , Hanseníase/microbiologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Mycobacterium leprae/química , Mycobacterium leprae/metabolismo , Ofloxacino/uso terapêutico , Potássio/análise , Pele/microbiologia , Pele/patologia , Sódio/análiseRESUMO
Laser microprobe mass analysis of single bacterial organisms allows the determination of their intrabacterial ratio of sodium-to-potassium ions and the registration of fragment ions originating from their organic bacterial cell matrices as mass fingerprint spectra. It has been established previously that the intrabacterial cation ratio provides information on the physiological state of an individual bacterial cell. In the present experiments it is also shown, with different cultivable mycobacterial species and strains (drug sensitive and resistant) exposed to various drugs, that data derived from the evaluation of the mass fingerprint spectra reflect changes in the degree of impairment. The analysis of Mycobacterium leprae derived from a limited number of skin biopsies of lepromatous/borderline lepromatous leprosy patients under World Health Organization-recommended multiple-drug therapy (WHO/MDT) showed impairment of the organisms with both of the methods of measurement in proportion to the duration of treatment except in one case. In one M. leprae population from a patient who had been treated for 19 months, the fingerprint evaluation gave the first evidence for an insufficient response to treatment. This was further confirmed by the unusual frequency distribution of the Na+,K+ ratios which revealed the existence of two subpopulations, one impaired and one unimpaired.
Assuntos
Antibacterianos/farmacologia , Hansenostáticos/farmacologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium/efeitos dos fármacos , Potássio/análise , Sódio/análise , Resistência Microbiana a Medicamentos , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Hansenostáticos/uso terapêutico , Hanseníase Dimorfa/tratamento farmacológico , Hanseníase Dimorfa/microbiologia , Hanseníase Virchowiana/tratamento farmacológico , Hanseníase Virchowiana/microbiologia , Espectrometria de Massas , Mycobacterium/análise , Mycobacterium leprae/análise , Polimixina B/farmacologia , Polimixina B/uso terapêutico , Rifampina/farmacologia , Rifampina/uso terapêutico , Trimetoprima/farmacologia , Trimetoprima/uso terapêuticoRESUMO
The application of the laser microprobe mass analyser LAMMA 500 to the solution of problems in the field of microbiology is reported. The special features of this instrument allow the analysis of single bacterial cells, and questions can be answered which are not accessible to the normally applied integral methods. Thus it is possible to establish distributions of, for instance, elemental concentrations within a bacterial population and of correlations between measured characteristics of a bacterium with its morphology. The mass spectrum of a single bacterial cell comprises information on its intracellular cation contents as well as on the organic matrix. The relation between the sodium and potassium contents can serve as a criterion of the physiological state of a cell and of its viability. The information from the organic matrix can be extracted from the complex spectra of fragment ions produced by the interaction of the laser with the cell by applying multivariate data analysis, thus rendering additional information. Examples will be given for in vitro drug screening and in vivo therapy control in leprosy, an infection which is caused by a mycobacterial species, Mycobacterium leprae; this organism does not multiply in artificial growth media so that only limited numbers of organisms are available for microbiological investigations.