Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.

A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.

Evaluation of microbial proteolysis in meat products by capillary electrophoresis.

AIMS: The available methods for evaluating proteolysis in meat products, particularly the contribution of micro-organisms, are expensive, time-consuming and require an unacceptable sample size. To minimize these problems, two capillary electrophoresis-based methods have been developed. METHODS AND RESULTS: Six Gram-positive, catalase-positive cocci, four moulds and three yeasts, isolated from dry-cured ham, were tested on sterile pork slices. Using the Capillary Gel Electrophoresis (CGE) method, changes in sarcoplasmic and myofibrillar proteins due to endogenous and microbial enzymes were detected. The Capillary Zone Electrophoresis (CZE) analysis allowed evaluation of bulk changes by micro-organisms in soluble nitrogen compounds. CONCLUSION: CGE analysis of myofibrillar proteins and CZE determination of soluble nitrogen compounds have proved to be valuable tools for evaluating proteolytic activity of endogenous and microbial origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The CGE and CZE methods developed can be used for a rapid and sensitive analysis of proteolysis in meat products.

Effects of substrate, water activity, and temperature on growth and verrucosidin production by Penicillium polonicum isolated from dry-cured ham.

Penicillium polonicum, a common mold on dry-cured meat products, is able to produce verrucosidin, a potent neurotoxin. The ability of P. polonicum isolated from dry-cured ham to grow and produce verrucosidin from 4 to 40 degrees C at water activities (a(w)) of 0.99, 0.97, and 0.95 on malt extract agar (MEA) and a medium made up with meat extract, peptone, and agar (MPA) was evaluated. Verrucosidin was quantified by high-pressure liquid chromatography and mass spectrometry. P. polonicum was able to grow on MEA and MPA at all the a(w) values tested from 4 to 37 degrees C but not at 40 degrees C. The optimal environmental conditions for growth were 20 degrees C, 0.99 a(w) on MEA and 20 to 25 degrees C, 0.97 a(w) on MPA, but the highest amount of verrucosidin was obtained at 25 degrees C, 0.99 a(w) in both media. No direct correlation between extension of mold growth and verrucosidin production was found. Temperature appears to be the most important factor ruling mycelial growth, whereas verrucosidin accumulation is mostly influenced by a(w). However, analysis of variance of the data showed that there was a complex interaction among all the environmental factors (medium, temperature, and a(w)) that significantly (P < 0.0001) affected growth and verrucosidin production. The reduction of a(w) to intermediates values of 0.95 has a stronger effect on growth on MEA than on MPA. Given that the meat-based medium proved to be an appropriate substrate for the biosynthesis of verrucosidin by P. polonicum, the ability of this mold to produce the toxin on meat products should be established.

[Non-compliance of the treatment with antibiotics in non-severe acute infections]. / Incumplimiento del tratamiento con antibióticos en infecciones agudas no graves.

BACKGROUND: To determine the nonfulfillment of antiinfectious therapy in clinical practice. MATERIAL AND METHODS: Fulfillment was quantified by tablet counting (TC) in the homes of 366 patients undergoing antibiotic treatment and the motives and predictive factors were identified. RESULTS: Nonfulfillment was of 61% (95% confidence interval [CI] 55.4-66.6%). Patient improvement was the main reason for discontinuation (54.5%). The predictive factors were greater length of treatment (p = 0.000004), dose (p = 0.0019) and number of tablets (p = 0.0000). CONCLUSIONS: Nonfulfillment of antiinfectious treatment in clinical practice is high, mainly due to clinical improvement and to the greater complexity and length of treatment.

Evaluation of proteolytic activity of micro-organisms isolated from dry cured ham.

In order to determine the possible contribution of micro-organisms to the ripening of meat products, 48 cocci, 18 moulds and 20 yeasts isolated from dry-cured Iberian ham were evaluated for proteolytic activity. Two specific methods were used: the ability to hydrolyse myosin in broth and, for those strains showing high activities, hydrolysis on both myofibrillar and sarcoplasmic proteins on pork slices. Moulds and cocci showed the highest proteolytic activity for myosin in broth. Both myofibrillar and sarcoplasmic proteins were recovered at lower rates from inoculated than from sterile incubated pork. The deepest changes in myofibrillar and sarcoplasmic proteins were originated by one strain each of Penicillium chrysogenum and Staphylococcus xylosus, respectively. Only small changes were observed in the concentrations of free amino acids from inoculated pork slices, except for the samples with P. chrysogenum, where there were increases in all free amino acids. Thus, P. chrysogenum makes a significant contribution to proteolysis during the ripening of dry-cured meat products.

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe.

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2.1-8.1 cfu ml-1) for types A and B, but rather low (10(4) cfu ml-1) for types E and F. However, after enrichment at 37 degrees C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g-1 of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.

Composition and toxigenic potential of the mould population on dry-cured Iberian ham.

The fungal population on dry-cured Iberian ham can be essential to the development of the product's unique characteristics, but health hazards due to mycotoxins may be significant. We examined the natural fungal population of Iberian hams during ripening at three different locations. Chloroform extracts from 59 selected isolates were tested for toxicity to brine shrimp larvae and VERO cells, for mutagenicity in the Ames test and for antimicrobial activity against Staphylococcus aureus. The diversity of moulds increased during ripening. Penicillium commune, Penicillium chrysogenum, Penicillium aurantiogriseum, Penicillium expansum and Penicillium echinulatum dominated most of the ripening time; however, the Eurotium species, particularly E. herbariorum and E. repens, increased in the final product. Using the above tests, most moulds were toxigenic. The toxigenic potential of the fungal population increased as the processing progressed. To minimize health hazards from uncontrolled fungal populations, we identified non toxigenic strains of Penicillium chrysogenum that could be used as starters in dry-cured hams.

Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential.

Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months. Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time. Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed. For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests. Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA. The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay. The majority of the isolates were identified as Staphylococcus spp. and Micrococcus spp. Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected. A great variety of staphylococcal strains were found within the different species at any sampling time. Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes. S. xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test. In addition, enterotoxin D was confirmed in the nonidentified strains. Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.

Yeast population during ripening of dry-cured Iberian ham.

The relationship between the superficial yeast population and the ripening conditions of Iberian dry cured hams has been studied for three different locations. Tentative identifications were carried out for 836 isolates. Candida zeylanoides was the dominating yeast in early stages, whereas more than 99% of isolates from the surface of matured hams were identified as Debaryomyces hansenii. A great diversity of strains of C. zeylanoides and D. hansenii was found. The characteristic pattern of isolates from the various locations and the selection of various strains of D. hansenii during ripening make the study of the yeasts useful for estimating the progress of maturation.

Characterization of Staphylococcus spp. and Micrococcus spp. isolated from Iberian ham throughout the ripening process.

The Iberian dry cured ham is an uncooked meat product highly appreciated because of its characteristic flavour. This product is obtained from highly marbled Iberian pig hindlegs after 18-24 months of maturation under natural environmental conditions. The role of Micrococcaceae in the development of the aroma characteristics of this products remains unclear. Identification of Gram-positive, catalase-positive cocci isolated from Mannitol Salt Agar plates showed that Staphylococcus xylosus followed by Staphylococcus equorum are the predominant organisms, even after 16 months of maturing. A remarkable variety of types of both staphylococci and micrococci are detected at any sampling time. The metabolic activities of these organisms could contribute to the characteristics of the final product.

Hydrolysis and loss of extractability of proteins during ripening of iberian ham.

To elucidate the extent of the hydrolysis and loss of extractability of protein during the traditional ripening of Iberian ham, the evolution during processing of non-protein nitrogen (NPN) and protein fractions soluble in 0·03 m pH 7·1 phosphate and 1·1 KI + 0·1 m phosphate pH 7·4 buffers and 6 m urea was followed from Semimembranosus and Biceps femoris muscles. The NPN steadily increased during processing, showing maximum intensity at salting and drying. Electrophoretic study of the proteins extracted, and microscopical examination of the pellet obtained after consecutive extractions with the above buffers, revealed that hydrolysis and insolubilization are more intense in myofibrillar than in sarcoplasmic proteins. Protein aggregation involves mainly the myofibrillar fraction, and occurs during the first stage of processing.

Phospholipids and their fatty acid composition in the muscle of trout fed on diets supplemented with olive oil bagasse or technical rendered fat.

A study to determine the effects of two by-products from the food industry (olive oil bagasse or technical rendered fat) on the phospholipid content and the fatty acid composition of the muscle of rainbow trout (Salmo gairdneri) has been made. Three batches of 150 trout were given for 100 days a commercial diet alone or supplemented either with 11% olive oil bagasse or technical rendered fat. The phospholipid content in the muscle of the three batches of trout ranged from 0.70 to 0.93% (wet weight). In this fraction, six different phospholipid classes were detected, phosphatidylcholine and phosphatidylethanolamine achieving average values of 55 and 25% of total phospholipids. Although differences in the fatty acid composition of the diet were observed, the only clear influence of diet was on the fatty acid C-22:6 of muscular phosphatidylethanolamine.

A method to assess the bacterial content of refrigerated meat.

A new method has been developed to estimate the levels of gram-negative bacteria on refrigerated meat. The method is based on the aminopeptidase activity of these bacteria, which cleaves L-alanine-p-nitroanilide to yield p-nitroaniline, which is easily determined spectrophotometrically. This method allows the determination of levels around 10(6) to 10(7) CFU cm-2 in about 3 h. Because of the yellow color of p-nitroaniline, bacterial loads around 10(7) CFU cm-2 develop a color intense enough to be detected with the naked eye.