Polycarboxylates enhance beetle antifreeze protein activity

Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N',N',N',N'-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn(2+) coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.     

Preparation of N-vanillyl chitosan and 4-hydroxybenzyl chitosan and their physico-mechanical, optical, barrier, and antimicrobial properties

Chitosan derivatives such as N-vanillyl chitosan and 4-hydroxybenzyl chitosan were prepared by reacting chitosan with 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxybenzaldehyde. Amino groups on chitosan reacts with these aldehydes to form a Schiff base intermediate, which is later on converted into N-alkyl chitosans by reduction with sodium cyanoborohydride. The chemical reaction was monitored by ¹H NMR spectroscopy and the absence of aldehydic proton at 9.83 ppm in NMR spectra was observed for both the modified chitosan derivatives confirming the reaction. Modified chitosan films were later prepared by solution casting method and their physico-mechanical, barrier, optical and thermal properties were studied. The results clearly indicated significant change in tensile strength, water vapour transmission rate, and haze properties of modified chitosans. Modified chitosan films were also studied for their antimicrobial activity against Aspergillus flavus. The results showed a marked reduction of aflatoxins produced by the fungus in the presence of the N-vanillyl chitosan and 4-hydroxybenzyl chitosan film discs to 98.9% and non-detectable levels, respectively.     

Preparation and performance evaluation of glucomannan–chitosan–nisin ternary antimicrobial blend film

The material behaviour and antimicrobial effect of konjac glucomannan edible film incorporating chitosan and nisin at various ratio or concentrations is discussed. This activity was tested against food pathogenic bacteria namely Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Mechanical and physical properties were determined and the results indicated that the blend film KC2 (mixing ratio konjac glucomannan 80/chitosan 20) showed the maximum tensile strength (102.8 ± 3.8 MPa) and a good transparency, water solubility, water vapor transmission ratio. The differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), etc. were used to characterize the structural change of the blend films. The results showed that the strong intermolecular hydrogen bonds took place between chitosan and konjac glucomannan. Incorporation of nisin at 42,000 IU/g of film for the selected blend film KC2 was found to have antimicrobial activity against S. aureus, L. monocytogenes, and B. cereus. The antimicrobial effect of chitosan or KC2 incorporating nisin was much better than that of konjac glucomannan incorporating nisin at each corresponding concentration and existed significant difference (p < 0.05), however, there was no significant difference on the antimicrobial effect between chitosan and KC2 both incorporating nisin. At all these levels, the ternary blend film KC2-nisin had a satisfactory mechanical, physical properties and antimicrobial activity, and could be applied as a potential ‘active’ packaging material.     

Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods

The objective of this study was to determine whether chitosan (poly-beta-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7 degrees C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of hummus, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7 degrees C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.     

Studies on the bacterial spore coat. IX. The role of surface charge in germination of Bacillus megaterium spores

The surface charge of Bacillus megaterium QM B1551 spores was estimated to be negative, -0.2 ad -0.4 mueq/mg by colloidal titration using glycol chitosan (GCh) and methylglycol chitosan (MGCh), respectively, as positive colloids. MGCh, which reacts with all of the negatively charged groups including carboxylate, inhibited the second stage of the germination to result in semirefractile spores, but GCh, which reacts only with strong acidic groups such as phosphate, did not. The spores produced in a medium with limited phosphate had coats with low phosphate content and carried less negative charge, and they were induced to germinate with 0.4 mM KNO3, which is one-tenth of the minimum concentration required for the germination of the control spores. A similar increase in germinability was observed in spores incubated with calcium acetate. The results suggest that the role of the surface charge in germination is as follows. Strong acidic groups (such as phosphate) in the coat may block the action of ionic germinants and act as a barrier against the initiation of ionic germination. Positively charged compounds (such as calcium) may compensate for this blocking effect. Weak acidic groups (such as carboxylate) may be involved in the later stage of germination.     

Antimicrobial Actions of Degraded and Native Chitosan against Spoilage Organisms in Laboratory Media and Foods

The objective of this study was to determine whether chitosan (poly-β-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7°C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of houmous, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7°C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.     

Modelling biofilm‐induced formation damage and biocide treatment in subsurface geosystems

Biofilm growth in subsurface porous media, and its treatment with biocides (antimicrobial agents), involves a complex interaction of biogeochemical processes which provide non‐trivial mathematical modelling challenges. Although there are literature reports of mathematical models to evaluate biofilm tolerance to biocides, none of these models have investigated biocide treatment of biofilms growing in interconnected porous media with flow. In this paper, we present a numerical investigation using a pore network model of biofilm growth, formation damage and biocide treatment. The model includes three phases (aqueous, adsorbed biofilm, and solid matrix), a single growth‐limiting nutrient and a single biocide dissolved in the water. Biofilm is assumed to contain a single species of microbe, in which each cell can be a viable persister, a viable non‐persister, or non‐viable (dead). Persisters describe small subpopulation of cells which are tolerant to biocide treatment. Biofilm tolerance to biocide treatment is regulated by persister cells and includes ‘innate’ and ‘biocide‐induced’ factors. Simulations demonstrate that biofilm tolerance to biocides can increase with biofilm maturity, and that biocide treatment alone does not reverse biofilm‐induced formation damage. Also, a successful application of biological permeability conformance treatment involving geologic layers with flow communication is more complicated than simply engineering the attachment of biofilm‐forming cells at desired sites.     

The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance

Aims: This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions. Methods and Results: The pET28 and pUC8 plasmids were separately used to transform E. coli JM109(DE3). Biofilm formation, removal and antimicrobial susceptibility to the cationic biocide benzyldimethyldodecylammonium chloride (BDMDAC) were assessed. Transformed cells formed thicker biofilms with higher cell densities, and the metabolic activity was higher whereas nontransformed cells had higher viabilities. Biocide treatment was not efficient for biofilm removal but was effective for cell killing. Biofilms formed by nontransformed cells were less affected by the treatment. Conclusions: Cell transformation with the tested plasmids has significant impacts on biofilm formation, cell viability, metabolic activity and resistance to biocide treatment. Our results show that in biofilm studies involving deletion/complementation experiments, a control with the strain carrying a plasmid devoid of the gene under investigation must be included so that the real effects of the genetic manipulation are not biased by the presence of the plasmid backbone. Significance and Impact of the Study: This is the first report where the presence of nonconjugative plasmids is assessed in flow conditions analysing biofilm formation, removal and antimicrobial susceptibility of high cell‐density biofilms.     

Effects of Spray Drying on Physicochemical Properties of Chitosan Acid Salts

The effects of spray-drying process and acidic solvent system on physicochemical properties of chitosan salts were investigated. Chitosan used in spray dryings was obtained by deacetylation of chitin from lobster (Panulirus argus) origin. The chitosan acid salts were prepared in a laboratory-scale spray drier, and organic acetic acid, lactic acid, and citric acid were used as solvents in the process. The physicochemical properties of chitosan salts were investigated by means of solid-state CP-MAS ¹³C nuclear magnetic resonance (NMR), X-ray powder diffraction (XRPD), differential scanning calorimetry, and Fourier transform infrared spectrometry (FTIR) and near-infrared spectroscopy. The morphology of spray-dried chitosan acid salts showed tendency toward higher sphericity when higher temperatures in a spray-drying process were applied. Analysis by XRPD indicated that all chitosan acid salts studied were amorphous solids. Solid-state ¹³C NMR spectra revealed the evidence of the partial conversion of chitosan acetate to chitin and also conversion to acetyl amide form which appears to be dependent on the spray-drying process. The FTIR spectra suggested that the organic acids applied in spray drying may interact with chitosan at the position of amino groups to form chitosan salts. With all three chitosan acid salts, the FTIR bands at 1,597 and 1,615 cm⁻¹ were diminished suggesting that –NH groups are protonated. The FTIR spectra of all chitosan acid salts exhibited ammonium and carboxylate bands at 1,630 and 1,556 cm⁻¹, respectively. In conclusion, spray drying is a potential method of preparing acid salts from chitosan obtained by deacetylation of chitin from lobster (P. argus) origin.     

Efficacy of biocides used in the modern food industry to control salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.     

Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers.

Polycationic polymers have been noted for their effects in promoting cell adhesion to various surfaces, but previous studies have failed to describe a mechanism dealing with this type of adhesion. In the present study, three polycationic polymers (chitosan, poly-L-lysine, and lysozyme) were tested for their effects on microbial hydrophobicity, as determined by adhesion to hydrocarbon and polystyrene. Test strains (Escherichia coli, Candida albicans, and a nonhydrophobic mutant, MR-481, derived from Acinetobacter calcoaceticus RAG-1) were vortexed with hexadecane in the presence of the various polycations, and the extent of adhesion was measured turbidimetrically. Adhesion of all three test strains rose from near zero values to over 90% in the presence of low concentrations of chitosan (125 to 250 micrograms/ml). Adhesion occurred by adsorption of chitosan directly to the cell surface, since E. coli cells preincubated in the presence of the polymer were highly adherent, whereas hexadecane droplets pretreated with chitosan were subsequently unable to bind untreated cells. Inorganic cations (Na+, Mg2+) inhibited the chitosan-mediated adhesion of E. coli to hexadecane, presumably by interfering with the electrostatic interactions responsible for adsorption of the polymer to the bacterial surface. Chitosan similarly promoted E. coli adhesion to polystyrene at concentrations slightly higher than those which mediated adhesion to hexadecane. Poly-L-lysine also promoted microbial adhesion to hexadecane, although at concentrations somewhat higher than those observed for chitosan. In order to study the effect of the cationic protein lysozyme, adhesion was studied at 0 degree C (to prevent enzymatic activity), using n-octane as the test hydrocarbon. Adhesion of E. coli increased by 70% in the presence of 80 micrograms of lysozyme per ml. When the negatively charged carboxylate residues on the E. coli cell surface were substituted for positively charged ammonium groups, the resulting cells became highly hydrophobic, even in the absence of polycations. The observed "hydrophobicity" of the microbial cells in the presence of polycations is thus probably due to a loss of surface electronegativity. The data suggest that enhancement of hydrophobicity by polycationic polymers is a general phenomenon.     

Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system.

Chitosan dissolved in acetic acid reacted with glutaraldehyde solution, ranging in concentration from 0.10 to 25.0 x 10(-2) mol dm3. The modified polymers were characterized by means of carbon, hydrogen and nitrogen elemental analysis, scanning electron microscopy, X-ray diffractometry, 13C nuclear magnetic resonance (NMR), infrared and Raman spectroscopies. The uptake of metallic cations in aqueous medium was checked through copper. The obtained data from 13C NMR, infrared and Raman spectroscopies evidenced the formation of an ethylenic double bond in the chitosan glutaraldehyde interaction. These data suggest that free pendant amine groups of chitosan polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds, due to the resonance established with adjacent double ethylenic bonds. The crosslinking is formed by the nonuniform length of chains and by terminal unities. The crosslinking formation can involve two chitosan unities belonging, or not, to the same polymeric chain. The sequence of reactions was established for a chitosan:glutaraldehyde molar proportion of 1:20. The degree of crystallinity and particle size decreased as the amount of glutaraldehyde was increased in the polymer. Physical and chemical properties are not just affected for the chitosan glutaraldehyde reaction, but are also affected strongly by the dissolution of the natural chitosan.     

Synthesis and self-assembly of chitosan-based copolymer with a pair of hydrophobic/hydrophilic grafts of polycaprolactone and poly(ethylene glycol)

Chitosan-based copolymers with binary grafts of hydrophobic polycaprolactone and hydrophilic poly(ethylene glycol) (CS-g-PCL&PEG) were prepared by a homogeneous coupling reaction of phthaloyl-protected chitosan with functional PCL-COOH and PEG-COOH, following deprotection to regenerate free amino groups back to chitosan backbone. They were characterized by ¹H NMR, Fourier transform infrared and X-ray diffraction analysis. These CS-g-PCL&PEG copolymers could form nano-size self-aggregates in acidic aqueous solution without a specific processing technique, which were investigated using dynamic light scattering and transmission electron microscopy. The formed self-aggregates become smaller with weakened stability upon pH increasing. Moreover, the aggregates of copolymer with higher content of PEG and PCL grafts could remain stable for over 30 days in both acid and neutral condition. A possible mechanism was proposed for the formation of self-aggregates from CS-g-PCL&PEG and their structural changes as pH. It is warranted to find promising application of these self-aggregates based on chitosan as drug carriers.     

Designing a Highly Efficient Chemical Chaperone System Using Chitosan-Coated Alginate

In the present work we prepared chitosan-coated alginate beads, to use as a chemical chaperone based on the electrostatic interaction between the carboxylate groups of alginate and the ammonium groups of chitosan. This procedure was an attempt for designing a highly efficient chemical chaperone to improve protein stability and refolding. Based on enzyme recovered activity, turbidity, far-UV CD and fluorescence data, alkaline phosphatase can be stabilized and refolded to a higher degree in the presence of alginate capsules compared with unassisted form and was further improved by including chitosan. Finally the maximum yield was obtained when the refolding process was achieved under a well worked out temperature program: incubation of the captured-enzyme for 20 min at 4 °C followed by overnight incubation at 22 °C, which showed that aggregation is a major limitation to refolding.     

Synthesis, structure and biological activity of copper(II) complexes with oxolinic acid

The neutral mononuclear copper complexes with the quinolone antibacterial drug oxolinic acid in the presence or not of a nitrogen donor heterocyclic ligand 1,10-phenanthroline, 2,2'-bipyridine or 2,2'-dipyridylamine have been synthesized and characterized with infrared, UV-visible and electron paramagnetic resonance spectroscopies. The experimental data suggest that oxolinic acid acts as a deprotonated bidentate ligand and is coordinated to the metal ion through the pyridone and one carboxylate oxygen atoms. The crystal structure of (chloro)(1,10-phenanthroline)(oxolinato) copper(II), 2, has been determined with X-ray crystallography. For all complexes a distorted square pyramidal environment around Cu(II) is suggested. The EPR (electron paramagnetic resonance) behavior of 2 in aqueous solutions indicates mixture of dimeric and monomeric species. The investigation of the interaction of the complexes with calf-thymus DNA has been performed with diverse spectroscopic techniques and showed that the complexes are bound to calf-thymus DNA. The antimicrobial activity of the complexes has been tested on three different microorganisms. The complexes show a decreased biological activity in comparison to the free oxolinic acid.     

A morphological study of heavy metal complexes of chitosan and crosslinked chitosans by SEM and WAXRD

Metal complexes of salts of Hg, Cu, Cd, Pb, Zn, and Mn with chitosan and crosslinked chitosans were prepared, and their morphologies were studied using scanning electron microscopy and wide angle X-ray diffraction. The metal ions which were specifically and strongly complexed to the amino functions of chitosans, like Hg, showed smooth surface morphology inspite of large number of ions complexed (372 mg/g of chitosan). The presence of metal ions on the surface of the chitosans could be detected with decrease in metal ion binding, in the following sequence Hg > Cu > Cd > Zn > Pb > Mn. Particularly in the case of Pb ions, the presence of these ions is clearly seen on the surface of the polymer by SEM. The number of ions of Mn complexed on the polymers was too few (5 mg/g of chitosan) to be visible. SEM of Hg and Cu complexes do not show the “holes” observed in the crosslinked polymers as they bind specifically to amino groups of chitosan, but for Cd, Zn, Mn, and Pb complexes, these “holes” are clearly visible. These results are also in agreement with the morphologies studied by WAXRD. The metal complexation data for each of these metal ions was also in the same sequence.     

Antimicrobial properties and death-inducing mechanisms of saccharomycin,a biocide secreted by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.