Marta Hugas

Amino acid-decarboxylase activity of bacteria isolated from fermented pork sausages.

The occurrence of amino acid-decarboxylase activity in 92 strains of lactic acid bacteria, coagulase-negative staphylococci, and Enterobacteriaceae isolated from Spanish fermented pork sausages was investigated. The presence of biogenic amines in a decarboxylase synthetic broth was determined by ion-pair high performance liquid chromatography with o-phtalaldehyde post-column derivatization. Among the 66 lactic acid bacteria strains tested, 21 lactobacilli (in particular, Lactobacillus curvatus) and all 16 enterococci were amine producers. Tyramine was the main amine produced by these bacteria, although they also produced phenylethylamine, tryptamine, and/or the diamines putrescine and cadaverine. None of the lactic acid bacteria produced histamine. Coagulase-negative staphylococci were found to be negative amine-producers. Aromatic monoamines, apart from histamine, were not formed by Enterobacteriaceae. This family was responsible for cadaverine and putrescine production. The results obtained for biogenic amine production by bacteria in a synthetic medium suggest that amino acid-decarboxylase activity is strain dependent rather than being related to specific species.

Functionalty of enterococci in meat products

The presence of enterococci in meat fermentation is a constant as reported in the literature. Despite the concern about pathogenicity of enterococci, recent studies point out that food and meat enterococci, especially Enterococcus faecium have a much lower pathogenicity potential than clinical strains. Enterococci possess a competitive advantage over other microbiota in meat fermentations, and many enterococci isolated from sausages have the ability to produce enterocins harbouring antimicrobial activity against pathogens and spoilage microorganisms of meat concern. The application of enterocins producing enterococci or their purified metabolites, as extra hurdles for preservation in sausage fermentation and in sliced-vacuum packed cooked meat products can be beneficial, preventing the outgrowth of Listeria monocytogenes and slime-producing lactic acid bacteria. Enterocins and bacteriocinogenic enterococci hold considerable promise as alternatives to traditional chemical preservatives and they could be exploited for the control of emergent pathogens in meat products. Their inhibitory effect can be increased when used in conjunction with particular physical and chemical processes, but current regulation is hampering the application of purified bacteriocins.

Biochemical characterization of lactobacilli from dry fermented sausages

The characterization of 254 strains of lactobacilli isolated from dry spontaneously fermented sausages from 15 different producers at two different stages of ripening time is reported. The species identified were Lactobacillus sake 55%, L. curvatus 26%, L. bavaricus 11% and L. plantarum 8%. The main criteria for the identification of isolates to species level were: production of lactic acid isomers, presence of mDpm acid in cell walls, deamination of arginine and fermentation of mannitol and melibiose. The composition of the populations of lactobacilli were the same for the two stages of ripening. The deamination of arginine was tested in aerobic and anaerobic cultures and in different media by checking the production of ammonia and detecting the production of citrulline. In 94% of strains tested both methods gave identical results. In two L. sake strains arginine catabolism was dependent on culture media; for two other L. sake strains the deamination of arginine only occurred when oxygen was scarce.

Inhibition of Listeria monocytogenes and Salmonella by Natural Antimicrobials and High Hydrostatic Pressure in Sliced Cooked Ham

The effectiveness of nisin, lactate salts, and high hydrostatic pressure to inhibit the growth of Listeria monocytogenes and Salmonella in sliced cooked ham was studied through a combination of PCR-based detection methods, most probable number, and classical microbial enumeration techniques (International Organization for Standardization protocols). A synergistic effect to inhibit a cocktail of Listeria monocytogenes CTC1010, CTC1011, and CTC1034 was observed between potassium lactate, high hydrostatic pressure (400 MPa, 17 degrees C, 10 min), and low storage temperature when sliced cooked ham was stored for 84 days at 1 degrees C. The high hydrostatic pressure treatment also proved to be useful to inhibit a cocktail of Salmonella enterica serotypes London CTC1003, Schwarzengrund CTC1015, and Derby CTC1022.

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

Aim:  To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects.

Application of enterocins as biopreservatives against Listeria innocua in meat products.

The antilisterial effect of enterocins A and B in meat and meat products (cooked ham, minced pork meat, deboned chicken breasts, pâté, and slightly fermented sausages [espetec]) have been shown. An infective dose of 5 to 10 most probable numbers (MPN)/g to simulate the counts of Listeria generally found in meat products was used. Enterocins at 4,800 AU/g reduced the numbers of Listeria innocua by 7.98 log cycles in cooked ham and by 9 log cycles in pâté when stored at 7 degrees C for 37 days. In deboned chicken breasts stored at 70 degrees C for 7 days, 4,800 AU/cm2 of enterocins diminished the L. innocua counts in 5.26 log cycles when compared to the control batch. In minced pork meat held at 7 degrees C for up to 6 days, 1,600 AU/g kept L. innocua counts under 3 MPN/g, while the control batch reached 50 CFU/g. In espetec sausages, 648 AU/g diminished the number of L. innocua under 50 CFU/g from the fifth day until the end of the process (12 days) while the control batch kept the initial counts (3 x 104 CFU/g). This is the first report on enterocins showing an antilisterial effect in different types of meat products.

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti‐listerial effect in dry fermented sausages

Enterocin A and B in Enterococcus faecium CTC492 were co‐induced by the different factors assayed in this study (r = 0·93) and followed primary metabolic kinetics. Enterocin production was significantly inhibited by sausage ingredients and additives, with the exception of nitrate. The addition of sodium chloride and pepper decreased production 16‐fold. The temperature and pH influenced enterocin production, with optima between 25 and 35 °C, and from 6·0 to 7·5 of initial pH. The maximum activity was achieved, under favourable growth conditions, with MRS supplemented with sucrose (2%) plus glucose (0·25%) and Tween‐80 (1%). MRS concentration, NaCl plus pepper addition, absence of Tween‐80 in the growth medium, incubation at 45 °C and an initial pH under 5·5 were detrimental to bacteriocin production. Stress conditions did not favour enterocin production. Desadsorption was Tween‐dependent. Enterocin A activity in the crude extracts stored at −80 °C was better preserved than enterocin B (when tested against their specific indicator strain), but anti‐listerial activity remained intact. Applied as anti‐listerial additives in dry fermented sausages, enterocins significantly diminished Listeria counts by 1·13 log (P < 0·001), while Enterococcus faecium CTC492 added as starter culture did not significantly reduce Listeria counts (P > 0·1) compared with the standard starter culture (Bac–). Enterocins A and B could be considered as extra biopreservative hurdles for listeria prevention in dry fermented sausages.

Rapid Quantitative Detection of Listeria monocytogenes in Meat Products by Real-Time PCR

ABSTRACT We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.

Genetic diversity and safety aspects of enterococci from slightly fermented sausages.

Aims:  To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects.