Intra- and inter-batch variability in raw pork challenge test studies and their consequences on model predictions: An intricate interplay between L. monocytogenes, the microbiome, and packaging atmosphere.

The purpose of this study was to conduct challenge studies in raw pork by strictly following all aspects of the 2014 EURL technical guidance document for conducting shelf-life studies on Listeria monocytogenes. Growth potential was assessed on three batches of self-cut pork chops and one batch of in-house prepared pure minced pork without any additives in air and MAP (70 % O2/30% CO2) packaging. Pork chops did not support the growth of the pathogen throughout the shelf-life, given the specific conditions used in this study, with growth potential values of 0.28 and 0.46 log CFU/g, respectively, for both air and MAP. Substantial growth (>0.5 log CFU/g) was obtained in minced pork after investigating only one batch, with growth potential values of 1.69 and 0.80 log CFU/g, for air and MAP. However, both intra- and inter-batch variability for pork chops and intra-batch variability for minced pork was observed; with elevated growth being evened out by the way growth potential is calculated in the EURL 2014 document, leading to underestimations and posing a potential risk to public health. Maximum growth rate in minced pork at a constant temperature of 7 °C was estimated at µmax = 0.680 log CFU/day and µmax = 0.489 log CFU/day in air and MAP, respectively. Model predictions for the growth potential showed acceptable results for air-packed minced pork with better accuracy when the lag phase was implemented as indicated in the renewed protocol (CRL EU, 2021). In MAP, all models used, including the Combase Growth model and to a lesser extent the DMRI dynamic safety model, overestimate the growth potential probably due to a lack of integration of the changing CO2 levels in the packages. The predictive models used in this study do not adequately account for the dynamics in the raw pig matrix, which may have an inhibitory effect on the growth of L. monocytogenes, including interaction with the microbiome and CO2, and emphasize the importance of remaining critical of predictive model outcomes. In addition, the experimental intra- and inter-batch variability raise questions about the sense or nonsense of using predictive microbiology in these raw pork products.

Time Is of the Essence-Early Activation of the Mevalonate Pathway in Apple Challenged With Gray Mold Correlates With Reduced Susceptibility During Postharvest Storage.

Apple is typically stored under low temperature and controlled atmospheric conditions to ensure a year round supply of high quality fruit for the consumer. During storage, losses in quality and quantity occur due to spoilage by postharvest pathogens. One important postharvest pathogen of apple is Botrytis cinerea. The fungus is a broad host necrotroph with a large arsenal of infection strategies able to infect over 1,400 different plant species. We studied the apple-B. cinerea interaction to get a better understanding of the defense response in apple. We conducted an RNAseq experiment in which the transcriptome of inoculated and non-inoculated (control and mock) apples was analyzed at 0, 1, 12, and 28 h post inoculation. Our results show extensive reprogramming of the apple's transcriptome with about 28.9% of expressed genes exhibiting significant differential regulation in the inoculated samples. We demonstrate the transcriptional activation of pathogen-triggered immunity and a reprogramming of the fruit's metabolism. We demonstrate a clear transcriptional activation of secondary metabolism and a correlation between the early transcriptional activation of the mevalonate pathway and reduced susceptibility, expressed as a reduction in resulting lesion diameters. This pathway produces the building blocks for terpenoids, a large class of compounds with diverging functions including defense. 1-MCP and hot water dip treatment are used to further evidence the key role of terpenoids in the defense and demonstrate that ethylene modulates this response.

Why mass allocation with representative allocation factor is preferential in LCA when using residual livestock products as organic fertilizers.

The production impact of agricultural residues are generally not accounted for in LCAs when they are given a second life as feedstocks for bio-fuels/plastics/fertilizers. Such is the case for the organic fertilizers manure and blood meal (stemming from livestock) used in organic cultivation. This raises questions on how realistic comparisons are between conventional cultivation systems and organic systems: the flows of resources and associated impacts are not represented equivalently in both systems. The aim of this study is to conduct sensitivity analyses for different possible allocation procedures and to select the most preferable one. The cultivation of organic apples in Flanders is used as case study. Considering no production impacts for organic fertilizers was firstly assessed as it is the generally used approach in LCA. In system expansion, the production impacts of two products: a mineral fertilizer and an organic plant-based fertilizer, are allocated to apple cultivation as a substitute for the organic fertilizers. For mass and economic allocation, the production impact is considered as a fraction of the impacts of the livestock system based on mass flows and economic value, respectively. Several possible allocation factors were assessed and price variations were considered. The different allocation procedures lead to diverging results, underlining the importance of selecting an appropriate procedure. Accounting no production impact is not advised since organic growers do not carry any environmental burden for a product they need for fertilization. System expansion causes too much uncertainties, needing to make speculative scenarios for factors lying outside the studied system. Economic allocation causes the impact of a product to change with its price while production stays the same. Therefore, mass allocation is preferable - though more harmonization research is needed - since no parameters from outside the system are needed, leading to a stable and close approximation of reality. For this case study, the amount of N available in fertilizers as a fraction of live weight and the mass of N in manure, is chosen as the least worse option. Our general recommendation is that the allocation factor needs to be chosen such that it is a representation of the function of the organic fertilizers and is comparable between different fertilizers.

Identifying Belgian protein consumption typologies by means of clustering and classification to move towards personalized advices for sustainable and nutritious food choices.

Protein sources are determinant for the environmental impact and nutritiousness of our food consumption patterns. Dietary advices, in terms of nutritional values and, more recently, the associated environmental impacts, are mostly formulated at nationwide level. However, actual food consumption patterns are variable between individuals within nations, leading to a need for more personalized dietary advices to ensure the feasibility of potential dietary changes. This research addresses the variability within Belgian food consumption patterns by identifying different "protein consumption typologies": consumer groups with different consumption frequencies of different protein sources. Hereto, both statistical clustering and classification were applied to food consumption data, the latter using predefined criteria related to meat consumption. The obtained typologies were further analyzed based on personal characteristics and compliance to nutritional guidelines. Five clusters and five classes were identified, with each survey participant belonging to one cluster and to one class. The clusters differed mainly in milk- and grain product intake, while the classes differed in the intake of meat products from the onset. Both clustering and classification showed that only a small part of the Belgian population frequently adopts plant-based protein sources, that meat products are predominant and that compliances to dietary guidelines are generally low. The typologies are the starting point for more personalized dietary advices to lower environmental impacts while ensuring adequate nutritional value, moving away from nationwide advices.

Genetic Listeria monocytogenes Types in the Pork Processing Plant Environment: From Occasional Introduction to Plausible Persistence in Harborage Sites.

The purpose of this study was to investigate the L. monocytogenes occurrence and genetic diversity in three Belgian pork cutting plants. We specifically aim to identify harborage sites and niche locations where this pathogen might occur. A total of 868 samples were taken from a large diversity of food and non-food contact surfaces after cleaning and disinfection (C&D) and during processing. A total of 13% (110/868) of environmental samples tested positive for L. monocytogenes. When looking in more detail, zone 3 non-food contact surfaces were contaminated more often (26%; 72/278) at typical harborage sites, such as floors, drains, and cleaning materials. Food contact surfaces (zone 1) were less frequently contaminated (6%; 25/436), also after C&D. PFGE analysis exhibited low genetic heterogeneity, revealing 11 assigned clonal complexes (CC), four of which (CC8, CC9, CC31, and CC121) were predominant and widespread. Our data suggest (i) the occasional introduction and repeated contamination and/or (ii) the establishment of some persistent meat-adapted clones in all cutting plants. Further, we highlight the importance of well-designed extensive sampling programs combined with genetic characterization to help these facilities take corrective actions to prevent transfer of this pathogen from the environment to the meat.

Study of the transfer of Listeria monocytogenes during the slaughter of cattle using molecular typing.

The introduction, transmission, and persistence of Listeria monocytogenes in Belgian beef slaughterhouses was investigated using genetic characterization. During slaughter, samples were taken of the hide, carcass, and environment to detect the pathogen. Remarkably, L. monocytogenes was massively present on the hide of incoming animals (93%; 112/120), regardless of their visual cleanliness, which implies high contamination pressure levels entering the slaughterhouses. Pathogen transfer via cross-contamination was conclusively confirmed in this study, with the same pulsotypes isolated from the hide, carcass, and environmental samples. Despite the important bacterial presence on the hide of incoming animals, most slaughterhouses succeeded in limiting the transfer as cause of carcass contamination. Persistence along the slaughter line seemed to be a more significant problem, as it was clearly linked to most of the L. monocytogenes positive carcasses. In one slaughterhouse, whole genome sequencing (WGS) revealed that the carcass splitter had been contaminating carcasses with the same strain belonging to CC9 for more than one year.

Modelling postmortem evolution of pH in beef M. biceps femoris under two different cooling regimes.

A model based on enzyme kinetics was developed to predict differences in postmortem pH change in beef muscles as affected by cooling rate. For the calibration and validation of the model, pH and temperature measurements were conducted at different positions in M. biceps femoris following conventional carcass cooling or faster cooling of the muscle after hot boning. The glycogen conversion, and, hence, the pH fall, was observed to significantly vary with position and cooling regime but only during the initial hours of cooling. Comparison of the cooling regimes indicated that fast cooling following hot boning avoids heat shortening induced by the combined effect of high temperature and low pH.

To disinfect or not to disinfect in postharvest research on the fungal decay of apple?

Postharvest losses of fruit and vegetables can reach up to 30%, the main cause being microbial decay. For apple fruit, mostly fungal pathogens, such as Penicillium expansum, Colletotrichum spp., Neofabraea spp. and Botrytis cinerea, are important. As such losses are unsustainable in many ways, it is necessary that research is conducted to prevent them. Generally, for plants and fruit grown under non-sterile field conditions, disinfection is carried out prior to the start of a phytopathological experiment. The motivation for this practice is the removal of background contamination so that it will not affect the experimental outcome and its interpretation. In literature, a plethora of disinfection methods exists, differing in disinfectant, strength and duration. The following two disinfectants are commonly used: sodium hypochlorite (NaOCl) and ethanol. This article presents a targeted investigation into the effects of these two disinfectants on apple fruit surface and physiology. The results clearly demonstrate that both were affected by both disinfectants. NaOCl caused oxidative damage to the apple's wax layer, causing it to crack. Ethanol affected a redistribution of the wax on the fruit surface and altered the wax composition and/or metabolism. Both NaOCl and ethanol treatment resulted in an increased respiration rate. Therefore, apple and possibly other fruit should not be disinfected in phytopathological studies. A negative control, as is typically used, is not solving this issue, as we clearly demonstrate that the living tissue shows metabolic effects following disinfection, and hence the study objects are changed, hampering a clear interpretation of the experimental outcomes. Moreover, fungal inoculation during experiments is typically taking place at rather large levels in wounded tissue (as infection success is the exception), outnumbering the variable levels of background population, if present.

Dynamic Labeling Reveals Temporal Changes in Carbon Re-Allocation within the Central Metabolism of Developing Apple Fruit.

In recent years, the application of isotopically labeled substrates has received extensive attention in plant physiology. Measuring the propagation of the label through metabolic networks may provide information on carbon allocation in sink fruit during fruit development. In this research, gas chromatography coupled to mass spectrometry based metabolite profiling was used to characterize the changing metabolic pool sizes in developing apple fruit at five growth stages (30, 58, 93, 121, and 149 days after full bloom) using 13C-isotope feeding experiments on hypanthium tissue discs. Following the feeding of [U-13C]glucose, the 13C-label was incorporated into the various metabolites to different degrees depending on incubation time, metabolic pathway activity, and growth stage. Evidence is presented that early in fruit development the utilization of the imported sugars was faster than in later developmental stages, likely to supply the energy and carbon skeletons required for cell division and fruit growth. The declined 13C-incorporation into various metabolites during growth and maturation can be associated with the reduced metabolic activity, as mirrored by the respiratory rate. Moreover, the concentration of fructose and sucrose increased during fruit development, whereas concentrations of most amino and organic acids and polyphenols declined. In general, this study showed that the imported compounds play a central role not only in carbohydrate metabolism, but also in the biosynthesis of amino acid and related protein synthesis and secondary metabolites at the early stage of fruit development.

Kinetic modelling: an integrated approach to analyze enzyme activity assays.

BACKGROUND: In general, enzyme activity is estimated from spectrophotometric data, by taking the slope of the linear part of the progress curve describing the rate of change in the substrate or product monitored. As long as the substrate concentrations are sufficiently high to saturate the enzyme and, the velocity of the catalyzed reaction is directly proportional to the enzyme concentration. Under these premises, this velocity can be taken as a measure of the amount of active enzyme present. Estimation of the enzyme activity through linear regression of the data should only be applied when linearity is true, which is often not the case or has not been checked. RESULTS: In this paper, we propose a more elaborate method, based on a kinetic modelling approach, to estimate the in vitro specific enzyme activity from spectrophotometric assay data. As a case study, kinetic models were developed to estimate the activity of the enzymes pyruvate decarboxylase and alcohol dehydrogenase extracted from 'Jonagold' apple (Malus x domestica Borkh. cv. 'Jonagold'). The models are based on Michaelis-Menten and first order kinetics, which describe the reaction mechanism catalyzed by the enzymes. CONCLUSIONS: In contrast to the linear regression approach, the models can be used to estimate the enzyme activity regardless of whether linearity is achieved since they integrally take into account the complete progress curve. The use of kinetic models to estimate the enzyme activity can be applied to all other enzymes as long as the underlying reaction mechanism is known. The kinetic models can also be used as a tool to optimize the enzyme assays by systematically studying the effect of the various design parameters.

A mechanistic modelling approach to understand 1-MCP inhibition of ethylene action and quality changes during ripening of apples.

BACKGROUND: 1-Methylcyclopropene (1-MCP) inhibits ripening in climacteric fruit by blocking ethylene receptors, preventing ethylene from binding and eliciting its action. The objective of the current study was to use mathematical models to describe 1-MCP inhibition of apple fruit ripening, and to provide a tool for predicting ethylene production, and two important quality indicators of apple fruit, firmness and background colour. RESULTS: A model consisting of coupled differential equations describing 1-MCP inhibition of apple ripening was developed. Data on ethylene production, expression of ethylene receptors, firmness, and background colour during ripening of untreated and 1-MCP treated apples were used to calibrate the model. An overall adjusted R2 of 95% was obtained. The impact of time from harvest to treatment, and harvest maturity on 1-MCP efficacy was modelled. Different hypotheses on the partial response of 'Jonagold' apple to 1-MCP treatment were tested using the model. The model was validated using an independent dataset. CONCLUSIONS: Low 1-MCP blocking efficacy was shown to be the most likely cause of partial response for delayed 1-MCP treatment, and 1-MCP treatment of late-picked apples. Time from harvest to treatment was a more important factor than maturity for 1-MCP efficacy in 'Jonagold' apples. © 2017 Society of Chemical Industry.

Cross-protection between controlled acid-adaptation and thermal inactivation for 48 Escherichia coli strains.

Given the importance of pH reduction and thermal treatment in food processing and food preservation strategies, the cross-protection between acid adaptation and subsequent thermal inactivation for 48 Escherichia coli strains was investigated. Those strains were selected among 188 E. coli strains according to their odds of growth under low pH conditions as determined by Haberbeck et al. (2015) [Haberbeck, L.U., Oliveira, R.C., Vivijs, B., Wenseleers, T., Aertsen, A., Michiels, C., Geeraerd, A.H., 2015. Variability in growth/no growth boundaries of 188 different Escherichia coli strains reveals that approximately 75% have a higher growth probability under low pH conditions than E. coli O157:H7 strain ATCC 43888. Food Microbiol. 45, 222-230]. E. coli cells were acid and nonacid-adapted during overnight growth in controlled acidic pH (5.5) and neutral pH (7.0), respectively, in buffered Lysogenic Broth (LB). Then, they were heat inactivated at 58°C in non-buffered LB adjusted to pH6.2 and 7.0. Thus, four conditions were tested in total by combining the different pH values during growth/thermal inactivation: 5.5/6.2, 5.5/7.0, 7.0/6.2 and 7.0/7.0. Acid adaptation in buffered LB at pH5.5 increased the heat resistance of E. coli strains in comparison with nonacid-adaptation at pH7.0. For instance, the median D58-value of strains inactivated at pH7.0 was approximately 6 and 4min for acid-adapted and nonacid-adapted strains, respectively. For the nonacid-adapted strains, the thermal inactivation at pH6.2 and 7.0 was not significantly (p=0.06) different, while for the acid-adapted strains, the thermal treatment at pH6.2 showed a higher heat resistance than at pH7.0. The correlation between the odds of growth under low pH previously determined and the heat resistance was significant (p<0.05). Remarkably, a great variability in heat resistance among the strains was observed for all pH combinations, with D58-values varying between 1.0 and 69.0min. In addition, highly heat resistant strains were detected. Those strains exhibited D58-values between 17.6 and 69.0min, while E. coli O157:H7 (ATCC 43888) showed D58-values between 1.2 and 3.1min. In summary, results clearly showed that adaptation of E. coli cells to constant acidic pH results in cross-protection against thermal inactivation.

Thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth versus agar surface.

The objective of the present study was to compare the thermal inactivation and sublethal injury kinetics of Salmonella enterica and Listeria monocytogenes in broth (suspended cells) and on solid surface (agar-seeded cells). A 3-strain cocktail of S. enterica or L. monocytogenes inoculated in broth or on agar was subjected to heating in a water bath at various set temperatures (55.0, 57.5 and 60.0°C for S. enterica and 60.0, 62.5 and 65°C for L. monocytogenes). The occurrence of sublethally injured cells was determined by comparing enumerations on nonselective (TSAYE) and selective (XLD or ALOA) media. Results showed that the inactivation curves obtained from selective media were log-linear, and significant shoulders (p<0.05) were observed on some of the inactivation curves from TSAYE media. The D-values derived from the total population were higher than those from the uninjured cells. Generally, cells on agar surface exhibited higher heat resistance than those in broth. For S. enterica, cell injury increased with the exposure time, no difference was observed when treated at temperatures from 55.0 to 60.0°C, while for L. monocytogenes, cell injury increased significantly with heating time and treatment temperature (from 60.0 to 65°C). Moreover, the degree of sublethal injury affected by thermal treatment in broth or on agar surface depended upon the target microorganism. Higher proportions of injured S. enterica cells were observed for treatment in broth than on agar surface, while the opposite was found for L. monocytogenes. The provided information may be used to assess the efficacy of thermal treatment processes on surfaces for inactivation of S. enterica and L. monocytogenes, and it provides insight into the sublethally injured survival state of S. enterica and L. monocytogenes treated in liquid or on solid food.

Slow softening of Kanzi apples (Malus×domestica L.) is associated with preservation of pectin integrity in middle lamella.

Kanzi is a recently developed apple cultivar that has an extremely low ethylene production, and maintains its crispiness during ripening. To identify key determinants of the slow softening behaviour of Kanzi apples, a comparative analysis of pectin biochemistry and tissue fracture pattern during different ripening stages of Kanzi apples was performed against Golden Delicious, a rapid softening cultivar. While substantial pectin depolymerisation and solubilisation was observed during softening in Golden Delicious apples, no depolymerisation or increased solubilisation was observed in Kanzi apples. Moreover, tissue failure during ripening was mainly by cell breakage in Kanzi apples and, in contrast, by cell separation in Golden Delicious apples. Kanzi apples had lower activity of beta-galactosidase, with no decline in the extent of branching of the pectin chain. A sudden decrease in firmness observed during senescence in Kanzi apples was not due to middle lamella dissolution, as tissue failure still occurred by cell breakage.

Thermal inactivation kinetics of surface contaminating Listeria monocytogenes on vacuum-packaged agar surface and ready-to-eat sliced ham and sausage.

The aim of this work was to study thermal inactivation kinetics of Listeria monocytogenes on vacuum-packaged food surfaces. The kinetics were first determined on model agar systems (BHI agar plates), mimicking cooked meat products, which have the same characteristics (pH, sodium chloride (NaCl) or sodium lactate (NaL) content and thickness) as the cooked meat products. Then, in order to validate how well the thermal inactivation on the model agar system simulated inactivation on real products, inactivation kinetics of L. monocytogenes on slices of cooked ham and cooked sausage were examined. BHI agar plates (pH6.2 or 7.2) were prepared with and without the addition of 3% NaCl or NaL. They were initially inoculated with approximately 109CFU/plate culture, aseptically packaged in linear low-density polyethylene pouches, and vacuum-sealed. Thermal treatments were performed by submerging packages in a water bath maintained at 60°C. For most of the conditions studied, the inactivation curves were linear; shoulders were only observed for curves at conditions of pH6.2 with 3.0% NaL. The t4D values (time needed to obtain an inactivation of four-log reduction) were calculated based on the best fitting models included in GInaFit. The observed t4D values for L. monocytogenes on agar surfaces ranged from 6.8 (pH6.2) to 13.7min (pH7.2 with 3.0% NaCl). At pH6.2 addition of NaCl or NaL significantly increased the heat resistance of L. monocytogenes while at pH7.2 this effect was not significant. NaL seemed to affect the heat resistance to the same extent as NaCl. Inactivation curves of L. monocytogenes on slices of cooked ham at pH6.2 with or without addition of NaCl or NaL appeared to be log-linear in shape. However, the curves obtained from cooked sausages were markedly concave and the Weibull model was used for fitting. Concerning heat resistance of L. monocytogenes on meat products, t4D values increased approximately two-fold compared to those corresponding on model agar surfaces. The addition of 3.0% NaCl and NaL in cooked ham increased t4D values of L. monocytogenes from 11.8 to 24.9min and 24.3min, respectively. Similar effects were observed on cooked sausage. Survival on the cooked sausage, containing about 33% of fat, was not significantly different from that on cooked ham. Meanwhile, the addition of NaCl or NaL decreased the average proportions of injured cells substantially. The results of this study can be used by food processors to validate thermal processes with regard to the expected inactivation of L. monocytogenes post-contaminating meat product surfaces.

Growth and inactivation of Salmonella enterica and Listeria monocytogenes in broth and validation in ground pork meat during simulated home storage abusive temperature and home pan-frying.

Ground pork meat with natural microbiota and inoculated with low initial densities (1-10 or 10-100 CFU/g) of Salmonella enterica or Listeria monocytogenes was stored under abusive temperature at 10°C and thermally treated by a simulated home pan-frying procedure. The growth and inactivation characteristics were also evaluated in broth. In ground pork meat, the population of S. enterica increased by less than one log after 12-days of storage at 10°C, whereas L. monocytogenes increased by 2.3 to 2.8 log units. No unusual intrinsic heat resistance of the pathogens was noted when tested in broth at 60°C although shoulders were observed on the inactivation curves of L. monocytogenes. After growth of S. enterica and L. monocytogenes at 10°C for 5 days to levels of 1.95 log CFU/g and 3.10 log CFU/g, respectively, in ground pork meat, their inactivation in the burger subjected to a simulated home pan-frying was studied. After thermal treatment S. enterica was undetectable but L. monocytogenes was recovered in three out of six of the 25 g burger samples. Overall, the present study shows that data on growth and inactivation of broths are indicative but may underestimate as well as overestimate behavior of pathogens and thus need confirmation in food matrix conditions to assess food safety in reasonably foreseen abusive conditions of storage and usual home pan-frying of meat burgers in Belgium.

Multi-objective experimental design for (13)C-based metabolic flux analysis.

(13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi-objective design should stimulate its application within the field of (13)C-based metabolic flux analysis.

Metabolic profiling reveals ethylene mediated metabolic changes and a coordinated adaptive mechanism of 'Jonagold' apple to low oxygen stress.

Apples are predominantly stored in controlled atmosphere (CA) storage to delay ripening and prolong their storage life. Profiling the dynamics of metabolic changes during ripening and CA storage is vital for understanding the governing molecular mechanism. In this study, the dynamics of the primary metabolism of 'Jonagold' apples during ripening in regular air (RA) storage and initiation of CA storage was profiled. 1-Methylcyclopropene (1-MCP) was exploited to block ethylene receptors and to get insight into ethylene mediated metabolic changes during ripening of the fruit and in response to hypoxic stress. Metabolic changes were quantified in glycolysis, the tricarboxylic acid (TCA) cycle, the Yang cycle and synthesis of the main amino acids branching from these metabolic pathways. Partial least square discriminant analysis of the metabolic profiles of 1-MCP treated and control apples revealed a metabolic divergence in ethylene, organic acid, sugar and amino acid metabolism. During RA storage at 18°C, most amino acids were higher in 1-MCP treated apples, whereas 1-aminocyclopropane-1-carboxylic acid (ACC) was higher in the control apples. The initial response of the fruit to CA initiation was accompanied by an increase of alanine, succinate and glutamate, but a decline in aspartate. Furthermore, alanine and succinate accumulated to higher levels in control apples than 1-MCP treated apples. The observed metabolic changes in these interlinked metabolites may indicate a coordinated adaptive strategy to maximize energy production.

Effectiveness of inactivation of foodborne pathogens during simulated home pan frying of steak, hamburger or meat strips.

In order to evaluate the effect of simulated home pan frying of raw meat and meat preparations of different animal species on the thermal inactivation of pathogens, the heat resistance (D-value) of three strains of Campylobacter jejuni, Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes and two strains of generic E. coli was validated in BHI and adjusted BHI (i.e. pH5.6 and 1.5% NaCl) at 60°C. The D-values were obtained of the linear phase of the survivor curves created in GInaFiT, a freeware tool to fit models to experimental data. The obtained D-values corresponded to those previously published in literature and confirmed L. monocytogenes to be the most heat resistant pathogen among them. Heat treatment in adjusted BHI significantly increased heat-resistance of E. coli O157:H7 and generic E. coli. Subsequently, the thermal inactivation of L. monocytogenes, Salmonella spp., C. jejuni and E. coli O157:H7 was evaluated using a standardized procedure simulating commonly used home pan frying of various types of meat including steaks or filets, hamburgers and meat strips from various animal species such as pork, beef, chicken, lamb and some turkey, horse, kangaroo and crocodile meat. Corresponding F70-values were calculated based upon measured core time/temperature profiles. It was noted that a core temperature of 70 °C was not always achieved and, moreover, a heat treatment equivalent to 2 min at 70 °C was also not always obtained. This was in particular noted in hamburgers although the meat was visually judged well done. On several occasions, residual survivors of the initial inoculated (4 logCFU/g) food borne pathogens could be recovered either by enumeration (limit of detection 1 logCFU/g) or by the presence/absence testing per 25 g. Pan frying of hamburgers yielded the highest number of surviving pathogenic bacteria (46%), followed by well-done filets and steaks (13%) and meat strips (12%). Taking only steaks (beef, horse, kangaroo, crocodile and turkey) into account, residual detection of pathogens occurred for all levels of doneness: 18% for well-done, 71% for medium and even 90% for rare steaks. Numbers of L. monocytogenes recovered after heat treatment ranged from <1 logCFU/g to 2.6 logCFU/g. Although, the prevalence of pathogens in meat might be low, and the numbers present in case of natural contamination are probably lower than the current used inoculum of 4 logCFU/g, consumers could still be exposed to surviving food borne pathogens in case of these commonly used pan frying of raw meat and meat preparations at consumer's home.

Formate hydrogen lyase mediates stationary-phase deacidification and increases survival during sugar fermentation in acetoin-producing enterobacteria.

Two fermentation types exist in the Enterobacteriaceae family. Mixed-acid fermenters produce substantial amounts of lactate, formate, acetate, and succinate, resulting in lethal medium acidification. On the other hand, 2,3-butanediol fermenters switch to the production of the neutral compounds acetoin and 2,3-butanediol and even deacidify the environment after an initial acidification phase, thereby avoiding cell death. We equipped three mixed-acid fermenters (Salmonella Typhimurium, S. Enteritidis and Shigella flexneri) with the acetoin pathway from Serratia plymuthica to investigate the mechanisms of deacidification. Acetoin production caused attenuated acidification during exponential growth in all three bacteria, but stationary-phase deacidification was only observed in Escherichia coli and Salmonella, suggesting that it was not due to the consumption of protons accompanying acetoin production. To identify the mechanism, 34 transposon mutants of acetoin-producing E. coli that no longer deacidified the culture medium were isolated. The mutations mapped to 16 genes, all involved in formate metabolism. Formate is an end product of mixed-acid fermentation that can be converted to H2 and CO2 by the formate hydrogen lyase (FHL) complex, a reaction that consumes protons and thus can explain medium deacidification. When hycE, encoding the large subunit of hydrogenase 3 that is part of the FHL complex, was deleted in acetoin-producing E. coli, deacidification capacity was lost. Metabolite analysis in E. coli showed that introduction of the acetoin pathway reduced lactate and acetate production, but increased glucose consumption and formate and ethanol production. Analysis of a hycE mutant in S. plymuthica confirmed that medium deacidification in this organism is also mediated by FHL. These findings improve our understanding of the physiology and function of fermentation pathways in Enterobacteriaceae.