Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico

A total of 82 strains of presumptive Aeromonas spp. were identified biochemically and genetically (16S rDNA-RFLP). The strains were isolated from 250 samples of frozen fish (Tilapia, Oreochromis niloticus niloticus) purchased in local markets in Mexico City. In the present study, we detected the presence of several genes encoding for putative virulence factors and phenotypic activities that may play an important role in bacterial infection. In addition, we studied the antimicrobial patterns of those strains. Molecular identification demonstrated that the prevalent species in frozen fish were Aeromonas salmonicida (67.5%) and Aeromonas bestiarum (20.9%), accounting for 88.3% of the isolates, while the other strains belonged to the species Aeromonas veronii (5.2%), Aeromonas encheleia (3.9%) and Aeromonas hydrophila (2.6%). Detection by polymerase chain reaction (PCR) of genes encoding putative virulence factors common in Aeromonas, such as aerolysin/hemolysin, lipases including the glycerophospholipid-cholesterol acyltransferase (GCAT), serine protease and DNases, revealed that they were all common in these strains. Our results showed that first generation quinolones and second and third generation cephalosporins were the drugs with the best antimicrobial effect against Aeromonas spp. In Mexico, there have been few studies on Aeromonas and its putative virulence factors. The present work therefore highlights an important incidence of Aeromonas spp., with virulence potential and antimicrobial resistance, isolated from frozen fish intended for human consumption in Mexico City.     

Changes in selected biochemical and sensory parameters as affected by the superficial inoculation of Penicillium camemberti on dry fermented sausages

An atoxigenic strain of Penicillium camemberti was superficially inoculated on fermented sausages in an attempt to improve their sensory properties. The growth of this mould on the surface of the sausages resulted in an intense proteolysis and lipolysis, which caused an increase in the concentration of free amino acids, free fatty acids (FFA) and volatile compounds. Many of these were derived from amino acid catabolism and were responsible for the "ripened flavour", i.e. branched aldehydes and the corresponding alcohols, acids and esters. The development of the fungal mycelia on the surface of the sausages also protected lipids from oxidation, resulting in both lower 2-thiobarbituric acid (TBARS) values and lipid oxidation-derived compounds, such as aliphatic aldehydes and alcohols. The sensory analysis of superficially inoculated sausages showed clear improvements in odour and flavour and, as a consequence, in the overall quality of the sausages. Therefore, this strain is proposed as a potential starter culture for dry fermented sausage production.     

Changes in wine yeast storage carbohydrate levels during preadaptation,rehydration and low temperature fermentations

The metabolism of glycogen and trehalose was analysed in a wine yeast strain fermenting at 25 and 13 degrees C. Trehalose and glycogen degradation were completed during the lag phase of fermentation. Ammonia was taken up rapidly and once it had been reduced to negligible amounts, the synthesis of trehalose started. Glycogen followed a similar pattern. If trehalose synthesis was taken as a stress indicator, the fermentation at 13 degrees C could not be considered stressful because the maximum concentrations are similar at both temperatures. In industrial fermentations, and after a preadaptation in grape must for several hours at 18 degrees C, the lag phase was reduced significantly, and this may be why trehalose and glycogen were completely depleted at the beginning of the low temperature fermentation. Various preadaptation conditions were tested so that their influence on trehalose and glycogen degradation could be determined. The presence of fermentable carbon sources, such as glucose or fructose, triggered the mobilisation and use of trehalose. However, just increasing the osmotic pressure did not reduce the trehalose content. No such differences were observed in glycogen metabolism.     

Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: the repetitive sequence is needed for binding to the cell wall beta-1,3-glucan

Identification of PIR/CIS3 gene was carried out by amino-terminal sequencing of a protein band released by beta-mercaptoethanol (beta-ME) from S. cerevisiae mnn9 cell walls. The protein was released also by digestion with beta-1,3-glucanases (laminarinase or zymolyase) or by mild alkaline solutions. Deletion of the two carboxyterminal Cys residues (Cys(214)-12aa-Cys(227)-COOH), reduced but did not eliminate incorporation of Pir4 (protein with internal repeats) by disulphide bridges. Similarly, site-directed mutation of two other cysteine amino acids (Cys(130)Ser or Cys(197)Ser) failed to block incorporation of Pir4; the second mutation produced the appearance of Kex2-unprocessed Pir4. Therefore, it seems that deletion or mutation of individual cysteine molecules does not seem enough to inhibit incorporation of Pir4 by disulphide bridges. In fks1Delta and gsc2/fks2Delta cells, defective in beta-1,3-glucan synthesis, modification of the protein pattern found in the supernatant of the growth medium, as well as the material released by beta-ME or laminarinase, was evident. However, incorporation of Pir4 by both disulphide bridges and to the beta-1,3-glucan of the cell wall continued. Deletion of the repetitive sequence (QIGDGQVQA) resulted in the secretion and incorporation by disulphide bridges of Pir4 in reduced amounts together with substantial quantities of the Kex2-unprocessed Pir4 form. Pir4 failed to be incorporated in alkali-sensitive linkages involving beta-1,3-glucan when the first repetitive sequence was deleted. Therefore, this suggests that this sequence is needed in binding Pir4 to the beta-1,3-glucan.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.     

Hydrolytic activity of Penicillium chrysogenum Pg222 on pork myofibrillar proteins

Moulds grow on many different dry-cured meat products and are able to hydrolyse muscle proteins. However, their contribution to proteolysis in these products is not well known. Only recently, the ability of just a few strains of Penicillium spp. to increase proteolysis in dry-cured meat products has been shown. For these strains to be used as starter cultures, their hydrolytic activity under standard conditions should be characterised. With this purpose, the effect of Penicillium chrysogenum Pg222 on pork myofibrillar proteins has been assayed in a culture medium containing 5% (w/v) NaCl. SDS-PAGE revealed that Pg222 was responsible for extensive hydrolysis of the main myofibrillar proteins except alpha-actinin. The proteolysis led to increases in free amino acids, reaching peak values at 84 h. Ala, Tyr and Lys were present in the greatest amount. These results suggest that P. chrysogenum Pg222 would contribute to development of desired texture and flavours in dry-cured meat products.     

Determination of fatty acids in the subcutaneous fat of Iberian breed swine by near infrared spectroscopy (NIRS) with a fibre-optic probe

A near infrared spectrometer equipped with a standard 1210/210 bundle remote reflectance fibre-optic probe, with a 5×5 cm quartz window, was used for the determination of fatty acids in the subcutaneous fat of Iberian pigs. A comparative study was made of the determination of fatty acids (C14:0, C16:0, C18:0, C18:1, C18:2, C18:3, C20: 1, Σpolyunsaturated, Σmonounsaturated and Σsaturated) in samples of subcutaneous fat from Iberian pigs by direct application of the fibre-optic probe on samples of whole subcutaneous fat and with cam-lock cups, assessing extracts of total lipids with diethyl ether. The regression method employed was modified partial least squares (MPLS). Calibration of 157 samples, using the fibre optic probe, allowed determination of fatty acids in the following ranges: C14:0 (0.78-1.77), C16:0 (15.87-29.74), C18:0 (4.61-15.90), C18:1 (43.50-61.27), C18:2 (2.03-13.94), C18:3 (0.13-1.14), C20:1 (0.45-2.32), Σpolyunsaturated (2.31-14.82), Σmonounsaturated (47.37-65.62), Σsaturated (22.09-47.31), with corrected standard errors of prediction SEP(C) of 0.093, 0.56, 0.67, 0.94, 0.42, 0.10, 0.20, 0.46, 0.94, 0.83, respectively. The robustness of the method using the fibre-optic probe was tested in a slaughterhouse using 23 samples for external validation, giving multiple correlation coefficients (RSQ) for C14:0, C16:0, C18:0, C18:1, C18:2, C18:3 C20:1, Σpolyunsaturated, Σmonounsaturated, Σsaturated acids of 0.72, 0.94, 0.72, 0.79, 0.88, 0.55, 0.17, 0.88, 0.74, and 0.90, respectively, and a corrected standard error of prediction [SEP(C)] for these acids (%) of 0.11, 0.60, 0.84, 1.20, 0.77, 0.11, 0.30, 0.76, 1.21, and 1.18, respectively.     

Correlation between DNA synthesis in the second, third and fourth generations of spermatogonia and the occurrence of apoptosis in both spermatogonia and spermatocytes

In the seminiferous epithelium, both DNA synthesis and apoptosis occur at equivalent stages in various species, with apoptosis taking place mainly at the same stages as DNA replication in the second, third and fourth spermatogonial generations. As preservation of the cellular associations found at these stages may have some functional significance, it is important to determine whether there is a correlation between these cellular events. In this study, pairs of immunoperoxidase-stained adjacent testis sections from rats, mice, rabbits and cats in which either bromodeoxyuridine incorporated into the newly synthesized DNA strand (BrdU labelling) or DNA 3' end labelling of the apoptotic DNA fragments (TUNEL assay) were detected were compared. In addition, both events were analysed in double-labelled sections. These two methods revealed a clear correlation between the occurrence of DNA replication in the second to fourth generations of spermatogonia and most physiological apoptosis taking place in both spermatogonia and spermatocytes in the three different mammalian orders (Rodentia, Lagomorpha and Carnivora). This correlation may result from the synchronization of mitotic spermatogonial and meiotic spermatocyte cell cycle checkpoints operating at these stages.     

Photochemistry of 4-chlorophenol on cellulose and silica

The photochemistry of 4-chlorophenol (4-CP) was studied on silica and cellulose, using time-resolved diffuse reflectance techniques and product degradation analysis. The results have shown that the photochemistry of 4-CP depends on the support, on the concentration, and also on the sample preparation method. Transient absorption and photoproduct results can be understood by assuming the formation of the carbene 4-oxocyclohexa-2,5-dienylidene in both supports. On cellulose, at concentrations lower than 10 micromol g(-1), the carbene leads to the unsubstituted phenoxyl radical, and phenol is the main degradation product. At higher concentrations a new transient resulting from phenoxyl radicals coupling was also observed, and dihydroxybiphenyls are also formed. The reaction of the carbene with ground-state 4-CP was also detected through the formation of 5-chloro-2,4'-dihydroxybiphenyl. 4-Chlorophenoxyl radical and degradations products resulting from its coupling were also detected. Oxygen has little effect on the photochemistry of 4-CP on cellulose. On silica the transient benzoquinone O-oxide was formed in the presence of oxygen. Benzoquinone and hydroquinone are the main degradation products. In well-dried samples the formation of hydroquinone is reduced. At higher concentrations the same products as detected on cellulose were observed. 4-CP undergoes slow photochemical decomposition under solar radiation in both supports. The same main degradation products were observed in these conditions.