B. Sanz

Effect of combined ultrasonic and heat treatment (thermoultrasonication) on the survival of a strain of Staphylococcus aureus

The combined effect of ultrasonic waves (20 kHz, 150 W) and heat treatment applied simultaneously (thermoultrasonication) on the survival of a strain of Staphylococcus aureus was studied in 0.05 M-phosphate buffer pH 6.8 and ultra-heat-treated whole milk. This combined process decreased by 63% the decimal reduction times for the heat treatment when the organism was suspended in buffer and by 43% when suspended in milk. These effects were much greater than the additive effect of the two agents considered independently.

Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages

Lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities under conditions that eliminated the effects of low pH and hydrogen peroxide. From 720 isolates tested 119 were inhibitory to Lactobacillus fermentum CECT285. The isolates showing the largest inhibitory activity exhibited an antagonistic effect against several other lactobacilli and the selected foodborne pathogens Staphylococcus aureus and Listeria monocytogenes. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. The isolates were tentatively characterized as Lactobacillus sake. One of the isolates, L. sake 148 was chosen for further study. The compound excreted by L. sake 148 was active against various lactobacilli and several Gram-positive foodborne bacteria, but not against the Gram-negative bacteria tested. The antagonistic effects were almost eliminated by treatment with proteases, whereas they were heat resistant and bacteriostatic rather than bacteriocidal.

Polymerase chain reaction-restriction fragment length polymorphism analysis of a short fragment of the cytochrome b gene for identification of flatfish species

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

Characteristics of lactobacilli isolated from dry fermented sausages

A study of the microflora of Spanish dry fermented sausages showed a similar pattern to that observed in other sausages during ripening. Lactic acid bacteria and organisms growing on Mannitol-salt-agar increased during the first five days, remaining at constant levels afterwards. From MRS agar, 368 strains were isolated and characterized. All Lactobacillus strains (343) could be separated into three groups according to their reactions in the API-50 L system. The biochemical characteristics of the Lactobacillus strains analyzed were those typical of isolates from sausages and other meat products. The extracellular and intracellular lipolytic activities of some strains of the three groups were also studied. It is concluded that lipolytic activity of lactobacilli is mainly directed against mono- and diglycerides and short-chain fatty acid triglycerides.

Effect of Carbon Dioxide and Oxygen Enriched Atmospheres on the Shelf-Life of Refrigerated Pork Packed in Plastic Bags

Changes in bacterial numbers, metmyoglobin percentage and 2-thiobarbituric acid number during the chill storage of pork longissimus dorsi packed with air, carbon dioxide, carbon dioxide and oxygen or vacuum-packed in plastic bags of high (polyethylene) and low (Cryovac BB-1) permeability to gases were studied. The fast increase of viable counts in polyethylene bags showed that plastic films of high permeability are not suitable to extend the shelf-life of meat using modified atmospheres. In Cryovac bags both carbon dioxide-enriched atmospheres and vacuum-packaging inhibited aerobic organisms, keeping the bacterial flora (mainly lactobacilli and Brochothrix thermosphacta ) below the level of 108/cm2 for about 3 weeks. Metmyoglobin formation was not affected by 20% carbon dioxide, whilst 80% oxygen significantly decreased its rate. Metmyoglobin accumulation in vacuum-packed samples was even slower than in the oxygen-enriched atmosphere, but meat color was less attractive in the former than in the latter. Lipid oxidation was not the limiting factor of shelf-life in either of these two atmospheres. Off-odors appeared in both at approximately 20-22 d of storage.

Sakacin M, a bacteriocin-like substance from Lactobacillus sake 148

The antagonistic activity of Lactobacillus sake 148 was evaluated during its growth on complex broth media and in a semisynthetic defined medium (SDM) with various supplements. The antagonistic activity was a growth-associated property, being detected and quantified when L. sake 148 was grown at either 4, 8, 16, 25 or 32 degrees C. The concentrated culture supernatant of L. sake 148 was subjected to purification by lyophilization and gel filtration. The purification procedure resulted in a small increase in its specific activity (7-fold) and in a low recovery of the original inhibitory activity (8%). Gel filtration analysis of the partially purified activity on Sephadex G-50 revealed an apparent molecular weight of 4640. The partially purified antagonistic activity of L. sake 148 was destroyed by treatment with proteolytic enzymes. However, the antagonistic activity was resistant to heat, having D-values at 121, 135 and 150 degrees C of 23.8, 17.4 and 15.2 min, respectively.

Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA.

Direct enumeration of Escherichia coli from oysters was achieved using a polymerase chain reaction (PCR) amplification of the lamB gene coupled with an enzyme‐linked immunosorbent assay (ELISA). Amplified PCR products generated using a digoxigenin‐labelled primer were heat denatured before being quantified by an ELISA. A biotinylated probe immobilized onto streptavidin‐coated microplates was used to capture the digoxigenin‐labelled fragments that were detected with a peroxidase antidigoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying oyster samples containing E. coli in the range 10–105 cfu g−1.

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk

We have developed a quantitative PCR‐ELISA for the rapid enumeration of bacteria inrefrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNAgene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment froma wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCRproducts generated using a digoxigenin‐labelled primer were heat‐denatured before beingquantified by an enzyme‐linked immunosorbent assay (ELISA). A biotinylated probe immobilizedonto streptavidin‐coated microplates was used to capture the digoxigenin‐labelled fragments thatwere detected with a peroxidase anti‐digoxigenin conjugate. Subsequent enzymic conversion ofsubstrate gave distinct absorbence differences when assaying milk samples containing bacteria inthe range 103–107 cfu ml−1. The detection threshold for thePCR‐ELISA assay developed in this work is 103 cfu ml−1.

Identification of Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss) by Using Polymerase Chain Reaction Amplification and Restriction Analysis of the Mitochondrial Cytochrome b Gene

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the identification of fresh and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Digestion of the 359-bp PCR product with the endonucleases EcoRV and TaqI yielded specific banding patterns for salmon and trout. This genetic marker can be very useful for detecting fraudulent substitution of the cheaper smoked trout for the more expensive smoked salmon.

Immunostick ELISA for Detection of Cow's Milk in Ewe's Milk and Cheese Using a Monoclonal Antibody against Bovine β-Casein

An immunostick enzyme-linked immunosorbent assay (ELISA) has been developed for the rapid detection of cows milk in ewes milk or cheese. The assay uses a monoclonal antibody (AH4) produced against bovine β-casein for the detection of cows milk or cheese bound to the paddles of immunostick tubes. This immunostick ELISA allows the visual identification of ewes milk containing more than 1% of cows milk or cheese samples containing more than 0.5% of cows cheese.