High pressure processing effect on different Listeria spp. in a commercial starter-free fresh cheese.

In this study, both microbial inactivation and growth of Listeria spp. inoculated in commercial free-starter fresh cheese was evaluated after high-pressure processing (HPP). HPP conditions (300, 400, 500 and 600 MPa at 6 °C for 5 min) and inoculum level (3-4 or 6-7 log CFU/g of cheese), as well as differences among strains inoculated (Listeria innocua, L. monocytogenes CECT 4031 and L. monocytogenes Scott A) were investigated. Inactivation and generation of sublethal injury were determined after HPP using ALOA (Agar Listeria according to Ottaviani and Agosti) and TAL (Thin Agar Layer) plating methods, respectively. Listeria inactivation increased with the pressure applied, presenting some statistical differences between the employed strains, inoculum level and sublethal injury. The highest lethality values were obtained at 600 MPa for the three strains tested, although the 500 MPa treatment presented high lethality for L. innocua and L. monocytogenes CECT 4031. After treatment, L. innocua and L. monocytogenes CECT 4031 counts in fresh cheese increased gradually during cold storage. By contrast, counts in cheeses inoculated with L. monocytogenes Scott A did not change significantly (p ≥ 0.05), being this strain the most pressure resistant and with the slowest growth rate. The manuscript present information supporting that, strains with high-level resistance should be employed during inactivation studies, instead of surrogate microorganisms. Application of HPP treatments of 500 MPa and especially 600 MPa on fresh cheeses would be effective to eliminate the most resistant microorganism to a level that should not present a public health risk under normal conditions of distribution and storage.

Inactivation of Bacillus spores inoculated in milk by Ultra High Pressure Homogenization.

Ultra High-Pressure Homogenization treatments at 300 MPa with inlet temperatures (Ti) of 55, 65, 75 and 85 °C were applied to commercial Ultra High Temperature treated whole milk inoculated with Bacillus cereus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus coagulans, Geobacillus stearothermophilus and Bacillus subtilis spores in order to evaluate the inactivation level achieved. Ultra High-Pressure Homogenization conditions at 300 MPa with Ti = 75 and 85 °C were capable of a spore inactivation of ∼5 log CFU/mL. Furthermore, under these processing conditions, commercial sterility (evaluated as the complete inactivation of the inoculated spores) was obtained in milk, with the exception of G. stearothermophilus and B. subtilis treated at 300 MPa with Ti = 75 °C. The results showed that G. stearothermophilus and B. subtilis have higher resistance to the Ultra High-Pressure Homogenization treatments applied than the other microorganisms inoculated and that a treatment performed at 300 MPa with Ti = 85 °C was necessary to completely inactivate these microorganisms at the spore level inoculated (∼1 × 10(6) CFU/mL). Besides, a change in the resistance of B. licheniformis, B. sporothermodurans, G. stearothermophilus and B. subtilis spores was observed as the inactivation obtained increased remarkably in treatments performed with Ti between 65 and 75 °C. This study provides important evidence of the suitability of UHPH technology for the inactivation of spores in high numbers, leading to the possibility of obtaining commercially sterile milk.

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.

Inactivation of Salmonella enterica serovar Senftenberg 775W in liquid whole egg by ultrahigh pressure homogenization.

Two batches of samples of liquid whole egg were inoculated with a load of approximately 3 and 7 log CFU/ml, respectively, of Salmonella enterica serovar Senftenberg 775W and submitted to different ultrahigh pressure homogenization (UHPH) treatments at 150, 200, and 250 MPa. The inlet temperature of the samples was 6 degrees C. Counts of viable and injured Salmonella cells were obtained 2 h after the UHPH treatments and after 5, 10, 15, and 20 days of storage at 4 degrees C. The level of pressure applied influenced the lethality attained significantly (P < 0.05). In the samples with an initial load of approximately 7 log CFU/ml, the highest lethality value of 3.2 log CFU/ml was obtained at 250 MPa, and it is similar to those values reported in other surveys for thermal pasteurization with this same Salmonella strain. When the initial load was approximately 3 log CFU/ml, total inactivation was apparently obtained after the 250-MPa treatment (2.7 log CFU/ml). After 10 days of storage at 4 degrees C, Salmonella counts decreased in UHPH-treated samples, and colonies were not observed in tryptone soy agar and yeast extract medium. Nevertheless, presence of viable Salmonella cells was detected with the VIDAS Salmonella immunoassay method during the entire storage period. These results encourage further investigation of UHPH processing of liquid whole egg, assaying the possibility of using higher pressures and fluid inlet temperatures.

Response of two Salmonella enterica strains inoculated in model cheese treated with high hydrostatic pressure.

The aim of this work was to determine the response to high hydrostatic pressure and the ability for survival, recovery, and growth of 2 strains of Salmonella enterica (Salmonella enteritidis and Salmonella typhimurium) inoculated in a washed-curd model cheese produced with and without starter culture. Inoculated samples were treated at 300 and 400 MPa for 10 min at room temperature and analyzed after treatment and after 1, 7, and 15 d of storage at 12 degrees C to study the behavior of the Salmonella population. Cheese samples produced with starter culture and treated at 300 and 400 MPa showed maximum lethality; no significant differences in the baroresistant behavior of both strains were detected. Nevertheless, when starter culture was not present, the maximum lethality was only observed in cheese samples treated at 400 MPa, in the case of S. enteritidis. Ability to repair and grow was not observed in model cheese produced with starter culture and cell counts of treated samples decreased after 15 d of storage at 12 degrees C. In cheese produced without starter culture, Salmonella cells showed the ability to repair and grow during the storage period, reaching counts over 3 log(10) (cfu/mL) in both applied treatments and serotypes. These results suggest that high hydrostatic pressure treatments are effective to reduce Salmonella population in this type of cheese, but the presence of the starter culture affects the ability of this microorganism to repair and grow during the storage period.

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.

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.

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.