Protocol: An updated integrated methodology for analysis of metabolites and enzyme activities of ethylene biosynthesis

<p>Abstract</p> <p>Background</p> <p>The foundations for ethylene research were laid many years ago by researchers such as Lizada, Yang and Hoffman. Nowadays, most of the methods developed by them are still being used. Technological developments since then have led to small but significant improvements, contributing to a more efficient workflow. Despite this, many of these improvements have never been properly documented.</p> <p>Results</p> <p>This article provides an updated, integrated set of protocols suitable for the assembly of a complete picture of ethylene biosynthesis, including the measurement of ethylene itself. The original protocols for the metabolites 1-aminocyclopropane-1-carboxylic acid and 1-(malonylamino)cyclopropane-1-carboxylic acid have been updated and downscaled, while protocols to determine <it>in vitro </it>activities of the key enzymes 1-aminocyclopropane-1-carboxylate synthase and 1-aminocyclopropane-1-carboxylate oxidase have been optimised for efficiency, repeatability and accuracy. All the protocols described were optimised for apple fruit, but have been proven to be suitable for the analysis of tomato fruit as well.</p> <p>Conclusions</p> <p>This work collates an integrated set of detailed protocols for the measurement of components of the ethylene biosynthetic pathway, starting from well-established methods. These protocols have been optimised for smaller sample volumes, increased efficiency, repeatability and accuracy. The detailed protocol allows other scientists to rapidly implement these methods in their own laboratories in a consistent and efficient way.</p

Heat adaptation of Escherichia coli K12: Effect of acid and glucose

AbstractThe objective of this work is to investigate the effect of the (possible) acid adaptation during growth in a glucose rich environment on the heat resistance of Escherichia coli K12 MG1655. E. coli cells were grown in TSB and/or TSB dextrose free broth until they reached the stationary phase. Afterwards, the stationary phase cells were added in TSB and/or TSB dextrose free broth and inactivation took place at 54oC and 58oC. It was observed that growth in a glucose rich environment leads to an increased heat resistance, most likely due to a certain level of acid and further heat adaptation via cross protection

Artificial neural networks as a tool for incorporating microbial stress adaptations in the quantification of microbial inactivation

Quantifying microbial adapted responses due to thermal stresses by an accurate methodology is imperative for assessing the efficacy of a heat process. Two different artificial neural network (ANN) models are constructed for studying the increased induction of the heat resistance of Escherichia coli K12 under a treatment of decreasing heating rates. In the first model structure there are two input vectors, namely, time t and temperature rate dT=dt, whereas in the second case is also added a third one, namely, the microbial load delayed with one time unit Nk¡1. For both models a minimal fully-connected feedforward architecture is used consisting of one hidden neuron and one output neuron. Results as based on the prediction capability of the model structures demonstrate the comparative advantage when an ANN architecture with a delay in its inputs is employed. Incorporation of past events seems to be an essential input for taking into account the observed induced microbial heat resistance.peer-reviewe

Tissue specific analysis reveals a differential organization and regulation of both ethylene biosynthesis and E8 during climacteric ripening of tomato

Background: Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. Results: All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. Conclusions: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening

Integrated approach on heat transfer and inactivation kinetics of microorganisms on the surface of foods during heat treatments: Software development

The objective of this work was to create a software application (Bugdeath 1.0) for the simulation of inactivation kinetics of microorganisms on the surface of foods, during dry and wet pasteurisation treatments. The program was developed under the Real Basic 5.2 application, and it is a user-friendly tool. It integrates heat transfer phenomena and microbial inactivation under constant and time-varying temperature conditions. On the basis of the selection of a heating regime of the medium, the program predicts the food surface temperature and the change in microbial load during the process. Input data and simulated values can be visualised in graphics or data tables. Printing, exporting and saving file options are also available. Bugdeath 1.0 includes also a useful database of foods (beef and potato) and related thermal properties, microorganisms (Salmonella and Listeria monocytogenes) and corresponding inactivation kinetic parameters. This software can be coupled to an apparatus developed under the scope of the European Project BUGDEATH (QLRT-2001-01415), which was conceived to provide repeatable surface temperature-time treatments on food samples. The program has also a great potential for research and industrial applications

Modelling the combined effects of structured food model system and lactic acid on Listeria innocua and Lactococcus lactis growth in mono- and coculture

A new class of predictive model developed in a liquid system is extended in order to quantify gelatin gel matrix structure effects on the growth of Listeria innocua and Lactococcus lactis (both in mono- and coculture, and both producing mainly lactic acid). It was observed that gelatin does not only act as a structuring agent but also alters the buffering capacity of the medium. Model extension occurs in two stages, describing chemical and microbiological processes, respectively. Firstly, equations relating undissociated lactic acid concentration and total lactic acid concentration on the one hand, and undissociated lactic acid concentration and pH on the other hand, are extended to account for the effects of gelatin concentration. Secondly, these equations are incorporated into the growth model to describe the combined effect of gelatin concentration, (undissociated) lactic acid and pH on the growth of either microorganism. The description of the model is in good agreement with the experimental data acquired in monoculture conditions. In a subsequent model validation step, when gelatin concentration and total lactic acid profile of the coculture experiments are used as inputs, the developed growth model consisting of condensed knowledge extracted from the monoculture experiments, is able to predict accurately the interaction effect occurring in coculture. The study suggests that, on the one hand, the extent of the effects of undissociated lactic acid and pH on microbial growth in structured food systems can be modified by the increase in buffering capacity, which can protect microorganisms and eventually promote higher levels of cell growth in comparison with liquid culture conditions. On the other hand, food matrix structure, in casu the gelatin, reduces the rate of microbial multiplication. Both effects are incorporated in the growth model developed in this research.[**]status: publishe

Sensitivity analysis of a two-dimensional quantitative microbiological risk assessment: keeping variability and uncertainty separated

The aim of quantitative microbiological risk assessment is to estimate the risk of illness caused by the presence of a pathogen in a food type, and to study the impact of interventions. Because of inherent variability and uncertainty, risk assessments are generally conducted stochastically, and if possible it is advised to characterize variability separately from uncertainty. Sensitivity analysis allows to indicate to which of the input variables the outcome of a quantitative microbiological risk assessment is most sensitive. Although a number of methods exist to apply sensitivity analysis to a risk assessment with probabilistic input variables (such as contamination, storage temperature, storage duration, etc.), it is challenging to perform sensitivity analysis in the case where a risk assessment includes a separate characterization of variability and uncertainty of input variables. A procedure is proposed that focuses on the relation between risk estimates obtained by Monte Carlo simulation and the location of pseudo-randomly sampled input variables within the uncertainty and variability distributions. Within this procedure, two methods are used-that is, an ANOVA-like model and Sobol sensitivity indices-to obtain and compare the impact of variability and of uncertainty of all input variables, and of model uncertainty and scenario uncertainty. As a case study, this methodology is applied to a risk assessment to estimate the risk of contracting listeriosis due to consumption of deli meats

Development of a novel approach for secondary modelling in predictive microbiology: incorporation of microbiological knowledge in black box polynomial modelling

This research deals with the development of a novel secondary modelling procedure within the framework of predictive microbiology. The procedure consists of three steps: (i) careful formulation of the available microbiological information, both from literature and from the experimental case study at hand, (ii) translation of these requirements in mathematical terms under the form of partial derivatives throughout the complete interpolation region of the experimental design, and (iii) determination of parameter values with suitable optimisation techniques for a flexible black box modelling approach, e.g., a polynomial model or an artificial neural network model. As a vehicle for this procedure, the description of the maximum specific growth rate of Lactobacillus sakei in modified BHI-broth as influenced by suboptimal temperature, water activity, sodium lactate and dissolved carbon dioxide concentration is under study. The procedure results in a constrained polynomial model with excellent descriptive and interpolating features in comparison with an extended Ratkowsky-type model and classical polynomial model, by combining specific properties of both model types. The developed procedure is illustrated on the description of the lag phase as well. It is stressed how the confrontation with experimental data is very important to appreciate the descriptive and interpolating capacities of new or existing models, which is nowadays not always carefully performed. Alternatively, the first two steps of the novel procedure can be used as a tool to demonstrate clearly (possible) interpolative shortcomings of an existing model with straightforward spreadsheet calculations. (C) 2003 Elsevier B.V. All rights reserved.status: publishe

Shelf life of modified atmosphere packed cooked meat products: addition of Na-lactate as a fourth shelf life determinative factor in a model and product validation

Cooked meat products are often post-contaminated because of a packaging and/or slicing step after the pasteurisation process. The shelf life is therefore limited and can be extended by adding Na-lactate. A previously developed model for the spoilage of gas packed cooked meat products, including temperature, water activity and dissolved CO2 as independent variables, was extended with a fourth factor: the Na-lactate concentration in the aqueous phase of the meat product. Models were developed for the maximum specific growth rate mu(max) and the lag phase lambda of the specific spoilage organism Lactobacillus sake subsp. carnosum. Quadratic response surface equations were compared with extended Ratkowsky models. In general, response surface equations fitted the experimental data best but in the case of mu(max), the response surface model predicted illogical growth behaviour at low water activities and high Na-lactate concentrations. A extensive product validation of the mathematical models was performed by means of inoculated as well as naturally contaminated industrially prepared cooked meat products. The deviations of the experimentally determined versus predicted growth parameters in inoculated cooked meat products were in general small. Both types of models were also able to predict the shelf life of naturally contaminated cooked meat products, except for pate where an under-estimation of the shelf life was predicted by the response surface equations. The validation studies revealed higher accuracy of the extended Ratkowsky models in comparison to the response surface equations. A significant shelf life extending effect of Na-lactate was predicted, which was more pronounced at low refrigerated temperatures. A synergistic effect has also been noticed between Na-lactate and carbon dioxide which, at least partly, could be explained by the pH-decreasing effect of CO2. (C) 2000 Elsevier Science B.V. All rights reserved.status: publishe

Modeling the fate of Escherichia coli O157:H7 and Salmonella enterica in the agricultural environment: current perspective

The significance of fresh vegetable consumption on human nutrition and health is well recognized. Human infections with Escherichia coli O157:H7 and Salmonella enterica linked to fresh vegetable consumption have become a serious public health problem inflicting a heavy economic burden. The use of contaminated livestock wastes such as manure and manure slurry in crop production is believed to be one of the principal routes of fresh vegetable contamination with E. coli O157:H7 and S. enterica at preharvest stage because both ruminant and nonruminant livestock are known carriers of E. coli O157:H7 and S. enterica in the environment. A number of challenge-testing studies have examined the fate of E. coli O157:H7 and S. enterica in the agricultural environment with the view of designing strategies for controlling vegetable contamination preharvest. In this review, we examined the mathematical modeling approaches that have been used to study the behavior of E. coli O157:H7 and S. enterica in the manure, manure-amended soil, and in manure-amended soil-plant ecosystem during cultivation of fresh vegetable crops. We focused on how the models have been applied to fit survivor curves, predict survival, and assess the risk of vegetable contamination preharvest. The inadequacies of the current modeling approaches are discussed and suggestions for improvements to enhance the applicability of the models as decision tools to control E. coli O157:H7 and S. enterica contamination of fresh vegetables during primary production are presented