Anna Gallone

Factors Affecting Quality and Safety of Fresh-Cut Produce

The quality of fresh-cut fruit and vegetable products includes a combination of attributes, such as appearance, texture, and flavor, as well as nutritional and safety aspects that determine their value to the consumer. Nutritionally, fruit and vegetables represent a good source of vitamins, minerals, and dietary fiber, and fresh-cut produce satisfies consumer demand for freshly prepared, convenient, healthy food. However, fresh-cut produce deteriorates faster than corresponding intact produce, as a result of damage caused by minimal processing, which accelerates many physiological changes that lead to a reduction in produce quality and shelf-life. The symptoms of produce deterioration include discoloration, increased oxidative browning at cut surfaces, flaccidity as a result of loss of water, and decreased nutritional value. Damaged plant tissues also represent a better substrate for growth of microorganisms, including spoilage microorganisms and foodborne pathogens. The risk of pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in fresh-cut produce are Listeria monocytogenes, pathogenic Escherichia coli mainly O157:H7, and Salmonella spp. This article describes the quality of fresh-cut produce, factors affecting quality, and various techniques for evaluating quality. In addition, the microbiological safety of fresh-cut produce and factors affecting pathogen survival and growth on fresh-cut produce are discussed in detail.

Bacterial Stressors in Minimally Processed Food

Stress responses are of particular importance to microorganisms, because their habitats are subjected to continual changes in temperature, osmotic pressure, and nutrients availability. Stressors (and stress factors), may be of chemical, physical, or biological nature. While stress to microorganisms is frequently caused by the surrounding environment, the growth of microbial cells on its own may also result in induction of some kinds of stress such as starvation and acidity. During production of fresh-cut produce, cumulative mild processing steps are employed, to control the growth of microorganisms. Pathogens on plant surfaces are already stressed and stress may be increased during the multiple mild processing steps, potentially leading to very hardy bacteria geared towards enhanced survival. Cross-protection can occur because the overlapping stress responses enable bacteria exposed to one stress to become resistant to another stress. A number of stresses have been shown to induce cross protection, including heat, cold, acid and osmotic stress. Among other factors, adaptation to heat stress appears to provide bacterial cells with more pronounced cross protection against several other stresses. Understanding how pathogens sense and respond to mild stresses is essential in order to design safe and effective minimal processing regimes.

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

Lactobacillus plantarum passage through an oro-gastro-intestinal tract simulator: carrier matrix effect and transcriptional analysis of genes associated to stress and probiosis.

Dietary probiotics should reach the intestine viable and in high numbers; therefore, they should tolerate the stress associated to the gastro-intestinal (GI) environment. Indeed, all along the different GI sections, probiotics are challenged by several sources of stress, including low pH, bile and digestive enzymes. Bacterial cells are equipped with various defense mechanisms to allow survival in hostile environments. The food matrix used to deliver beneficial bacteria may contribute to their probiotic action, e.g. by enhancing survival to stress and gut colonization. In this study, the survival of the lactic acid bacterium Lactobacillus plantarum WCFS1, a model probiotic strain, was examined in a human oro-gastric-intestinal (OGI) in vitro system, using different carrier matrices to compare protective and buffering properties. Higher survival was observed in complex and/or nutrient-rich matrices, and when potential prebiotics were added. The molecular response of L. plantarum to the OGI transit was analyzed by studying the transcriptional levels of genes involved in stress response and probiosis. The OGI steps of higher mortality corresponded to greater induction of stress genes, thus implying their involvement in adaptation to the gut environment. Plantaricins were significantly upregulated all along the different OGI sections; adhesion genes were mainly induced by gastric environment.

Probiotic features of Lactobacillus plantarum mutant strains.

In this study, the probiotic potential of Lactobacillus plantarum wild-type and derivative mutant strains was investigated. Bacterial survival was evaluated in an in vitro system, simulating the transit along the human oro-gastro-intestinal tract. Interaction with human gut epithelial cells was studied by assessing bacterial adhesive ability to Caco-2 cells and induction of genes involved in innate immunity. L. plantarum strains were resistant to the combined stress at the various steps of the simulated gastrointestinal tract. Major decreases in the viability of L. plantarum cells were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to small intestine poorly affected bacterial viability. All the bacterial strains significantly adhered to Caco-2 cells, with the ΔctsR mutant strain exhibiting the highest adhesion. Induction of immune-related genes resulted higher upon incubation with heat-inactivated bacteria rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with different L. plantarum strains, evidencing a possible role of the knocked out bacterial genes in the modulation of host cell response. In particular, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. Our study highlights the relevance of microbial genetic background in host–probiotic interaction and might contribute to identify candidate bacterial genes and molecules involved in probiosis.

ULTRASTRUCTURAL AND FUNCTIONAL STUDY OF THE LIVER PIGMENT CELLS FROM RANA ESCULENTA L.

SummaryA study of the liver pigment cells of Rana esculenta L. has been performed on both liver in toto and cells in culture. Ultrastructural and cytochemical analyses showed a close relationship between this visceral pigment cell system and the cells of hepatic macrophage lineage. Like the latter, the liver pigment cells present phagocytic activity, in the sinusoids and in vitro, and give a positive response to tests for peroxidase and lipase. The liver pigment cells are isolated, together with the Kupffer cells, from the sinusoidal cell fraction of the liver. In culture, they maintain their melanogenetic ability, demonstrated by the presence of dopaoxidase activity in the soluble, membranous, and melanosome fractions. Analysis of the cultures showed that as culture time increased, so did melanosome dopaoxidase activity, the number of pigmented fields, and the level of pigmentation of the cells. The values of dopaoxidase activity of the pigment cells in culture show the same seasonal oscillations as the system in toto, indicating that the cells maintain an internal clock, at least in the first 72 h of culture. There is evidence that the pigment cells are macrophages which can express a melanogenetic function. Our results and other experimental data provide a basis for hypothesizing that the pigment cells in Rana esculenta L. liver may derive from, or have a common origin with, the Kupffer cells.

MORPHO-FUNCTIONAL CHARACTERIZATION OF CULTURED PIGMENT CELLS FROM RANA ESCULENTA L. LIVER

SummaryA simple method to isolate and culture liver pigment cells fromRana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells fromRana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance.

Melanogenesis in visceral tissues of Salmo salar. A link between immunity and pigment production

Melanogenesis is mostly studied in melanocytes and melanoma cells, but much less is known about other pigment cell systems. Liver, spleen, kidney, and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes. In teleosts, melanin-containing cells occur in the reticulo-endothelial system and are mainly in the kidney and spleen. The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature adversely affects the quality of fish products and is a problem for the aquaculture industry. With the aim to reveal novel functions and behaviour of the salmonid extracutaneous pigment system, we investigated aspects of the melanogenic systems in the tissues of Atlantic salmon, as well as in SHK-1 cells, which is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney. We demonstrate that a melanogenic system is present in SHK-1 cells, head-kidney, and spleen tissues. As teleosts lack lymph nodes and Peyers patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs. The detection of tyrosinase activity in lymphoid organs indicates that a link exists between the extracutaneous pigmentary system and the immune system in salmon.

A multidisciplinary study of the extracutaneous pigment system of European sea bass (Dicentrarchus labrax L.). A possible relationship between kidney disease and dopa oxidase activity level

Infectious diseases and breeding conditions can influence fish health status. Furthermore it is well known that human and animal health are strongly correlated. In lower vertebrates melano-macrophage centres, clusters of pigment-containing cells forming the extracutaneous pigment system, are widespread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. Hence, they are employed as biomarker of fish health status. We have investigated this cell system in the European sea bass (Dicentrarchus labrax L.) following the enzyme activities involved in melanin biosynthesis. We have found a possible relationship between kidney disease of farmed fishes and dopa oxidase activity level, suggesting it as an indicator of kidney disease. Moreover variations of dopa oxidase activity in extracutaneous pigment system have been observed with respect to environmental temperature. At last, for the first time, using femtosecond transient absorption spectroscopy (Femto-TA), we pointed out that pigment-containing cells of fish kidney tissue present melanin pigments.

Effects of copper on the tyrosinase of liver pigment cells from Rana esculenta L.

1. The liver pigment cells of R. esculenta L. constitute a peculiar pigment cell system of histiocytic nature and contain a tyrosinase-like activity localized in the protein component of melanosomes. 2. The effects of addition and/or removal of Cu on the DOPA-oxidase activity of the system were studied. 3. It was concluded that: (a) this tyrosinase behaves as a Cu-enzyme; (b) Cu could be involved in the regulation of the enzyme activity; and (c) mixtures of apoenzyme and active enzyme coexist in the melanosomes.