A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Clostridium sporogenes as surrogate for proteolytic C. botulinum - Development and validation of extensive growth and growth-boundary model.

An extensive cardinal parameter growth and growth boundary model for C. sporogenes, as a surrogate for proteolytic C. botulinum, was developed to include the inhibitory effect of 11 environmental factors. 626 maximum specific growth rates (µmax) in broth were generated to determine cardinal parameter values for the growth inhibiting effect of temperature, pH, NaCl/water activity (aw), organic acids (acetic, benzoic, citric, lactic, sorbic) and phosphate melting salts (ortho-, di- and tri-phosphates). µmax-values for C. sporogenes growing in well-characterized processed cheeses were used for product calibration (n = 10) and for product evaluation of the developed broth-model (n = 29). 112 growth/no-growth responses and including 104 µmax-values from the scientific literature for 58 different isolates of proteolytic and toxigenic C. botulinum (Group I) were used for further model evaluation. The developed model had less bias and a higher percentage of correct predictions than available models and was acceptable for processed cheese and good for meat products. The new and extensive model can predict combinations of environmental factors that prevent growth of C. sporogenes and of proteolytic C. botulinum. These predictions are expected to facilitate development or re-formulation of processed cheese and meat products where growth is prevented.

Microbial Safety and Sensory Analyses of Cold-Smoked Salmon Produced with Sodium-Reduced Mineral Salts and Organic Acid Salts.

Cold-smoked (CS) salmon contains high levels of sodium salts, and excess dietary sodium intake is associated with an array of health complications. CS salmon may also represent a food safety risk due to possible presence and growth of the foodborne pathogen Listeria monocytogenes which may cause fatal human infections. Here we determine how reformulated CS salmon using commercial sodium-reduced salt replacers containing KCl (e.g., Nutek, Smart Salt, SOLO-LITE) and acetate-based preservative salts (Provian K, proviant NDV) affect sensory properties, quality, and microbial safety. Initial sensory screening of sodium-reduced CS salmon was followed by L. monocytogenes growth analyses in selected variants of reformulated CS salmon, and finally by analyses of CS salmon variants produced in an industrial smokehouse. Projective mapping indicated overall minor sensory changes in sodium-replaced samples compared with a conventional product with NaCl. Growth of L. monocytogenes was temperature-dependent (4 °C vs. 8 °C storage) with similar growth in sodium-reduced and conventional CS salmon. The addition of 0.9% of the preservative salts Provian K or Provian NDV gave up to 4 log lower L. monocytogenes counts in both sodium-reduced and conventional cold-smoked salmon after 29 days of chilled storage. No changes in pH (range 6.20−6.33), aw levels (range 0.960−0.973), or weight yield (96.8 ± 0.2%) were evident in CS salmon with salt replacers or Provian preservative salts. Analyses of CS salmon produced with selected mineral salt and preservative salt combinations in an industrial salmon smokery indicated marginal differences in sensory properties. Samples with the preservative salt Provian NDV provided L. monocytogenes growth inhibition and low-level total viable counts (<2.8 log/g) dominated by Photobacterium and Carnobacterium during storage. Production of sodium-reduced CS salmon with inhibiting salts provides a simple method to achieve a healthier food product with increased food safety.

Extensive growth and growth boundary model for non-proteolytic Clostridium botulinum - Evaluation and validation with MAP and smoked foods.

The growth inhibiting effect of lactic acid bacteria (LAB) on non-proteolytic Clostridium botulinum was studied. LAB had no significant effect on growth of C. botulinum and their effect was not included in the model to be evaluated. An available cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum (Koukou et al., 2021; https://doi.org/10.1016/j.ijfoodmicro.2021.109162) was evaluated using a total of 822 time-to-toxin (TTT) formation data extracted from the scientific literature for seafood, poultry, vegetables and meat products. These data included smoked products and food stored in air, vacuum or modified atmosphere packaging (MAP) with added CO2. The available extensive model predicted TTT formation without bias (Bf-TTT value = 0.99) and with a reasonable accuracy (Af-TTT value = 1.76). The model was successfully validated for seafood and poultry products. This study substantially increased the range of applicability of the available growth and growth boundary model for non-proteolytic C. botulinum. The performed evaluation showed this model can be used to predict environmental conditions to prevent growth in seafood and poultry products including smoked fish and MAP foods. It is expected that this validated model will contribute to product development and innovation including new sodium reduced foods.

Cardinal parameter growth and growth boundary model for non-proteolytic Clostridium botulinum - Effect of eight environmental factors.

A new cardinal parameter growth and growth boundary model for non-proteolytic C. botulinum was developed and validated for fresh and lightly preserved seafood and poultry products. 523 growth rates in broth were used to determine cardinal parameter values and terms for temperature, pH, NaCl/water activity, acetic, benzoic, citric, lactic and sorbic acids. The new growth and growth boundary model included the inhibiting interactive effect between these factors and it was calibrated using growth curves from 10 challenge tests with unprocessed seafood. For model evaluation, 40 challenge tests with well characterized fresh and lightly preserved seafood were performed. Comparison of these observed growth curves and growth rates (µmax-values) predicted by the new model resulted in a bias factor (Bf) of 1.12 and an accuracy factor (Af) of 1.40. Furthermore, the new model was evaluated with 94 growth rates and 432 time to toxin formation data extracted from the scientific literature for seafood, poultry, meat, pasta and prepared meals. These data included responses for 36 different toxigenic strains of non-proteolytic C. botulinum. The obtained Bf-/Af-values were 0.97/2.04 for µmax-values and 0.96/1.80 for time to toxin formation. The model correctly predicted 93.8% of the growth responses with 5.6% being fail-safe and <1% fail-dangerous. A cocktail of four non-toxin producing Clostridium spp. isolates was used to develop the new model and these isolates had more than 99.8% 16S rRNA gene similarity to non-proteolytic C. botulinum (Group II). The high number of environmental factors included in the new model makes it a flexible tool to facilitate development or reformulation of seafood and poultry products that do not support the growth of non-proteolytic C. botulinum. Further, evaluation of the new model with well characterized products is desirable particularly for meat, vegetables, pasta and prepared meals as well as for dairy products that was not included in the present study.

Antimicrobial effect of nisin in processed cheese - Quantification of residual nisin by LC-MS/MS and development of new growth and growth boundary model for Listeria monocytogenes.

This study tested the hypothesis that growth of Listeria monocytogenes in processed cheese with added nisin can be predicted from residual nisin A concentrations in the final product after processing. A LC-MS/MS method and a bioassay were studied to quantify residual nisin A concentrations and a growth and growth boundary model was developed to predict the antilisterial effect in processed cheese. 278 growth rates were determined in broth for 11 L. monocytogenes isolates and used to determine 13 minimum inhibitory concentration (MIC) values for nisin between pH 5.5 and 6.5. To supplement these data, 67 MIC-values at different pH-values were collected from the scientific literature. A MIC-term was developed to describe the effect of pH on nisin MIC-values. An available growth and growth boundary model (doi: https://doi.org/10.1016/j.fm.2019.103255) was expanded with the new MIC-term for nisin to predict growth in processed cheese. To generate data for model evaluation and further model development, challenge tests with a total of 45 growth curves, were performed using processed cheese. Cheeses were formulated with 11.2 or 12.0 ppm of nisin A and heat treated to obtain residual nisin A concentrations ranging from 0.56 to 5.28 ppm. Below 15 °C, nisin resulted in extended lag times. A global regression approach was used to fit all growth curves determined in challenge tests. This was obtained by combining the secondary growth and growth boundary model including the new term for the inhibiting effect of nisin on µmax with the primary logistic growth model with delay. This model appropriately described the growth inhibiting effect of residual nisin A and showed that relative lag times depended on storage temperatures. With residual nisin A concentrations, other product characteristics and storage temperature as input the new model correctly predicted all observed growth and no-growth responses for L. monocytogenes. This model can support development of nisin A containing recipes for processed cheese that prevent growth of L. monocytogenes. Residual nisin A concentrations in processed cheese were accurately quantified by the developed LC-MS/MS method with recoveries of 83 to 110% and limits of detection and quantification being 0.04 and 0.13 ppm, respectively. The tested bioassay was less precise and nisin A recoveries varied for 53% to 94%.

Predicting growth of Listeria monocytogenes at dynamic conditions during manufacturing, ripening and storage of cheeses - Evaluation and application of models.

Mathematical models were evaluated to predict growth of L. monocytogenes in mould/smear-ripened cheeses with measured dynamic changes in product characteristics and storage conditions. To generate data for model evaluation three challenge tests were performed with mould-ripened cheeses produced by using milk inoculated with L. monocytogenes. Growth of L. monocytogenes and lactic acid bacteria (LAB) in the rind and in the core of cheeses were quantified together with changes in product characteristics over time (temperature, pH, NaCl/aw, lactic- and acetic acid concentrations). The performance of nine available L. monocytogenes growth models was evaluated using growth responses from the present study and from literature together with the determined or reported dynamic product characteristics and storage conditions (46 kinetics). The acceptable simulation zone (ASZ) method was used to assess model performance. A reduced version of the Martinez-Rios et al. (2019) model (https://doi.org/10.3389/fmicb.2019.01510) and the model of Østergaard et al. (2014) (https://doi.org/10.1016/j.ijfoodmicro.2014.07.012) had acceptable performance with a ASZ-score of 71-70% for L. monocytogenes growth in mould/smear-ripened cheeses. Models from Coroller et al. (2012) (https://doi.org/10.1016/j.ijfoodmicro.2011.09.023) had close to acceptable performance with ASZ-scores of 67-69%. The validated models (Martinez-Rios et al., 2019; Østergaard et al., 2014) can be used to facilitate the evaluation of time to critical L. monocytogenes growth for mould/smear-ripened cheeses including modification of recipes with for example reduced salt/sodium or to support exposure assessment studies for these cheeses.

Effect of the sodium reduction and smoking system on quality and safety of smoked salmon (Salmo salar).

Excessive sodium (Na) intake has been associated with high blood pressure and cardiovascular diseases. Therefore, sodium reduction is a public health challenge worldwide. The aim of this study was to develop smoked salmon with a reduced Na content. Sodium chloride (NaCl) was replaced by potassium chloride (KCl) at 25% and 50% (molar replacement) and studied in combination with two smoking procedures (natural wood and liquid smoke) as well as two smoking temperatures (18-19 °C or 56 °C). Smoked salmon samples were characterized by physicochemical, sensory and microbiological analyses. No major differences were observed regarding physicochemical properties in the studied treatments. Smoked samples with 50% of NaCl replaced by KCl were slightly more bitter than those with 25% whereas samples with 25% of replacement did not show differences to those with non-reduced Na content (5 g of added NaCl per 100 g of salmon). Molar Na:K ratio decreased from 4,3 in controls to 1,4 and 0,6 in samples with a NaCl reduction level of 25% and 50% respectively. Microbiological assessment indicates that 2-week shelf-life would be appropriate and safe in terms of accomplishment of the EU regulation, taking into account foreseeable storage temperatures (up to 8 °C). Thus, it is possible to achieve a reduction of 25-50% of NaCl in smoked salmon by replacing NaCl by KCl and considerer this product as a "source" of K.

Superchilling in combination with modified atmosphere packaging resulted in long shelf-life and limited microbial growth in Atlantic cod (Gadus morhua L.) from capture-based-aquaculture in Greenland.

Sensory, chemical and microbial changes for Atlantic cod (Gadus morhua L.) filets from capture-based-aquaculture in Greenland were studied. The objective was to determine shelf-life and indices of spoilage for iced or superchilled fillets when stored in air, or modified atmosphere packed (MAP; 40% CO2 and 60% N2). MAP iced storage extended the sensory shelf-life from 15 days to 21 days compared to storage in air. With superchilling at -1.7 °C sensory shelf-life was above 32 days, and no formation of total volatile nitrogen (TVN) was observed irrespective of storage in air or MAP. pH of ≥7.0, TVN (≥35 mg-N/100 g) and trimethylamine (≥20 mg-N TMA/100 g) were promising indices of spoilage. Aerobic viable counts were less valuable indices of spoilage as the dominating microbiota of cod in air (Pseudomonas spp., Photobacterium spp., Shewanella spp., Acinetobacter spp.) changed to Photobacterium spp. in MAP cod. Spoilage activity determined as the yield factor for TVN formation was 6-200 folds higher for Photobacterium spp. compared to Shewanella spp. and Pseudomonas spp. Photobacterium carnosum was responsible for TVN formation in iced cod irrespective of storage in air or MAP, and it was identified at the specific spoilage organism that limited iced product shelf-life.

Growth and growth boundary model with terms for melting salts to predict growth responses of Listeria monocytogenes in spreadable processed cheese.

The aim of this study was to develop and validate a growth and growth boundary model with terms for melting salts to predict growth of Listeria monocytogenes in spreadable processed cheese. Cardinal parameter terms for phosphate salts and citric acid were developed in broth studies and used to expand an available growth and growth boundary model. The expanded model includes the effect of nine environmental factors (temperature, pH, aw, lactic acid, acetic acid, citric acid, orthophosphate, di-phosphate and tri-phosphate). To generate growth data for model evaluation challenge tests with inoculated commercial (n = 10) and customized (n = 10) spreadable processed cheeses were performed. Evaluation of the new model by comparison of observed and predicted µmax-values resulted in a bias factor of 1.12 and an accuracy factor of 1.33 (n = 42). Prediction of growth and no-growth responses in processed cheese (n = 60) were 89% correct with 11% fail-safe and 0% fail-dangerous predictions. The developed model can be used to support product development, reformulation or risk assessment for spreadable processed cheese.

New Term to Quantify the Effect of Temperature on pH min -Values Used in Cardinal Parameter Growth Models for Listeria monocytogenes.

The aim of this study was to quantify the influence of temperature on pH min -values of Listeria monocytogenes as used in cardinal parameter growth models and thereby improve the prediction of growth for this pathogen in food with low pH. Experimental data for L. monocytogenes growth in broth at different pH-values and at different constant temperatures were generated and used to determined pH min -values. Additionally, pH min -values for L. monocytogenes available from literature were collected. A new pH min -function was developed to describe the effect of temperatures on pH min -values obtained experimentally and from literature data. A growth and growth boundary model was developed by substituting the constant pH min -value present in the Mejlholm and Dalgaard (2009) model (J. Food. Prot. 72, 2132-2143) by the new pH min -function. To obtain data for low pH food, challenge tests were performed with L. monocytogenes in commercial and laboratory-produced chemically acidified cheese including glucono-delta-lactone (GDL) and in commercial cream cheese. Furthermore, literature data for growth of L. monocytogenes in products with or without GDL were collected. Evaluation of the new and expanded model by comparison of observed and predicted µ max -values resulted in a bias factor of 1.01 and an accuracy factor of 1.48 for a total of 1,129 growth responses from challenge tests and literature data. Growth and no-growth responses of L. monocytogenes in seafood, meat, non-fermented dairy products, and fermented cream cheese were 90.3% correctly predicted with incorrect predictions being 5.3% fail-safe and 4.4% fail-dangerous. The new pH min -function markedly extended the range of applicability of the Mejlholm and Dalgaard (2009) model from pH 5.4 to pH 4.6 and therefore the model can now support product development, reformulation or risk assessment of food with low pH including chemically acidified cheese and cream cheese.

Predicting growth of Listeria monocytogenes in fresh ricotta.

Challenge tests with eight brands of fresh ricotta showed rapid growth of Listeria monocytogenes and significant variability in physical-chemical characteristics. Thus, two cardinal parameters models were developed for the growth of L. monocytogenes in ricotta including, respectively, terms for temperature (Model 1) and temperature and pH (Model 2). Also an extensive, existing growth model including the effect of organic acids (Model 3) was product recalibrated to predict growth of L. monocytogenes in ricotta. Interestingly, a lack of anti-listerial effect of organic acids in ricotta was observed in this study. The range of applicability of Models 1 and 2 in ricotta (characterized by absence of competitive microbiota) included storage at temperatures from 4.1 to 20.6 °C, pH from 5.5 to 6.6, moisture contents from 72% to 82%, NaCl from 0.38% to 0.60%, concentrations of acetic acid from 579 to 1559 ppm in the water phase, citric acid from 14,774 to 46,116 ppm in the water phase, and lactic acid from 0 to 4146 ppm in the water phase. Comparing observed and predicted maximum specific growth rates of L. monocytogenes in ricotta showed a bias-factor significantly above 1, for existing models developed for broth and these models were thus fail-safe with predicted growth being faster than observed, while typically below 1 for models developed for other food types. The models developed in the present study showed bias-factors of 1.10, 1.06 and 0.78, respectively, for Model 1, 2, and 3. In particular, Model 1 and 2 developed and successfully validated could allow an easy determination of safe shelf-life of ricotta and facilitated the reformulation the product with the aim to increase shelf-life or safety.

Modeling Growth of Listeria and Lactic Acid Bacteria in Food Environments.

Predictive food microbiology models can facilitate the assessment and management of microbial food safety. Importantly, the combined effect of storage conditions and product characteristics can be predicted by successfully validated models. This makes it easier and faster to develop or reformulation safe food recipes and predictions can be used to documents safety of available foods. The effect of various product characteristics and storage conditions must be taken into account and extensive mathematical models including the effect of these environmental factors are needed. Here the development, evaluation and application of an extensive growth and growth boundary model for Listeria monocytogenes including the effect of 12 environmental factors as well as the growth dampening effect of lactic acid bacteria is described. The Food Spoilage and Safety Predictor software is used to illustrate how predictions can be applied.

New insights on Listeria monocytogenes growth in pressurised cooked ham: A piezo-stimulation effect enhanced by organic acids during storage.

The aim of the present study was to understand growth and survival responses of Listeria monocytogenes during the storage of high pressure processed (HPP) cooked ham formulated with organic acids to inhibit growth of the pathogen. Cooked ham batches were manufactured without organic acids (control), with potassium lactate (2.8% or 4%) or with potassium lactate and sodium diacetate (2.0% + 0.11% or 2.0% + 0.45%). Products were aseptically sliced and inoculated with 107 cfu/g or 102 cfu/g of either L. monocytogenes CTC1034 (a meat isolate) or a cocktail of three isolates (12MOB045Lm, 12MOB089Lm and Scott A). Vacuum-packed samples with 107 cfu/g were HPP at 600 MPa for 3 min, whereas samples with 102 cfu/g were not HPP. Growth or survival of L. monocytogenes was determined during subsequent storage at 8, 12 and 20 °C. Growth or survival was characterized by fitting the experimental data using the primary logistic model and the log-linear with shoulder model, respectively. Secondary models were fitted to characterize the effect of temperature on growth kinetic parameters without or with HPP. For cooked ham without organic acids, growth rates of L. monocytogenes were slightly increased by HPP and lag times were longer. Interestingly, for cooked ham with organic acids, the HPP had a significant stimulating effect on subsequent growth of L. monocytogenes (piezo-stimulation). At 20 °C, the growth rates of L. monocytogenes in cooked ham with lactate were up to 4-fold higher than those of the same product without HPP. The observed enhancement of the piezo-stimulating effect of organic acids on growth rates during storage of HPP cooked ham represents a challenge for the use of organic acids as antimicrobials in these products. A predictive model available as part of the Food Spoilage and Safety Predictor (FSSP) software seemed useful to predict growth and growth boundary of L. monocytogenes in non-pressurised cooked ham. This model was calibrated to take into account the observed piezo-stimulating effect and to predict growth of L. monocytogenes in HPP cooked ham with organic acids.

Predicting outgrowth and inactivation of Clostridium perfringens in meat products during low temperature long time heat treatment.

With low temperature long time (LTLT) cooking it can take hours for meat to reach a final core temperature above 53°C and germination followed by growth of Clostridium perfringens is a concern. Available and new growth data in meats including 154 lag times (tlag), 224 maximum specific growth rates (µmax) and 25 maximum population densities (Nmax) were used to developed a model to predict growth of C. perfringens during the coming-up time of LTLT cooking. New data were generate in 26 challenge tests with chicken (pH6.8) and pork (pH5.6) at two different slowly increasing temperature (SIT) profiles (10°C to 53°C) followed by 53°C in up to 30h in total. Three inoculum types were studied including vegetative cells, non-heated spores and heat activated (75°C, 20min) spores of C. perfringens strain 790-94. Concentrations of vegetative cells in chicken increased 2 to 3logCFU/g during the SIT profiles. Similar results were found for non-heated and heated spores in chicken, whereas in pork C. perfringens 790-94 increased less than 1logCFU/g. At 53°C C. perfringens 790-94 was log-linearly inactivated. Observed and predicted concentrations of C. perfringens, at the time when 53°C (log(N53)) was reached, were used to evaluate the new growth model and three available predictive models previously published for C. perfringens growth during cooling rather than during SIT profiles. Model performance was evaluated by using mean deviation (MD), mean absolute deviation (MAD) and the acceptable simulation zone (ASZ) approach with a zone of ±0.5logCFU/g. The new model showed best performance with MD=0.27logCFU/g, MAD=0.66logCFU/g and ASZ=67%. The two growth models that performed best, were used together with a log-linear inactivation model and D53-values from the present study to simulate the behaviour of C. perfringens under the fast and slow SIT profiles investigated in the present study. Observed and predicted concentrations were compared using a new fail-safe acceptable zone (FSAZ) method. FSAZ was defined as the predicted concentration of C. perfringens plus 0.5logCFU/g. If at least 85% of the observed log-counts were below the FSAZ, the model was considered fail-safe. The two models showed similar performance but none of them performed satisfactorily for all conditions. It is recommended to use the models without a lag phase until more precise lag time models become available.

Modelling and predicting growth of psychrotolerant pseudomonads in milk and cottage cheese.

Mathematical models were developed and evaluated for growth of psychrotolerant pseudomonads in chilled milk and in cottage cheese with cultured cream dressing. The mathematical models include the effect of temperature, pH, NaCl, lactic acid and sorbic acid. A simplified cardinal parameter growth rate model was developed based on growth in broth. Subsequently, the reference growth rate parameter µref25°C-broth of 1.031/h was calibrated by fitting the model to a total of 35 growth rates from cottage cheese with cultured cream dressing. This resulted in a µref25°C-cottage cheese value of 0.621/h. Predictions from both growth rate models were evaluated by comparison with literature and experimental data. Growth of psychrotolerant pseudomonads in heat-treated milk (n=33) resulted in a bias factor (Bf) of 1.08 and an accuracy factor (Af) of 1.32 (µref25°C-broth), whereas growth in cottage cheese with cultured cream dressing and in non-heated milk (n=26) resulted in Bf of 1.08 and Af of 1.43 (µref25°C-cottage cheese). Lag phase models were developed by using relative lag times and data from both the present study and from literature. The acceptable simulation zone method showed the developed models to successfully predict growth of psychrotolerant pseudomonads in milk and cottage cheese at both constant and dynamic temperature storage conditions. The developed models can be used to predict growth of psychrotolerant pseudomonads and shelf life of chilled cottage cheese and milk at constant and dynamic storage temperatures. The applied methodology and the developed models seem likely to be applicable for shelf life assessment of other types of products where psychrotolerant pseudomonads are important for spoilage.

Volatile organic compounds and Photobacterium phosphoreum associated with spoilage of modified-atmosphere-packaged raw pork.

Accumulation of volatile organic compounds was monitored in association with sensory quality, bacterial concentrations and culture-independent microbial community analyses in raw pork loin and pork collar during storage under high-oxygen modified atmosphere at +4 °C. Of the 48 volatile compounds detected in the pork samples, the levels of acetoin, diacetyl and 3-methyl-1-butanol had the highest correlations with the sensory scores and bacterial concentrations. These compounds accumulated in all of the four monitored lots of non-sterile pork but not in the sterilized pork during chilled storage. According to the culture-dependent and culture-independent characterization of bacterial communities, Brochothrix thermosphacta, lactic acid bacteria (Carnobacterium, Lactobacillus, Lactococcus, Leuconostoc, Weissella) and Photobacterium spp. predominated in pork samples. Photobacterium spp., typically not associated with spoilage of meat, were detected also in 8 of the 11 retail packages of pork investigated subsequently. Eleven isolates from the pork samples were shown to belong to Photobacterium phosphoreum by phenotypic tests and sequencing of the 16S rRNA and gyrB gene fragments. Off-odors in pork samples with high proportion of Photobacterium spp. were associated with accumulation of acetoin, diacetyl and 3-methyl-1-butanol in meat, but these compounds did not explain all the off-odors reported in sensory analyses.

Stochastic modelling of Listeria monocytogenes single cell growth in cottage cheese with mesophilic lactic acid bacteria from aroma producing cultures.

A stochastic model was developed for simultaneous growth of low numbers of Listeria monocytogenes and populations of lactic acid bacteria from the aroma producing cultures applied in cottage cheese. During more than two years, different batches of cottage cheese with aroma culture were analysed for pH, lactic acid concentration and initial concentration of lactic acid bacteria. These data and bootstrap sampling were used to represent product variability in the stochastic model. Lag time data were estimated from observed growth data (lactic acid bacteria) and from literature on L. monocytogenes single cells. These lag time data were expressed as relative lag times and included in growth models. A stochastic model was developed from an existing deterministic growth model including the effect of five environmental factors and inter-bacterial interaction [Østergaard, N.B, Eklöw, A and Dalgaard, P. 2014. Modelling the effect of lactic acid bacteria from starter- and aroma culture on growth of Listeria monocytogenes in cottage cheese. International Journal of Food Microbiology. 188, 15-25]. Growth of L. monocytogenes single cells, using lag time distributions corresponding to three different stress levels, was simulated. The simulated growth was subsequently compared to growth of low concentrations (0.4-1.0 CFU/g) of L. monocytogenes in cottage cheese, exposed to similar stresses, and in general a good agreement was observed. In addition, growth simulations were performed using population relative lag time distributions for L. monocytogenes as reported in literature. Comparably good predictions were obtained as for the simulations performed using lag time data for individual cells of L. monocytogenes. Therefore, when lag time data for individual cells are not available, it was suggested that relative lag time distributions for L. monocytogenes can be used as a qualified default assumption when simulating growth of low concentrations of L. monocytogenes.

Development of a real-time PCR method coupled with a selective pre-enrichment step for quantification of Morganella morganii and Morganella psychrotolerans in fish products.

Histamine fish poisoning is common and due to toxic concentrations of histamine often produced by Gram-negative bacteria in fin-fish products with a high content of the free amino acid histidine. The genus Morganella includes two species previously reported to cause incidents of histamine fish poisoning. Morganella morganii and Morganella psychrotolerans are both strong producer of histamine. However, little is known about the occurrence and critical stages for fish contamination with these bacteria. To elucidate contamination routes of Morganella, specific real-time quantitative PCR (RTi qPCR) methods for quantification of M. morganii and M. psychrotolerans have been developed. Selective primers amplified a 110 bp region of the vasD gene for M. psychrotolerans and a 171 bp region of the galactokinase gene for M. morganii. These primer-sets showed high specificity as demonstrated by using purified DNA from 23 other histamine producing bacteria and 26 isolates with no or limited histamine production. The efficiency of the qPCR reactions on artificially contaminated fish samples were 100.8% and 96.3% respectively. The limit of quantification (LOQ) without enrichment was 4 log CFU/g. A quantitative enrichment step with a selective medium was included and improved the sensitivity of the methods to a LOQ of below 50 CFU/g in seafood. RTi qPCR methods with or without enrichment were evaluated for enumeration of Morganella species in naturally contaminated fresh fish and lightly preserved seafood from Denmark. These new methods will contribute to a better understanding of the occurrence and histamine production by Morganella species in fish products, information that is essential to reduce the unacceptably high frequency of histamine fish poisoning.