Proteomic analysis by two-dimensional differential gel electrophoresis (2D DIGE) of a high-pressure effect in Bacillus cereus.

High hydrostatic pressure (HHP) is a new method used to reduce or eliminate microorganisms that are present in food. Proteins are known to be the most important target of high pressure in living organisms. The main goal of this investigation was focused on the changes that occur on the proteins of Bacillus cereus under HHP stress conditions. The two-dimensional differential gel electrophoresis (2D DIGE) technique allows for a simultaneous resolution of thousands of proteins based on fluorescent prelabeling of the samples with spectrally resolvable fluorescent CyDyes. The results of proteomics profiling show an average of 1300 spots being detected. The analysis revealed 75 spot proteins whose abundance is modified after the application of high pressure, of which 66 were decreased after the HHP treatment. Among them, flagellin was the protein that changed the most. The differential expression of some proteins after HHP treatment at 700 MPa may suggest a reduction of virulence and protective response against oxidative stress in flagellated Bacillus .

High pressure processing for food safety.

Food preservation using high pressure is a promising technique in food industry as it offers numerous opportunities for developing new foods with extended shelf-life, high nutritional value and excellent organoleptic characteristics. High pressure is an alternative to thermal processing. The resistance of microorganisms to pressure varies considerably depending on the pressure range applied, temperature and treatment duration, and type of microorganism. Generally, Gram-positive bacteria are more resistant to pressure than Gram-negative bacteria, moulds and yeasts; the most resistant are bacterial spores. The nature of the food is also important, as it may contain substances which protect the microorganism from high pressure. This article presents results of our studies involving the effect of high pressure on survival of some pathogenic bacteria -- Listeria monocytogenes, Aeromonas hydrophila and Enterococcus hirae -- in artificially contaminated cooked ham, ripening hard cheese and fruit juices. The results indicate that in samples of investigated foods the number of these microorganisms decreased proportionally to the pressure used and the duration of treatment, and the effect of these two factors was statistically significant (level of probability, P

Reaction of B. cereus bacteria and peroxidase enzymes under pressures >400 MPa.

It is known that B. cereus (Gram-positive bacteria) and peroxidase enzymes are resistant to pressures of approximately 400 MPa in fruit and vegetable products among others. The aim of the present work is to have knowledge about their behavior when using pressures >400 MPa without other combined treatments. The results showed that B. cereus was not inactivated at pressures of 1000 MPa for 15 min at 20 degrees C. In peroxidase enzymes the results remained similar and the pressure of 1000 MPa for 30 min was not enough to inactivate them. The apple cell structure at these high-pressure levels revealed that it changed and the cells were less cemented. The treated apple presented a translucent aspect, and some fluids migrated from the inside to the outside of the cell.

Soybean vegetable protein (Tofu) preserved with high pressure.

Tofu is a soybean vegetable protein that Asians have long consumed; its intake is increasing in other countries. Tofu was purchased at a local shop. The tofu samples were already preserved in plastic bags subjected to vacuum and storage (5 C). Tofu samples were subjected to high pressure (HP) of 400 MPa at 5 C for 5, 30, and 45 mm. Microbial analysis, sensorial evaluation, and structure were determined. HP treatment in tofu reduces the microbial population. Most of the microorganisms found in the initial population belonged to the Enterobacteriaceae family, bacteria Gram-negative (no Enterobacteriaceae), and bacteria Gram-positive. Alter HP treatment, Hafnia alvei and Bacillus cereus were found. After HP treatment, tofu is a pasteurized product, which is safer in terms of secondary pathogenic microbial contamination. Sensorial test results revealed that treated tofu was acceptable to consumers. The micrographs on the cryofracture observed with a cryoscanning electron microscope revealed a more compact structure after pressure compared with that of untreated samples, but the aggregates in the treated samples were more disperse.

Biological response of rats fed with tofu treated with high hydrostatic pressure.

Emerging technologies for food preservation have arisen in recent years, such as high-pressure (HP) hydrostatic treatment, and the biological response for this kind of food preservation is not well-known. Forty female rats (six weeks old) were used in the experiment to evaluate the biological effects of HP treatment of tofu. The animals were divided into groups that were fed with tofu (untreated), tofu treated with HP, and conventional food (as control) for 28 days. The glucose level, mineral content (calcium, potassium, zinc, and magnesium), shinbone maximum shear force, weight of the body, and weight of organs (heart, liver, spleen, and kidneys) were analyzed. The biological response for the rats was that significant differences were found in the calcium amount determined on the serum of the rats fed with untreated tofu and those fed with tofu treated with HP, and the calcium amount was lower on the rats fed with tofu treated with HP. Also, there were significant differences in the weight of the liver, and it was lower in the rats fed with tofu treated with HP. It was quite remarkable how the weight of the body and organs were smaller in the rats fed with tofu in comparison to the weight of the control rats. In the other components assayed no significant differences were found. HP produces a potential effect on tofu as it is observed in the rats response to the tofu treated with HP.

Protective effect of ascorbic acid against the browning developed in apple fruit treated with high hydrostatic pressure.

Apple Reineta variety was used as an apple dessert. The 1-1.5-cm cubes were immersed in a sucrose solution (30% w/v) and subjected to high pressure (HP) of 400 MPa for 30 min at 5 degrees C. Different ascorbic acid concentrations were used to protect the fruit from the browning developed after the HP treatment. After 2 months of storage at 5 degrees C, no brown color was observed in the samples treated with 20 mM ascorbic acid, and they were acceptable to consumers. However, untreated samples presented fermentation, and they were not acceptable to consumers. The electric conductivity and potassium content were found to be good indicators of the metabolites released from the fruit to the solution in samples treated with high pressure. HP did not affect the peroxidase activity but eliminated the microbial population.

Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy.

Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min. Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L. monocytogenes, than in the other test micro-organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.

High pressure response of fruit jams contaminated with Listeria monocytogenes.

High pressure is an alternative to thermal processing and is used to preserve food. Listeria monocytogenes is a bacterium which grows at low temperature, is able to multiply under vacuum, and is responsible for food poisoning. Pressures of 100, 200, 300 and 400 MPa were used for 5, 10 and 15 min at 20 degrees C on pure culture, and on apple and plum jam baby food artificially contaminated with Listeria. Pure culture was also to test pressures of 200, 300, 350 and 400 MPa at 5 degrees C for 30 min. The results were analysed statistically and showed that there were no significant differences between pressures of 100 and 200 MPa at 5, 10 and 15 min. However, at 300 MPa, there were significant differences at 15 min. When the pressure treatment was 400 MPa, significant differences were observed at pressure times of 5, 10 and 15 min. The results were fitted to a linear curve. In pure culture, no viable cells were detected after high pressure treatment of 350 MPa for 30 min at 5 degrees C. The use of low temperature helps to maintain the sensory properties of the product.

Ultrastructural distribution of a MAP kinase and transcripts in quiescent and cycling plant cells and pollen grains.

Mitogen-activated protein kinases (MAPKs) are components of a kinase module that plays a central role in the transduction of diverse extracellular stimuli, including mitogens, specific differentiation and developmental signals and stress treatments. This shows that reversible protein phosphorylation cascades play a pivotal role in signal transduction in animal cells and yeast, particularly the entry into mitosis of arrested cells. Homologues of MAPKs have been found and cloned in various plant species, but there have been no data about their in situ localization at the subcellular level and their expression in plant cells so far. In the present paper we report the first data on the ultrastructural in situ localization of MAPK and their mRNAs in various plant cells. Proliferating and quiescent meristematic plant cells were studied to evaluate whether changes in MAPK presence, distribution and expression accompany the entry into proliferation of dormant cells. Moreover, MAPK localization was analyzed in vacuolate microspores. Polyclonal antibodies against the deduced MAPK from the tobacco Ntf6 clone were able to recognize homologue epitopes by immunocytochemical techniques in the cell types studied. The pattern of protein distribution is similar in all the cases studied: it is localized in the cytoplasm and in the nucleus, mainly in the interchromatin region. The quantitative study of the density showed that MAPK labelling is more abundant in cycling than in quiescent cells, also suggesting that, in plants, MAPK pathways might play a role in cell proliferation. RNA probes for conserved regions of the catalytic domain of plant MAPK homologue genes were used to study MAPK expression in those plant cells. In situ hybridization (ISH) showed the presence of MAPK transcripts in the three plant cell types studied, but levels were very low in quiescent cells compared to those in cycling cells. The quantification of labelling density of ISH signals strongly suggests a higher level of MAPK expression in proliferating cells, but also some basal messenger presence and/or expression in the quiescent ones. Immunogold and ISH results show the presence and distribution of MAPK proteins and mRNAs in vacuolate microspores. This represents a very dynamic stage during pollen development in which the cell nucleus is being prepared for an asymmetrical mitotic division, giving rise to both the generative and the vegetative nuclei of the bicellular pollen grain. Taken together, the data indicate a role played by MAPK in the re-entry into proliferation in plant cells.

Effect of high pressure on the reduction of microbial populations in vegetables.

Resistance of micro-organisms to high pressure is variable and directly related to extrinsic and intrinsic factors. Pressures of 100, 200, 300, 350 and 400 MPa were applied at 20 degrees C for 10 min and at 10 degrees C for 20 min using strains of Gram-positive and Gram-negative bacteria, moulds and yeasts, as well as spores of Gram-positive bacteria. The results showed that at pressures of 100 and 200 MPa, decreases in microbial populations were not significant, whereas the populations of all the micro-organisms tested decreased considerably at a pressure of 300 MPa. A pressure of 300 MPa at 10 degrees C for 20 min was required to completely reduce the population of Saccharomyces cerevisiae, and a pressure of 350 MPa was needed to reduce most of the Gram-negative bacteria and moulds. The Gram-positive bacteria were more resistant, and pressures of 400 MPa were unable to completely reduce their populations. The different pressures employed had little effect on the initial numbers of spores. The initial populations of viable aerobic mesophiles and moulds and yeasts in vegetables (lettuce and tomatoes) decreased 1 log unit at pressures of 300 MPa and above under both sets of experimental treatment conditions. However, treatment at that pressure also resulted in alterations in the organoleptic properties of the samples. In the tomatoes, the skin loosened and peeled away, though the flesh remained firm, and colour and flavour were unchanged. The lettuce remained firm but underwent browning; flavour was unaffected. In vegetables use of moderate pressures in combination with other treatment conditions would appear to be required to reduce the populations of contaminating micro-organisms while avoiding the undesirable alterations in organoleptic properties that take place at 300 MPa.

Purification and characterization of two ribonucleases from developing tomato fruit.

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.