M.T. Mora‐Ventura

Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies (Engraulis encrasicholus).

This study was performed to investigate halotolerant and halophilic histamine-producing bacteria isolated during the ripening of salted anchovies. Of the isolates obtained during the ripening of anchovies, 1.37% showed histamine-forming activity, most of them (70%) belonging to the Staphylococcus genus. S. epidermidis showed a powerful histamine-forming activity, producing more than 1,000 microg/ml in the presence of 3% and 10% NaCl. Another powerful histamine-producing bacterium isolated during the ripening of salted anchovies was S. capitis. It was able to produce about 400 microg/ml of histamine in 10% NaCl under experimental conditions. Most of these species might be expected to be found as a result of contamination of fish during capture and subsequent unhygienic handling. However, no increase in histamine content was found in any batches through the ripening process. Histamine content always was acceptable in accordance with the maximum allowable levels of histamine fixed by the Spanish and European Union regulations.

Sensory quality and histamine formation during controlled decomposition of tuna (Thunnus thynnus)

Histamine production was studied during controlled tunafish decomposition at 0, 8, and 20°C. The influence of the location of the anatomic section on the amount of histamine formed and the incidence of histidine decarboxylating bacteria were also considered. By the time of sensory rejection, histamine levels in tunafish sections stored at 0 and 20°C were still below the hazard levels and the allowable levels established by both the U.S. Food and Drug Administration (FDA) and the European Union. Toxic amounts were only formed after the tunafish was considered organoleptically unsuitable for human consumption. However, at 8°C, levels of histamine between 100 and 200 mg/l00 g of fish were found before tuna reached the rejection point. Hence, physical appearance was not a good criterion for estimating the shelf life and especially the histamine-related health hazard when tuna was stored at 8°C, a common temperature in many home refrigerators.

Bacteriological Quality of Tuna Fish (Thunnus thynnus) Destined for Canning: Effect of Tuna Handling on Presence of Histidine Decarboxylase Bacteria and Histamine Level

The present paper analyzes the bacteriological quality and histamine content of tuna fish samples destined for canning. Raw material used in the canning process was of good quality. Histamine-producing bacteria counts were only found in three samples from the last step of the canning process before sterilization. Most of the bacteria identified as histamine formers were gram negative, and nearly all of which belonged to the Enterobacteriaceae family. Morganella morganii was the most frequent and active histamine former in tuna fish destined for canning. Other powerful histamine-producing bacteria isolated during the canning operation were Klebsiella oxytoca , Klebsiella pneumoniae and some strains of Enterobacter cloacae and Enterobacter aerogenes . They all were able to produce more than 500 ppm of histamine in experimental conditions. Most of these species might be expected to be found as a result of contamination of fish during capture and subsequent unhygienic handling in the canning plant. An increase in histamine content in tuna meat was not expected through the canning process. Histamine content always was acceptable in accordance with the maximum allowable levels of histamine fixed by both the European Economic Community and Food and Drug Administration.

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.

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.

Evaluation of histidine decarboxylase activity of bacteria isolated from sardine (Sardina pilchardus) by an enzymic method

A fast and simple enzymic method has been adapted to measure histidine decarboxylase activity in bacteria isolated from fish. It was possible to quantify the presence of histamine at levels higher than 1.03 times 10‐2μmol (3 ppm) per ml of culture medium. There was a good correlation (γ=0.99) between the histamine content and the increase of absorbance in the concentration range of 3 and 30 ppm. The method can be used for quantitation of the amount of histamine produced by a selected strain, and has the advantages of being low in cost, reliable, easy to use and fast to perform. Plesiomonas shigelloides, a bacterium frequently isolated from fish and aquatic environments, has been identified as a new histamine‐former in fish.

Histidine decarboxylase activity of bacteria isolated from raw and ripened Salchichón, a Spanich cured sausage

Twenty samples of raw sausages (before the ripening process) and 10 of ripened sausages of two different sizes of salchichón , one of the most-consumed ripened meat products in Spain, were microbiologically analyzed. Histidine decarboxylase activity of different isolates obtained from different culture media was evaluated by a decarboxylase agar medium and confirmed afterwards by an enzymatic method. Of the isolates obtained from raw sausages, 15.8% showed histamine-forming activity, most of them (78.1%) belonging to the Enterobacteriaceae family. Klebsiella oxytoca , Enterobacter aerogenes (one isolate), and Enterobacter cloacae (six isolates) showed a powerful histamine-forming activity, producing more than 3,000 μg/ml. In spite of histamine-forming activity being detected in eight of ten analyzed samples of ripened sausages, only one histamine-forming bacterium, identified as Lactobacillus curvatus , was isolated; it showed an ability to form 1,994 μg/ml of histamine in experimental conditions.

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).

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 micro‐organisms. However, all three media failed to detect one histamine‐positive strain of lactic acid bacteria used as a control.

Histidine decarboxylase activity of Enterobacter cloacae S15/19 during the production of ripened sausages and its influence on the formation of cadaverine

The histidine decarboxylase activity of Enterobacter cloacae S15/19 was studied during the production process of salchichón , a Spanish ripened sausage. Counts of fecal coliform and histidine decarboxylase bacteria decreased during the production process, showing a good correlation in both inoculated and control samples. In the samples inoculated with Enterobacter cloacae S15/19, fecal coliforms were undetectable the last day of the survey, while the population of histidine decarboxylase bacteria was over 2 log MPN/g. Despite the fact that inoculation with Enterobacter cloacae S15/19 increased histidine decarboxylase bacteria counts, no differences were observed in the histamine concentration reached, which was undetectable in most of the control and inoculated samples. In contrast, cadaverine concentration increased significantly (P < 0.01) in the inoculated samples, suggesting that cadaverine could be used as a hygienic-quality indicator of the raw materials employed in sausage processing.