Effect of UHPH on indigenous microbiota of apple juice: a preliminary study of microbial shelf-life.

The effect of ultra high pressure homogenisation (UHPH) at 100, 200 and 300 MPa on apple juice at two different inlet temperatures (4 and 20 degrees C) was studied. Raw and conventional heat treated (PA) apple juice was compared with UHPH treated juice after treatment and during 60 days of storage at 4 degrees C. The microbial quality was studied by enumerating aerobic mesophilic counts (AM), psychrotrophs (PS), moulds and yeasts (MY), lactobacilli (LB), enterobacteriaceae (EB), and faecal coliforms (FC). PA samples were below detection level (< or = -2 log cfu/mL) for all groups, the same as UHPH treated juices at 200 MPa and above but for around 1 log cfu/mL for AM. Those AM counts did not change during 60 days storage at 4 degrees C and proved to be spores. Juices treated at 100 MPa shown little reductions in microbial counts, and surviving microorganisms significantly increased their numbers during storage. This research work showed that non-thermal methods such a UHPH technology may give new opportunities to develop "fresh like" apple juice with a shelf-life equivalent to PA in terms of microbiological characteristics without affecting the product quality.

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.

Fate of Staphylococcus aureus in cheese treated by ultrahigh pressure homogenization and high hydrostatic pressure.

We evaluated the influence of ultrahigh pressure homogenization (UHPH) treatment applied to milk containing Staphylococcus aureus CECT 976 before cheese making, and the benefit of applying a further high hydrostatic pressure (HHP) treatment to cheese. The evolution of Staph. aureus counts during 30 d of storage at 8 degrees C and the formation of staphylococcal enterotoxins were also assessed. Milk containing approximately 7.3 log(10) cfu/mL of Staph. aureus was pressurized using a 2-valve UHPH machine, applying 330 and 30 MPa at the primary and the secondary homogenizing valves, respectively. Milk inlet temperatures (T(in)) of 6 and 20 degrees C were assayed. Milk was used to elaborate soft-curd cheeses (UHPH cheese), some of which were additionally submitted to 10-min HHP treatments of 400 MPa at 20 degrees C (UHPH+HHP cheese). Counts of Staph. aureus were measured on d 1 (24 h after manufacture or immediately after HHP treatment) and after 2, 15, and 30 d of ripening at 8 degrees C. Counts of control cheeses not pressure-treated were approximately 8.5 log(10) cfu/g showing no significant decreases during storage. In cheeses made from UHPH treated milk at T(in) of 6 degrees C, counts of Staph. aureus were 5.0 +/- 0.3 log(10) cfu/g at d 1; they decreased significantly to 2.8 +/- 0.2 log(10) cfu/g on d 15, and were below the detection limit (1 log(10) cfu/g) after 30 d of storage. The use of an additional HHP treatment had a synergistic effect, increasing reductions up to 7.0 +/- 0.3 log(10) cfu/g from d 1. However, for both UHPH and UHPH+HHP cheeses in the 6 degrees C T(in) samples, viable Staph. aureus cells were still recovered. For samples of the 20 degrees C T(in) group, complete inactivation of Staph. aureus was reached after 15 d of storage for both UHPH and UHPH+HHP cheese. Staphylococcal enterotoxins were found in controls but not in UHPH or UHPH+HHP treated samples. This study shows a new approach for significantly improving cheese safety by means of using UHPH or its combination with HHP.

Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening.

Two commercial starters were investigated for their potential ability to decarboxylate amino acids during goat cheese ripening. Two batches of goat cheese were produced with identical pasteurized milk but different starter cultures. One of them contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and the other Lactococcus lactis subsp. lactis. The amine contents, microbial counts, proteolysis-related parameters, pH, total solids and salt content were studied in raw materials and cheeses. In raw materials, polyamines were the prevailing amines, whereas the main amines in cheeses were putrescine, tryptamine and, in particular, tyramine (94.59 mg/kg). Aerobic mesophilic microorganisms and Lactococcus counts increased throughout ripening, while Enterobacteriaceae were no longer detectable in cheese after 30 days of ripening. Amine concentration rose during cheese ripening in both batches. Moreover, the decarboxylase activity of microorganisms isolated from samples during cheese ripening was assayed and discussed.

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.

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).