Traditional dry fermented sausages produced in small-scale processing units in Mediterranean countries and Slovakia. 1: Microbial ecosystems of processing environments.

Microbial ecosystems were surveyed in 314 environmental samples from 54 Southern and Eastern European small-scale processing units (PUs) manufacturing traditional dry fermented sausages. The residual microflora contaminating the surfaces and the equipment were analysed after cleaning and disinfection procedures. All the PU environments were colonised at various levels by spoilage and technological microflora with excessive contamination levels in some of the PUs. Sporadic contamination by pathogenic microflora was recorded. Salmonella and Listeria monocytogenes were detected in 4.8% and 6.7% of the samples, respectively, and Staphylococcus aureus was enumerated in 6.1% of the samples. Several critical points were identified, such as the machines for S. aureus and the tables and the knives for L. monocytogenes; this knowledge is crucial for the improvement of hygiene control systems in small and traditional meat processing industries. The variability of the residual contamination emphasized the different cleaning, disinfecting and manufacturing practices routinely followed by these small-scale processing units.

Diversity of microorganisms in the environment and dry fermented sausages of small traditional French processing units.

Naturally fermented sausages produced in nine traditional French processing units and their environmental surfaces were characterised by microbial and physico-chemical analyses. Salmonella and Staphylococcus aureus were not detected in the environment whereas Listeria monocytogenes was detected in four samples. Staphylococcus/Kocuria, lactic acid bacteria (LAB), Enterobacteriaceae, Pseudomonas, yeasts/moulds and enterococci contaminated the surfaces of two processing units, indicating insufficient cleaning and disinfection procedures. The final sausages did not present any health risk in seven of the processing units. In two of the processing units, the final sausages were contaminated with S. aureus and L. monocytogenes, respectively, at levels exceeding the maximum tolerable limit. Staphylococcus/Kocuria and LAB grew well in the products. Biogenic amines were found in the majority of the final products. Their occurrence was associated with high numbers of lactic acid bacteria and enterococci. The study outlined the processing and microbial diversities of French naturally fermented sausages.

Combined physico-chemical and water transfer modelling to predict bacterial growth during food processes.

The quality and safety of food products depend on the microorganisms, the food characteristics and the process. The prediction of conditions that prevent growth in complex situations due to the characteristics of the process and of the food cannot be obtained by predictive models of bacterial growth only. Thus, a combined modelling approach was developed by integrating three models, which were selected in a first step: (1) a bacterial model that predicts the bacterial growth from the physico-chemical properties of the media; (2) a water transfer model that predicts the effects of the drying process variables on the medium characteristics; and (3) a thermodynamic model that predicts the water activity aw and the pH of the media from its composition. A second step consisted in separately validating each selected model in which all of the physical, chemical or biological parameters appearing in the equations were previously measured. The third step combined the three knowledge models. The global model was validated on the basis of experimental results concerning the growth of Listeria innocua on the surface of a gelatine gel, the surface of which was submitted to a drying process (changes in relative humidity and air velocity). It was shown that bacterial growth models had to be modified: a specific model was set up to predict the maximum growth rate and another for the lag. Additionally, growth models set up in broth could not be applied in gelatine, leading to the development of a specific growth model on a solid surface. The thermodynamic model accurately predicted the pH and aw of bacterial broth in which high concentrations of solutes were added, and those of the solid media, the gelatine. The water transfer model was applied on gelatine data to predict the evolution of its surface aw during the drying process. The three models-bacterial, water transfer and thermodynamic, separately validated-were combined according to an integrated modelling strategy. The water transfer model coupled with the thermodynamic model predicted the aw on the gel surface. The predicted surface aw explained why growth inhibition was observed. Indeed, growth stopped at a predicted surface aw <0.94, corresponding to L. innocua minimum aw during the drying process. The global model satisfactorily predicted L. innocua growth on the surface of the gel. This study proves the validity of the approach and shows that the combination of the water transfer and thermodynamic models compensates for the lack of aw measurement techniques.

Serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables.

For consumers, tenderness is the most important sensory attribute of beef meat and, though to a lesser extent, of pork. Tenderness is therefore by far the most common cause of its unacceptability. The major challenge for the beef industry is to evaluate the toughness of the meat as soon as possible after death. In this context, the aim of the present work was to develop an equation to predict the myofibrillar ultimate resistance of raw meat. The study was done on the Longissimus muscle from twenty three 19 months-old Charolais bulls grown in the same INRA farm. Muscles excised within 1h post-mortem were vacuum packed and stored at 15°C during 24h and then transferred to 4°C until used. The activities of lactate dehydrogenase, citrate synthase and myofibrillar Mg-Ca dependent ATPase, the levels of lactate dehydrogenase enzyme, myoglobin, myosin types I, IIa and IIb, cysteine and serine peptidase inhibitors, the pH, the osmolarity, the expressible juice, µ-calpain, m-calpain and calpastatin and meat toughness were measured. According to the physical method used here, the force measured on raw meat represents the resistance of the myofibillar structure. Stepwise linear regression was used to determine the best equation (p<0.05) for predicting toughness at 6 days post-mortem. A 6-variables predictive equation including serine peptidase inhibitors (partial R(2)=0.4), the rate (partial R(2)=0.25), and the extent of pH decline (partial R(2)=0.03), the at death LDH activity (partial R(2)=0.24), the extent of increase in osmotic pressure (partial R(2)=0.13), and the rate of µ-calpain activity loss (partial R(2)=0.09), explained 70% of the variability in meat toughness at 6 days post-mortem. This equation was developed from 20 animals and the other 3 animals, chosen randomly, were used to validate it. The absolute need for a predictive model of meat toughness and the nature of the serine peptidase inhibitors together with their potential target enzymes are discussed.

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation.

Staphylococcus carnosus strain 833, inoculated into sausage, increased the levels of methyl ketones which contributed to the cured aroma. These ketones were predicted to arise from incomplete beta-oxidation followed by a decarboxylation. To check this hypothesis, we measured the beta-decarboxylase activity in resting cells of S. carnosus grown in complex or in synthetic medium, using as substrate a beta-ketoacid, which can be an intermediate of the beta-oxidation pathway. This activity was present throughout the growth period. The enzyme appeared to be constitutive because no induction was observed. High aeration, a pH of 5 and the presence of nitrate promoted the production of methyl ketones.

Roles of superoxide dismutase and catalase of Staphylococcus xylosus in the inhibition of linoleic acid oxidation.

Staphylococcus xylosus used as starter culture in sausages decreases the level of volatile organic compounds arising from lipid oxidation and so contributes to the aroma by avoiding rancidity. The aim of this study was to characterize the roles of catalase and superoxide dismutase (SOD) in the inhibition of free fatty acid oxidation by comparing antioxidant capacity of the S. xylosus wild-type strain with those of the katA mutant and the sod mutant. Antioxidant capacity was determined by measuring the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation. The three strains inhibited the oxidation of linoleic acid. However, the katA mutant, and especially the sod mutant, had less antioxidant capacity than the S. xylosus wild-type strain. Thus both catalase and SOD of S. xylosus contributed to the inhibition of lipid oxidation.

Modelling the growth of Listeria monocytogenes in dynamic conditions.

A recurrent neural network for the prediction of Listeria monocytogenes growth under pH and a(w) variable conditions was developed. The use of this model offered the possibility to take into account the consequences of the variations of the factors on L. monocytogenes growth. The effects of solutions, such as NaCl, acetic acid and NaOH, and their interactions on the response of L. monocytogenes cells were studied. Furthermore, the results showed the capacity of the recurrent neural network to predict growths carried out in different experimental conditions without using those used for its elaboration.

Application of recurrent neural network to predict bacterial growth in dynamic conditions.

A combination of a factorial design and two central composite designs was used to assess quantitatively the effects of acid pH (5.6-7.0) or alkaline pH (7.0-9.5) and NaCl (0-8%) variations on the growth of Listeria monocytogenes in a meat broth, at 20 degrees C and lower temperature 10 degrees C. Two principal phenomena were observed when bacteria were submitted to abrupt change of pH and a(w) during growth, whatever the growth temperature: (i) large environmental variations induced a lag phase following the fluctuation, and (ii) the growth continued with a generation time value different from that observed before the change or that associated to the new environment. A dynamic model, based on recurrent neural network (RNN), was developed to describe the growth of L. monocytogenes as a function of temperature and fluctuating conditions of acid pH, alkaline pH and concentration of NaCl. The results showed that the neural network model can be used to represent the complex effects of environmental variable conditions on the microorganism behaviour.

Growth of Pseudomonas fluorescens and Pseudomonas fragi in a meat medium as affected by pH (5.8-7.0), water activity (0.97-1.00) and temperature (7-25 degrees C).

A total of 59 strains of Pseudomonas, isolated from meat products, were grown in micro-titer plates in a meat medium over a range of pH (5.8-7.0), alpha w (0.97-1.00) and temperature (7-25 degrees C). Growths were performed in a meat broth with an automated turbidimeter (Bioscreen C, Labsystem, France). The growth curves obtained in this study did not have sigmoidal shapes making it impossible to calculate growth parameters using the Gompertz equation. The medium was weakly oxygenated in the micro-titer plates and reached 0%-dissolved oxygen at the beginning of the exponential phase. Strains were separated into two groups: P. fragi and P. fluorescens. Strains of P. fragi had shorter lag times than those of P. fluorescens. The impact of such results is interesting in that these could assist to explain the succession of flora that is observed during the processing of meat: P. fluorescens is the dominant bacteria among Pseudomonas spp. at the beginning of a slaughter line and P. fragi becomes dominant during the chilling process.

Application of polynomial models to predict growth of mixed cultures of Pseudomonas spp. and Listeria in meat.

Three models for one rapid and one slow growing strain of Pseudomonas fragi and one slow growing strain of P. fluorescens were developed in a meat broth; they were designed to take account of variations in growth and to provide a growth response interval. These models, and another for Listeria monocytogenes (Lm14 model), were used to predict the growth of spoilage Pseudomonas spp. and pathogenic Listeria in meat products. The Pseudomonas and Listeria models provided satisfactory predictions concerning inoculated strains grown in decontaminated beef meat. It was also possible to use the Pseudomonas models to predict the growth of the natural flora (mainly Pseudomonas spp.) of refrigerated meat stored under aerobic conditions. In experiments with mixed populations, three situations were observed: (1) in decontaminated meat, L. monocytogenes inoculated alone grew well at 6 degrees C, and this result was correctly predicted by the model; (2) in decontaminated meat inoculated with Listeria and Pseudomonas strains, L. innocua grew well and was not affected by the presence of Pseudomonas, and the growth of both organisms was correctly predicted by the models; (3) in naturally contaminated meat inoculated with Listeria, the strain did not grow until Pseudomonas had reached the stationary phase. The models satisfactorily predicted the growth of Pseudomonas spp. but not that of Listeria. In conclusion, the Lm14 model cannot be used for refrigerated meat stored aerobically as the results suggest a 'fail-safe' level which may be too high: meat had already reached a spoilage state even though no increase in the level of Listeria was observed. The Pseudomonas models accurately predicted the growth of naturally occurring Pseudomonas spp.

Effect of a(w), controlled by the addition of solutes or by water content, on the growth of Listeria innocua in broth and in a gelatine model.

The effect of a(w) on the growth of Listeria innocua was investigated in broth and on the surface of a gelatine food model. In broth, a(w) was controlled from 0.91 to 0.99 by the addition of solutes such as NaCl, KCl, glucose, sucrose and glycerol. In the gelatine food model, a(w) was controlled by removal of water. In the a(w) range, 0.92-0.99, the generation times observed in broth in the presence of NaCl, KCl, sucrose and glucose were similar but were longer than those in glycerol. For lag times, the inhibition of L. innocua growth followed the order: NaCl = KCl = sucrose>glucose>glycerol. When comparing growth at a(w) 0.95 for the three media--broth + NaCl, gelatine gel (a(w) controlled by removal of water) and gelatine gel with NaCl (gel + NaCl, a(w) controlled by NaCl)--the shortest generation time was observed in broth + NaCl, followed by gel + NaCl and, finally, on gel with a larger gap between the last two. The generation time on gel was five times greater than the generation time in broth + NaCl and 2.5 times greater on gel + NaCl. It was concluded that not only the structure of the media (solid or liquid) had an effect on Listeria inhibition but also and mainly the way the a(w) was adjusted. Removal of water was more stressful to Listeria than the addition of NaCl.

Effects of pH or a(w) stress on growth of Listeria monocytogenes.

The growth of three strains of Listeria monocytogenes at 20 degrees C in a meat broth of different pH or water activity was investigated. At inoculation or at the beginning of the exponential phase, cells were exposed to stress by the addition of NaOH or NH4+, acetic acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or an a(w) of 0.950 or 0.965, respectively. The effects of the exposure to stress on the generation and lag times of each strain were analysed by turbidity measurements for cultures in micro-titer plates. Results were confirmed by conducting the same experiments in a fermentor, except for the maximal population reached. The three strains showed similar behaviour. Cells were able to overcome the alkaline stress rapidly whereas acid and osmotic shocks induced important changes of the growth parameters. Cells exposed to acid or osmotic conditions from the time of inoculation were less affected than cells exposed at the beginning of the mid-exponential phase.

Comparison between two statistical models for prediction of turkey breast meat colour.

The aims of the present study were: (1) to determine the relevant objective measurements which could express visual assessment of turkey meat colour; and (2) to use these variables for the early prediction of the colour development of turkey breast meat. The colour of the meat was assessed subjectively by an expert at a processing plant at 24 hr post mortem, using a four-category scale (score a: light-pale meat, score b: light pink meat or normal meat, score c: dark meat, score d: very dark meat). Objective measurements included meat pH, temperature, dielectric loss factor, pigment concentration, L(∗) (lightness), a(∗) (redness) and b(∗) (yellowness) colour coordinates determined at different times post mortem. Colour coordinates and pH were chosen as relevant variables when measured at 1 and 4 hr post mortem and were used in prediction models. Linear analysis (canonical discriminant analysis) showed that the efficiency of prediction was 15%. A non-linear analysis (neural network) gave better prediction; the colour of the meat being correctly predicted for 70% of the muscles.

Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis.

The object of this study was to determine muscle characteristics which might predict meat toughness. Eleven Charolais cattle were slaughtered at approximately 26 months of age and the Longissimus lumborum et thoracis muscle was taken 1 hr post mortem and stored at 12 °C for 24 hr and then at 4 °C. The average half-life for ageing in these raw muscles was 4.6 days but the toughness varied widely between the animals. Toughness varied 3-fold and the rate of ageing varied 20-fold between animals. Correlations were done to determine which characteristics might explain this variability. Toughness was correlated positively with increase in oxidative status of muscle and the initial levels of calpastatin. Toughness was correlated negatively with the initial levels of µ- and m-calpains and cysteine and serine proteinase inhibitors, the initial pH values and the rates of their decline. The rates of ageing were highly correlated positively with the initial levels of proteinase inhibitors and the rates of decline of calpastatin and negatively with the ultimate amounts of expressible juice. There was a wide variability in tenderness in M. Longissimus lumborum et thoracis from similar animals. Variations in metabolism and enzyme activity controlled by inhibitors and calpains appear to be largely responsible for this variability.

[Research-action on medical and social management of uninsured pregnant women]. / Recherche-Action sur la prise en charge médico-sociale des femmes enceintes sans couverture sociale.

INTRODUCTION: Perinatal morbidity is increased among women without health insurance and/or receiving poor antenatal care. The objectives of this study were to estimate the prevalence of such situations, to analyze underlying social conditions, to identify possible means to secure health insurance and to assess the impact of the absence of health insurance on antenatal care and pregnancy outcomes. MATERIAL AND METHODS: All pregnant women without health insurance during one calendar year were included. Those who delivered a live child were compared with a control group of women with health insurance who delivered a live child until the date of the last delivery for uninsured women. Statistical analyses were conducted to describe the characteristics of uninsured women and to identify risk factors for poor antenatal care and pregnancy outcomes. RESULTS: A total of 259 uninsured women were enrolled, including 201 (78%) foreigners the majority of whom had resided in France for more than one year; 205 were followed-up and delivered in our hospital, including 192 live births. The social context of uninsured French women was worse than that of foreign uninsured women. Health insurance was obtained for 85% (173/205). Overall, the absence of health insurance was associated with increased risks for poor antenatal care. Being uninsured and receiving poor antenatal care was associated with an increase in the incidence of intra-uterine growth failure. Post-term deliveries were more frequent among women with poor antenatal care. CONCLUSIONS: Improving antenatal care and health insurance coverage could help decrease perinatal morbidity, given the current increase in the number of women with social risk factors. It seems essential that all women obtain access to public maternity wards, and that these wards develop effective social services working in concert with ambulatory health and social workers.

Effect of temperature and pH on growth of Staphylococcus aureus in co-culture with Lactococcus garvieae.

Staphylococcus aureus growth and enterotoxin production in co-culture with Lactococcus garvieae were studied in laboratory medium as a function of incubation temperature and pH values. Doehlert experimental design was used to study the effect of L. garvieae concentration, temperature, and pH on S. aureus growth in laboratory medium. The mathematical model obtained was validated in cheeses. The inhibition of S. aureus growth by L. garvieae was more important during the first 6 hours of incubation, and its effect increased when its concentration increased. After 24 and 48 hours, the effect of L. garvieae decreased, and the growth of S. aureus was positively influenced by higher temperature and pH values. Staphylococcal enterotoxins were detected in only one experimental set after 48 hours of incubation at 30 degrees C at pH 6.8. Our results argue in favor of adding antagonist strain early in the cheese-making process.

Modelling the behaviour of Listeria monocytogenes in ground pork as a function of pH, water activity, nature and concentration of organic acid salts.

AIMS: to study and model the effect of sodium acetate, sodium lactate, potassium sorbate and combination of acid salts on the behaviour of Listeria monocytogenes in ground pork. METHODS AND RESULTS: Water activity (a(w)), pH and concentration of acid salt of the meat were adjusted. The behaviour of inoculated L. monocytogenes was studied and modelled according to physicochemical parameters values. Whatever the acid salt concentration used, we observed an inhibition of the growth of L. monocytogenes at pH 5.6 and a(w) 0.95. At pH 6.2 and a(w) 0.97, addition of 402 mmol l(-1) of sodium lactate or 60 mmol l(-1) of potassium sorbate was required to observe a slower growth. CONCLUSIONS: The inhibitory effect of acid salts was a function of pH, a(w), as well as of the nature and concentration of acid salts added. When one acid salt was added, the Augustin's model (Augustin et al. 2005) yielded generally correct predictions of either the survival or growth of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment concerning L. monocytogenes in pork products.

Effects of physico-chemical factors influencing tyramine production by Carnobacterium divergens.

Tyramine production by a strain of Carnobacterium divergens was tested in relation to different conditions of pH, temperature, glucose, oxygen availability, potassium nitrate and sodium chloride content, using a combination of a Doehlert and Plackett-Burman experimental design. A second degree polynomial model was chosen to describe tyramine production which was quantified by high performance liquid chromatography. Maximal tyramine production occurred during the stationary phase in acidic conditions obtained by low initial pH (< 5) or addition of glucose (0.6%) to the medium. Production was slower at 5 degrees C than at 23 degrees C and 10% sodium chloride inhibited this production. However, the formation of tyramine was not affected by the presence of potassium nitrate or oxygen availability.