M.M. Hernández-Herrero

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.

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.

Electrochemical detection of Salmonella using gold nanoparticles.

A disposable immunosensor for Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S) detection using a magneto-immunoassay and gold nanoparticles (AuNPs) as label for electrochemical detection is developed. The immunosensor is based on the use of a screen-printed carbon electrode (SPCE) that incorporates a permanent magnet underneath. Salmonella containing samples (i.e. skimmed milk) have been tested by using anti-Salmonella magnetic beads (MBs-pSAb) as capture phase and sandwiching afterwards with AuNPs modified antibodies (sSAb-AuNPs) detected using differential pulse voltammetry (DPV). A detection limit of 143 cells mL(-1) and a linear range from 10(3) to 10(6) cells mL(-1) of Salmonella was obtained, with a coefficient of variation of about 2.4%. Recoveries of the sensor by spiking skimmed milk with different quantities of Salmonella of about 83% and 94% for 1.5×10(3) and 1.5×10(5) cells mL(-1) were obtained, respectively. This AuNPs detection technology combined with magnetic field application reports a limit of detection lower than the conventional commercial method carried out for comparison purposes in skimmed milk samples.

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.

Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua)

Collagenase activity and changes in muscular protein of iced Atlantic cod stored for 9 days were studied. The crude fish muscle extract showed maximum collagenase-like activity against bovine insoluble tendon collagen at 48 h of incubation at 37 °C. Collagenase activity against synthetic substrate increased (P<0.05), especially for fish in initial and advanced stages of decomposition. These results suggest that endogenous collagenases and other proteases may be responsible for the destruction of fine collagenous fibrils in the skeletal muscle of cod. The content of titin 1 decreased when decomposition was advanced. Moreover, a progressive degradation of sarcoplasmic proteins with a molecular weight of 100, 94, 85 and 80 kDa was observed. Results suggest that softening of cod muscle during iced storage is caused more by collagenase activity than by proteolysis of myofibrils.

Comparing the effects of ultra-high-pressure homogenization and conventional thermal treatments on the microbiological, physical, and chemical quality of almond beverages.

UNLABELLED The effects of ultra-high-pressure homogenization (UHPH) at 200 and 300 MPa, in combination with different inlet temperatures (55, 65, and 75 °C) on almond beverages with lecithin (AML) and without lecithin (AM), were studied. UHPH-treated samples were compared with the base product (untreated), pasteurized (90 °C, 90 s), and ultra-high-temperature (UHT, 142 °C, 6 s) samples. Microbiological analysis, physical (dispersion stability, particle size distribution, and hydrophobicity), and chemical (hydroperoxide index) parameters of special relevance in almond beverages were studied. Microbiological results showed that pressure and inlet temperature combination had a significant impact on the lethal effect of UHPH treatment. While most UHPH treatments applied produced a higher quality of almond beverage than the pasteurized samples, the combination of 300 MPa and 65 and/or 75 °C corresponded to a maximum temperature after high pressure valve of 127.7 ± 9.7 and 129.3 ± 12.6 °C, respectively. This temperature acted during less than 0.7 s and produced no bacterial growth in almond beverages after incubation at 30 °C for 20 d. UHPH treatments of AML samples caused a significant decrease in particle size, resulting in a high physical stability of products compared to conventional heat treatments. UHPH treatment produced higher values of hydroperoxide index at day 1 of production than heat-treated almond beverage. Hydrophobicity increased in AML-UHPH-treated samples compared to AM and conventional treatments. PRACTICAL APPLICATION  Ultra-high-pressure-homogenization (UHPH) is an emerging technology, a potential alternative to conventional heat treatments. It is a simple process consisting of single step. When liquid food (almond beverage in this study) passes through the high-pressure valve, a very good stability and reduction of microorganisms is achieved, both effects due to the particle breakdown. Specific UHPH conditions could produce commercial sterilization of almond beverage.

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk.

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0 log(10) cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0 log(10) cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400 MPa. Microbiological analyses were performed 2 h after the UHPH treatments and after 5, 8, and 15 d of storage at 4 degrees C. Maximum lethality values were observed in samples treated at 400 MPa with 15 and 10% fat (7.95 and 7.46 log(10) cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4 degrees C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200 MPa but not in the milk samples treated at 300 and 400 MPa. When the load of L. monocytogenes was approximately 3.0 log(10) cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.

Behavior of Yersinia enterocolitica strains inoculated in model cheese treated with high hydrostatic pressure.

The effects of high hydrostatic pressure treatment and the ability for survival, repair, and growth of three human pathogenic serotypes (O:1, O:3, O:8) of Yersinia enterocolitica were investigated in washed-curd model cheese made with pasteurized bovine milk. Samples were treated at 300, 400, and 500 MPa for 10 min at 20 degrees C and analyzed at 0, 1, 7, and 15 days to assess the viability of the Yersinia population. A long-term study (up to 60 days of ripening after high hydrostatic pressure treatment) was also undertaken. Treatments at 400 and 500 MPa caused maximum lethality, and only the treatment at 300 MPa showed significant differences (P < 0.05) between serotypes; the most baroresistant was O:3. Ability to repair and grow was not observed after 15 days of storage at 8 degrees C. Yersinia counts in untreated cheese samples also decreased below the detection limit at day 45 in the long-term study. These results suggest that the cheese environment did not allow recovery of injured cells or growth. A primary contributing factor to this effect seemed to be the low pH resulting from the production of lactic acid during cheese ripening.