Teresa Aymerich

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.

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.

Inhibition of Salmonella sp. Listeria monocytogenes and Staphylococcus aureus in cooked ham by combining antimicrobials, high hydrostatic pressure and refrigeration.

Recontamination of ready-to-eat products such as cooked ham during post-processing may be the cause of outbreaks of food-borne disease. The effectiveness of the combination of high pressure processing (HPP) at 600MPa with the natural antimicrobials nisin and potassium lactate has been evaluated in sliced cooked ham spiked with 4LogCFU/g of Salmonella sp., Listeria monocytogenes and Staphylococcus aureus after 3-months of storage at 1 and 6°C. In non-HPP sliced cooked ham, the addition of nisin plus lactate inhibited the growth of L. monocytogenes during the entire storage period while the refrigerated storage inhibited the growth of Salmonella sp. and S. aureus. The application of an HPP reduced the levels of Salmonella and L. monocytogenes to levels below 10CFU/g. These levels continued until the end of storage at both 1 and 6°C. HPP produced a reduction of less than 1LogCFU/g to S. aureus. The combination of HPP, nisin and refrigeration at 6°C was necessary to decrease the levels of S. aureus by 2.4LogCFU/g after 3-months of storage.

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.

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 degrees C or at room temperature. AS-48 (148 AU g(-1)) caused a drastic 5.5 log cfu g(-1) decrease in L. monocytogenes (P<0.001) and a significant (P<0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g(-1) in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.

High hydrostatic pressure and biopreservation of dry-cured ham to meet the Food Safety Objectives for Listeria monocytogenes

This work aimed to evaluate the effect of nisin application (biopreservation) combined with high hydrostatic pressure processing (HHP) on the behavior of Listeria monocytogenes CTC1034 intentionally inoculated (at ca. 10(7)cells/g) onto the surface of ready-to-eat (RTE) sliced dry-cured ham. Two types of dry-cured ham, which had different water activities and fat contents were studied (a(w) of 0.92 and 14.25% fat and a(w) of 0.88 and 33.26% fat). Three batches were prepared for each type of product: (C) control, without nisin; (N) nisin directly applied (200 AU/cm(2)) and (F) nisin applied through active packaging, polyvinyl alcohol films with 200 AU/cm(2). Half of the samples were pressurized at 600 MPa for 5min. Counts of L. monocytogenes were periodically monitored throughout 60 days of storage at 8°C. The physico-chemical characteristics of the products enabled the survival of L. monocytogenes, but it was significantly reduced by the presence of nisin. The effect of biopreservation was greater when applied directly to the surface and in the product with lower water activity in comparison with the active packaging and the high water activity products, respectively. The immediate inactivation of L. monocytogenes by HHP ranged from 1.82 to 3.85 Log units, depending on the type of dry-cured ham. The lower the water activity, the less was the inactivation induced by HHP, both immediately and during storage. The reduction of L. monocytogenes immediately after HHP and during storage was more evident in batches with nisin applied directly to the surface of the product. The pathogen was not detected in some samples from day 5 of storage in the product with higher water activity. The effect of nisin applied through active packaging was lower than the direct application. The results of the present study indicated that HHP, as post-processing listericidal treatment, is more effective (both immediately and long term) than the use of nisin as an antimicrobial measure. However, the both hurdles combined (i.e. biopreservation and HHP) provided a wider margin of safety in the control of L. monocytogenes during the storage of RTE cured meat products.