Protein hydrolysis and proteinase activity during the ripening of salted anchovy (Engraulis encrasicholus l.). A microassay method for determining the protein hydrolysis.

The protein hydrolysis and proteinase activity during the ripening of salted anchovy were studied. A rapid, simple, and inexpensive microassay method for determining the protein hydrolysis by trinitrobenzenesulfonic acid (TNBS) has been developed. A linear relationship was observed between proteolysis determination by the TNBS method and ripening time in the fish muscle and in the brine (r = 0.99). A linear relationship was also observed between the ratio nonprotein nitrogen and total nitrogen (NPN/TN) and ripening time (r = 0.98). Proteolysis by the TNBS method and NPN/TN determination could be considered as objective methods to follow and assess the ripening process of an anchovy. A value of proteolysis by the TNBS method of 240 mM leucine in the fish muscle and/or 200 mM leucine in the brine would indicate the ripening point. The crude enzyme prepared of fish muscle and brine showed that alkaline proteinases dominate.

Magnetic immunoseparation PCR assay (MIPA) for detection of hepatitis A virus (HAV) in American oyster (Crassostrea virginica).

In order to detect the low numbers of hepatitis A viral (HAV) particles which may potentially be present in food and cause a serious illness, an original procedure which combines immunomagnetic separation and PCR is described. The use of streptavidin magnetic beads coated with biotinylated human anti-HAV IgG allows virus capture and the removal of the RT-PCR inhibitory compounds which usually are present in shellfish extracts. Following immunomagnetic capture, the separated HAV were lysed, the beads discarded, and the supernatant containing the viral RNA subjected to the RT-PCR protocol. Levels of HAV ranging from 10 to 10(5) pfu were successfully detected in artificially contaminated samples of shucked American oyster (Crassostrea virginica).

Evaluation of three decarboxylating agar media to detect histamine and tyramine-producing bacteria in ripened sausages.

Histidine- and tyrosine-decarboxylase activity of 175 strains of bacteria isolated from eight retail samples of Spanish ripened sausages was tested in three decarboxylating agars (Niven medium, Joosten and Northolt medium and modified decarboxylating agar of Maijala) and confirmed by an enzymic method (histamine) and thin-layer chromatography (tyramine). Enterobacteria and pseudomonads showed the highest percentage of positive responses to histamine and tyramine in the three decarboxylating agars, but only enterobacteria were subsequently confirmed as histamine-producing. Confirmed tyramine-producing strains were all identified as enterococci or lactic acid bacteria. The medium described by Joosten and Northolt was more sensitive and faster at detecting tyramine-producing microorganisms. However, all three media failed to detect one histamine-positive strain of lactic acid bacteria used as a control.

Incidence of histamine-forming bacteria and histamine content in scombroid fish species from retail markets in the Barcelona area.

Incidence and diversity of histidine decarboxylating bacteria were determined in samples of tunafish, bonito and mackerel purchased at different retail markets. Histamine-forming bacteria occurred in a low proportion and always accounted for less than 0.1% of the total bacterial load in the fish samples studied. Similarly, histamine content in fish samples also was low ( < 25 ppm) and all of them met current histamine standards established by the European Union. Histamine was found in 83.3% of the tested tunafish samples with an average of 8.9 ppm. In contrast, none of mackerel samples and only 2 out of 12 of bonito showed detectable amounts of histamine. Morganella morganii and Klebsiella oxytoca were the most active histamine formers under experimental conditions, and produced on average 2765 and 1415 ppm of histamine, respectively, after incubation at 37 degrees C for 18 h. Some new histamine formers, such as Plesiomonas shigelloides, Enterobacter intermedium, Serratia marcescens, Serratia plymuthica and Serratia fonticola, have been identified. Especially Plesiomonas shigelloides would have an important role within histidine decarboxylating bacteria because it was the sole histamine former isolated that has frequently been associated with the marine aquatic environment. However, only 8-340 ppm of histamine was formed by these strains in laboratory trials.

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat.

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4 degrees C, 0 degrees C, -18 degrees C and -24 degrees C) and bacterial levels (10(2), 10(4), 10(5) and 10(7) ml-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.

Histidine, Lysine and Ornithine Decarboxylase Bacteria in Spanish Salted Semi-preserved Anchovies.

We have studied the histidine decarboxylase activity in 118 strains of bacteria isolated from commercial samples of Spanish semi-preserved anchovies. The lysine and ornithine qualitative decarboxylase activity was also studied. The microorganism that presented the highest histamine activity was Morganella morganii , with 2123.26 ± 414.00 ppm of histamine after 24 h of incubation at 37°C. Two strains of Bacillus spp. and a strain of Staphylococcus xylosus were isolated with the capacity to form 10.54 and 110.00 ppm of histamine, respectively. However, the histidine decarboxylase activity of Bacillus spp. is not likely to be significant to human health. The microbic species with capacity to form histamine and those with capacity to form other biogenic amines were similar. Therefore, the prevention of the proliferation of microorganisms able to form histamine would also mean avoiding amine accumulation that leads to histamine food poisoning. The Niven medium was an efficient test to valutate the histamine production of isolated strains after an incubation of 24 h at 37°C and using a backwards technique for quantification and detecting the false positives. This incubation time should be longer (48 h) when Bacillus is detected, with the finality to eliminate false negatives on the initial screening. The application of the enzymic technique for histamine quantification was excellent. In our research, we have observed that the number of microorganisms is an important factor in the accumulation of histamine, but other factors exist which also influence such accumulation, probably depending on the kind of enzyme decarboxylase.

Evolution of histidine decarboxylase bacterial groups during the ripening of Spanish semi-preserved anchovies.

We have studied the count evolutions of total aerobic mesophilic microorganisms, psychotrophic microorganisms, enterobacteria, faecal coliforms, sulphite-reducing bacteria and vibrio in spanish semi-preserved anchovies. These microorganisms are a sanitary index of the product and may produce high concentrations of histamine in both fresh and processed fish. The influence of NaCl concentration, redox potential, oxygen concentration and pH on bacterial growth have also been studied. With the exception of the sulphite-reducers and vibrio, the counts of the different bacterial groups decreased during the first two weeks of ripening, but later stabilized. The faecal coliforms did not appear in the culture media after these first two weeks, and the enterobacteria, what initially did not appear after first two weeks too, are detected at final phases probably for the final manipulation of elaboration processes. The count of the sulfite-reducers remained unchanged during the whole ripening process. Vibrio were not detected in any of the samples studied. NaCl and oxygen concentrations were the main factors influencing the decreasing bacterial counts. According to our results, the accumulation of high histamine concentrations in salted fish could be due to poor quality of the raw material, to inadequate handling or to other causes during its shelf life. The relationship with the histamine activity is probably due more to the presence of the halophilic or halotolerant microorganisms.

Determination of histamine in fish using an enzymic method.

The present paper describes a quick and simple enzymic method for evaluating histamine content in fish. After preparing a perchloric acid extract, it was neutralized to pH 6.8 with KOH. The action of a diamine oxidase on the histamine caused the formation of hydrogen peroxide. The addition of a second enzyme, a peroxidase, in the presence of hydrogen peroxide and a chromogen (leuco crystal violet) in reduced form (colourless) facilitated its oxidation into crystal violet (coloured). Following a two-hour incubation period, absorbance was measured at 596 nm. The correlation between histamine level and absorbance was acceptable in the concentration range from 3 to 30 mg/kg of histamine (r = 0.9946; p < 0.001). The accuracy of the method, expressed by CV, varied between 2.6% and 4.9%, and the recovery between 95.7% and 100.1%, depending on the concentration of analyte in the sample. The diamine oxidase used was very selective in relation to the histamine. Only the presence of tyramine, when histamine concentration was low (< 10 mg/kg), tended to interfere to a slight degree in the technique. Using a regression analysis, no statistically significant differences were found between the results obtained from the analysis of 18 tuna fish samples by HPLC and the enzymic method (r = 0.9867; p < 0.001).