Characterization of Desmodesmus pleiomorphus isolated from a heavy metal-contaminated site: biosorption of zinc.

Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn(2+) concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn(2+) uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.

Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus.

Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.

Antimicrobial effects of chitosans and chitooligosaccharides, upon Staphylococcus aureus and Escherichia coli, in food model systems.

The objective of this study was to elucidate the controversial relationship between the molecular weight (MW) of chitosans and their antibacterial activity (upon different inoculum levels, at several concentrations). The influence of food components on the activity was also ascertained, as well as acceptance by a sensory panel. All the compounds tested exhibited antibacterial activity against Staphylococcus aureus and Escherichia coli. This activity was shown to be closely dependent on the inoculum level, MW and concentration used. Within 4h at 10(3) cells/mL, all five compounds, at every concentration (0.5%, 0.25% and 0.1%, w/v), proved to be bactericidal; for higher inocula, 0.1% (w/v) was only bacteriostatic; at 10(7) or 10(5) cells/mL, and independently of the inoculum level, 0.25% (w/v) of any chitooligosaccharide (COS) mixture was sufficient to reduce the E. coli initial population by at least 3 log cycles; COS never exhibited bactericidal action over S. aureus, unlike high and medium MW chitosans-which, at 0.5% (w/v), presented a bactericidal effect even against 10(7) cells/mL. When incorporated in liquid food matrices, medium and high MW chitosans maintained their activity, for both matrices and bacteria, although a slower activity was noticeable in milk; however, COS lost their activity upon both bacteria in milk after 4-8h. Furthermore, addition of chitosans to apple juice led to several unpleasant off-flavors, such as astringency and after taste--which increased in magnitude with MW.

Development of iron-rich whey protein hydrogels following application of ohmic heating - Effects of moderate electric fields.

The influence that ohmic heating technology and its associated moderate electric fields (MEF) have upon production of whey protein isolate cold-set gels mediated by iron addition was investigated. Results have shown that combining heating treatments (90°C, 5min) with different MEF intensities let hydrogels with distinctive micro and macro properties - i.e. particle size distribution, physical stability, rheological behavior and microstructure. Resulting hydrogels were characterized (at nano-scale) by an intensity-weighted mean particle diameter of 145nm, a volume mean of 240nm. Optimal conditions for production of stable whey protein gels were attained when ohmic heating treatment at a MEF of 3V∙cm-1 was combined with a cold gelation step using 33mmol∙L-1 of Fe2+. The consistency index of hydrogels correlated negatively to MEF intensity, but a shear thickening behavior was observed when MEF intensity was increased up to 10V∙cm-1. According to transmission electron microscopy, ohmic heating gave rise to a more homogenous and compact fine-stranded whey protein-iron microstructure. Ohmic heating appears to be a promising technique, suitable to tailor properties of whey protein gels and with potential for development of innovative functional foods.

Study of the antibacterial effects of chitosans on Bacillus cereus (and its spores) by atomic force microscopy imaging and nanoindentation.

Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely distributed in nature. Its intrinsic thermal resistance coupled with the extraordinary resistance against common food preservation techniques makes it one of the most frequent food-poisoning microorganisms causing both intoxications and infections. In order to control B. cereus growth/sporulation, and hence minimize the aforementioned hazards, several antimicrobial compounds have been tested. The aim of this work was to assess by atomic force microscopy (AFM) the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon both vegetative and resistance forms of B. cereus. The use of AFM imaging studies helped us to understand how chitosans with different MW act differently upon B. cereus. Higher MW chitosans (628 and 100kDa) surrounded both forms of B. cereus cells by forming a polymer layer-which eventually led to the death of the vegetative form by preventing the uptake of nutrients yet did not affect the spores since these can survive for extended periods without nutrients. Chitooligosaccharides (COS) (<3kDa), on the other hand, provoked more visible damages in the B. cereus vegetative form-most probably due to the penetration of the cells by the COS. The use of COS by itself on B. cereus spores was not enough for the destruction of a large number of cells, but it may well weaken the spore structure and its ability to contaminate, by inducing exosporium loss.