Jean-Paul Chacornac

Safety improvement and preservation of typical sensory qualities of traditional dry fermented sausages using autochthonous starter cultures.

Traditional dry fermented sausages are manufactured without addition of starter cultures in small-scale processing units, their fermentation relying on indigenous microflora. Characterisation and control of these specific bacteria are essential for the sensory quality and the safety of the sausages. The aim of this study was to develop an autochthonous starter culture that improves safety while preserving the typical sensory characteristics of traditional sausages. An autochthonous starter composed of Lactobacillus sakei, Staphylococcus equorum and Staphylococcus succinus isolated from a traditional fermented sausage was developed. These strains were tested for their susceptibility to antibiotics and their production of biogenic amines. This starter was evaluated in situ at the French traditional processing unit where the strains had been isolated. Effects of the autochthonous starter were assessed by analysing the microbial, physico-chemical, biochemical and sensory characteristics of the sausages. Inoculation with the chosen species was confirmed using known species-specific PCR assays for L. sakei and S. equorum and a species-specific PCR assay developed in this study for S. succinus. Strains were monitored by pulse-field gel electrophoresis typing. Addition of autochthonous microbial starter cultures improved safety compared with the traditional natural fermentation of sausages, by inhibiting the pathogen Listeria monocytogenes, decreasing the level of biogenic amines and by limiting fatty acid and cholesterol oxidation. Moreover, autochthonous starter did not affect the typical sensory quality of the traditional sausages. This is the first time to our knowledge that selection, development and validation in situ of autochthonous starter cultures have been carried out, and also the first time that S. equorum together with S. succinus have been used as starter cultures for meat fermentation. Use of autochthonous starter cultures is an effective tool for limiting the formation of unsafe compounds in traditional sausage while preserving their original and specific sensory quality.

Low occurrence of safety hazards in coagulase negative staphylococci isolated from fermented foodstuffs

Some coagulase negative staphylococci (CNS) species play an important role in the fermentation of meat and milk products and are considered as food-grade. However, the increasing clinical significance of CNS and the presence of undesirable and unsafe properties in CNS question their presence or use in food. Our goal was to assess the safety of CNS by developing a diagnostic microarray targeting 268 genes corresponding to safety hazards in a food context i.e. toxins (especially enterotoxins) and determinants of antibiotic resistance and biogenic amine production. Target genes were selected among staphylococci and Gram-positive species that may be in contact with CNS in foodstuffs. The diagnostic microarray was used to screen 129 strains belonging to the 2 dominant species isolated from foodstuffs (S. equorum and S. xylosus) and the 2 main species isolated both in foodstuffs and clinical samples (S. epidermidis and S. saprophyticus). Microarray data were further completed by antibiograms and measurement of biogenic amine production. Safety hazards associated with CNS were mostly limited to the presence of antibiotic resistance. Seventy-one percent of the strains possessed at least one gene encoding antibiotic resistance, while only one strain carried an enterotoxin gene. Most strains did not carry any genes encoding staphylococcal toxins (68%), non-staphylococcal toxins (95%) or decarboxylases involved in biogenic amine production (78%). Food safety hazards were more pronounced in S. epidermidis than in the three other species regardless the food or clinical origin of the strains. Seventy-six percent of the strains carrying genes encoding staphylococcal toxin and 69% of strains carrying 5 or more antibiotic determinants belonged to S. epidermidis species. The dominant antibiotic resistance targeted erythromycin, tetracycline and penicillin and were generally traced back to the presence of tetK and blaZ in the two latest cases. Six percent of the food-related strains produced significant amounts of biogenic amines in vitro without any of the corresponding genes detected, reflecting a lack of knowledge on genetic determinants of such production in staphylococci. This work gives a first picture of safety hazards within four species of CNS frequently isolated from food or clinical environment.

Biodiversity of indigenous staphylococci of naturally fermented dry sausages and manufacturing environments of small-scale processing units.

The staphylococcal community of the environments of nine French small-scale processing units and their naturally fermented meat products was identified by analyzing 676 isolates. Fifteen species were accurately identified using validated molecular methods. The three prevalent species were Staphylococcus equorum (58.4%), Staphylococcus saprophyticus (15.7%) and Staphylococcus xylosus (9.3%). S. equorum was isolated in all the processing units in similar proportion in meat and environmental samples. S. saprophyticus was also isolated in all the processing units with a higher percentage in environmental samples. S. xylosus was present sporadically in the processing units and its prevalence was higher in meat samples. The genetic diversity of the strains within the three species isolated from one processing unit was studied by PFGE and revealed a high diversity for S. equorum and S. saprophyticus both in the environment and the meat isolates. The genetic diversity remained high through the manufacturing steps. A small percentage of the strains of the two species share the two ecological niches. These results highlight that some strains, probably introduced by the meat, will persist in the manufacturing environment, while other strains are more adapted to the meat products.

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.

Development of a New Oligonucleotide Array To Identify Staphylococcal Strains at Species Level

ABSTRACT The genus Staphylococcus is made up of 36 validated species which contain strains that are pathogenic, saprophytic, or used as starter cultures for the food industry. An oligonucleotide array targeting the manganese-dependent superoxide dismutase (sodA) gene was developed to overcome the drawbacks of the conventional methods of identification. Divergences of the sodA gene were used to design oligonucleotide probes, and we showed that each of the 36 species had a characteristic pattern of hybridization. To evaluate the array, we analyzed 38 clinical and 38 food or food plant Staphylococcus isolates identified by the phenotype-based system VITEK 2 (bioMérieux). This commercial kit failed to identify 8 (21%) of the clinical isolates and 32 (84%) of the food and food plant isolates. In contrast, the oligonucleotide array we designed provided an accurate and rapid method for the identification of staphylococcal strains, isolated from clinical, environmental, or food samples, at species level.

Genetic diversity and biofilm formation of Staphylococcus equorum isolated from naturally fermented sausages and their manufacturing environment

S. equorum is often isolated from naturally fermented sausages and from the environment of processing units. The aim of this work was first to characterize the genetic diversity of this species in a single small processing unit manufacturing traditional sausages without the use of starter cultures. One hundred and eighteen S. equorum isolates were collected from meat products and surfaces of this unit. Secondly, the capacity to form biofilm of 57 isolates of S. equorum selected from pulsed-field gel electrophoresis (PFGE) profiles was assessed to determine if this property conferred an advantage for the colonization of surfaces in the processing unit. Characterization of the isolates by PFGE analysis revealed a high diversity of the strains with 52 distinct PFGE patterns detected in this limited environment. It showed also that the exchanges between meat products and environmental surfaces could be limited or that the strains could be adapted to a specific niche as only four strains out of the 52 identified colonized both niches. The majority of the S. equorum strains formed biofilm; this was determined using a validated test on polystyrene microplates. This ability was not correlated with their origin, meat products or environmental surfaces.

The influence of high dietary protein, energy and mineral intake on deficient young camel (Camelus dromedarius)—II. Changes in mineral status☆

1. Mangrove Avicennia marina is poor in some trace elements such as copper, zinc and manganese. In a trial we used 32 young camels divided into four groups. 2. Groups 1 and 3 were supplemented with copper and zinc in drinking water after 1 month of mangrove feeding. 3. Groups 2 and 3 received concentrate rich in protein and energy. The supplementation was stopped after 2 months. 4. All the camels were deficient in trace elements at the beginning of mineral supplementation. 5. The plasma concentration of copper increased significantly up to normal levels (less than 70 micrograms/100 ml) in energy protein supplemented groups, but the quantity supplied (100 mg of copper sulphate/day) was not sufficient to maintain this level after the end of supplementation. 6. The original zinc deficiency was too severe to observe a significant effect of the mineral supplementation. 7. Calcium, magnesium and phosphorus levels were improved during the supplementation period in protein-energy supplemented groups. 8. A high interaction between mineral absorption and quality of the diet was observed. A well-balanced diet seems essential to avoid deficient mineral status.

METABOLIC PROFILES AND RISKS OF DISEASES IN CAMELS IN TEMPERATE CONDITIONS

A survey of 65 female camels has been conducted over a 1-year period in France to determine their metabolic profiles and to study the correlations between this profile and the feeding and health status in temperate conditions. The following parameters were measured: protein (albumin, globulin, total protein), urea, glucose, free fatty acid, liver enzymes (GLDH, GGT, GOT), minerals (Ca, Mg, Cu, Zn) and ceruloplasmin. The values obtained were similar to those reported in desert areas throughout the world, but the standard deviation was generally higher. This important variation might be due to the large variability of feeding conditions: albumin (36.4 +/- 4.7 g/l), total globulin (32.7 +/- 5.1 g/l), total protein (69.2 +/- 6.1 g/l), urea (30.0 +/- 14.8 mg/100 ml), glucose (111.0 +/- 12.2 mg/100 ml), FFA (0.15 +/- 0.15 mmol/l), GLDH (5.8 +/- 10.8 IU/l), GGT (10.1 +/- 5.8 IU/l), GOT (48.1 +/- 14.3 IU/l), calcium (10.2 +/- 6.5 mg/100 ml), magnesium (2.6 +/- 0.3 mg/100 ml), copper (65.4 +/- 20.2 micrograms/100 ml), zinc (34.6 +/- 7.8 micrograms/100 ml), ceruplasmin (41.4 +/- 2.6 UO). The season, the mineral supplementation and the health status had a significant effect on the metabolic profile of the she-camels.

Staphylococcal ecosystem of kitoza, a traditional malagasy meat product

Kitoza is a traditional meat product from Madagascar manufactured with strips of pork or beef. The process includes a first step of salting and mixing with spices followed by sun-drying or smoking step. As salting and drying select coagulase-negative staphylococci (CNS), our aim was to identify the CNS species in kitoza with the objective in the future of developing indigenous starters. Microbial analyses revealed that the only pathogenic bacterium enumerated was Staphylococcus aureus, which was found in 54% of the samples. The level of Enterobacteriaceae revealed a rather good hygienic quality of these products. CNS were confirmed in all the samples at high levels ranging from 5 to 7logcfu/g. Identification of CNS species in a large collection of 829 isolates revealed 9 identified species, 7 for beef and 8 for pork kitoza. There were significant difference in the distribution of CNS species according to the type of meat and the process. Staphylococcus saprophyticus was the dominant species for sun-dried or smoked beef and sun-dried pork kitoza (73-75%), while for smoked pork kitoza Staphylococcus equorum (26%), S. saprophyticus (23%), Staphylococcus succinus (23%) and Staphylococcus epidermidis (17%) co-dominated. Some CNS could be used as indigenous starters in particular to compete against S. aureus.

Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives

As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored.