Sibel Roller

Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria

The mode of antimicrobial action of chitosan (polymeric beta-1,4-N-acetylglucosamine) on gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM). Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium. The effect was reduced (E. coli, salmonellae) or abolished (P. aeruginosa) by MgCl2. No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2. Chitosan also sensitized P. aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl2 and was reversible by washing chitosan-treated cells prior to SDS exposure. Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E. coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids. However, chitosan rendered E. coli more sensitive to the inhibitory action of dyes and bile acids used in selective media. Highly cationic mutants of S. typhimurium were more resistant to chitosan than the parent strains. Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures. Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function. This property makes chitosan a potentially useful indirect antimicrobial for food protection.

The antifungal properties of chitosan in laboratory media and apple juice.

The antimicrobial properties of chitosan glutamate, a derivative of chitin, were investigated in laboratory media and apple juice against 15 yeasts and moulds associated with food spoilage in order to assess the potential for using chitosan as a natural food preservative. Of the seven strains of filamentous fungi studied, chitosan reduced the growth rate of Mucor racemosus at 1 g/l whilst concentrations of 5 g/l were required to completely prevent growth of three strains of Byssochlamys spp. on agar plates incubated at 25 degrees C for 3 weeks. Three strains of filamentous fungi were resistant to the antifungal effects of chitosan at 10 g/l. The presence of chitosan in apple juice (pH 3.4) at levels ranging from 0.1 to 5 g/l inhibited growth at 25 degrees C of all eight spoilage yeasts examined in this study. The initial effect of chitosan in apple juice was biocidal with viable numbers reduced by up to 3 log cycles. Following an extended lag phase, some strains recovered and resumed growth to levels similar to those observed in unsupplemented apple juice. The most sensitive strain was an isolate of Zygosaccharomyces bailii obtained from a spoiled carbonated beverage; this yeast was completely inactivated by chitosan at 0.1 and 0.4 g/l for 32 days of storage at 25 degrees C. The most resistant strain was Saccharomycodes ludwigii, an isolate from spoiled cider: a level of addition of 5 g/l of chitosan was required to inactivate this strain and to maintain yeast-free conditions in apple juice for 14 days at 25 degrees C. Growth inhibition and inactivation of filamentous moulds and yeasts, respectively, was concentration-, pH- and temperature-dependent. It was concluded that chitosan was worthy of further study as a natural preservative for foods prone to fungal spoilage.

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

ABSTRACT 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.

Antimicrobial Action of Carvacrol at Different Stages of Dual-Species Biofilm Development by Staphylococcus aureus and Salmonella enterica Serovar Typhimurium

ABSTRACT The effects of carvacrol, a natural biocide, on dual-species biofilms formed by Staphylococcus aureus and Salmonella enterica serovar Typhimurium were investigated with a constant-depth film fermentor. Biofilm development reached a quasi-steady state in 12 days at 25°C with S. aureus predominance (≈99%). Cryosectional analysis detected viable S. aureus and S. enterica serovar Typhimurium at depths of 320 and 180 μm from the film surface, respectively. Carvacrol pulses (1.0 mmol/h) inhibited S. aureus by 2.5 log CFU/biofilm during the early stages of film formation, ultimately causing a significant reduction (P < 0.001) of the staphylococcal population at quasi-steady state. Initial carvacrol pulsing elicited a 3 log CFU/biofilm reduction in viable S. enterica serovar Typhimurium, and additional periodic carvacrol pulses instigated significant inhibition of salmonellae (1 to 2 log CFU/biofilm) during biofilm development. Carvacrol pulsing reduced protein levels fivefold (P < 0.001) during initial biofilm development. Comparative studies with a peroxide-based commercial sanitizer (Spor-Klenz RTU) revealed that this commercial sanitizer was more biocidal than carvacrol during early biofilm development. When the biofilm reached quasi-steady state, however, periodic pulses with 1 mmol of carvacrol per h (P = 0.021) elicited a significantly higher inhibition than Spor-Klenz RTU (P = 0.772). Dual-species microcolonies formed under the influence of continuously fed low carvacrol concentrations (1.0 mmol/h) but failed to develop into a mature quasi-steady-state biofilm and did not reach any stage of film formation in the presence of high concentrations (5.0 mmol/h). These data show that carvacrol is an effective natural intervention to control dual-species biofilm formation.

Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut melon and kiwifruit at 4° and 8°C

Aim: To establish whether or not carvacrol and cinnamic acid delay microbial spoilage of fresh‐cut fruit.

Novel bioemulsifiers from microorganisms for use in foods

The main objective of this study was to test the range of microorganisms for production of extracellular, high molecular weight emulsifiers for potential use in foods. A standard emulsification assay developed specifically for assessing food emulsifiers was used to examine 24 extracellular microbial products from bacteria, yeasts and algae. Of the 24 products tested, nine had emulsification ability that was as good as and eight had emulsifying properties that were better than those of the commonly used food emulsifiers gum arabic and carboxymethylcellulose. The eight good producer organisms included the yeasts Candida utilis, Candida valida, Hansenula anomala, Rhodospiridium diobovatum and Rhodotorula graminis, the red alga Porphiridium cruentum, and the bacteria Klebsiella spp. and Acinetobacter calcoaceticus. Of these, C. utilis was selected for further study due to the excellent emulsification properties of its extracellular products and food-grade status of the organism. Crude preparations of the bioemulsifier from C. utilis exhibited low viscosity and had a carbohydrate content of over 80%. Preliminary trials showed that the bioemulsifier from this organism had potential for use in salad cream.

Novel combinations of chitosan, carnocin and sulphite for the preservation of chilled pork sausages

The aim of this study was to develop novel preservation systems for fresh pork sausages based on combinations of chitosan (polymeric β-1,4-N-acetylglucosamine) carnocin (a bacteriocin produced by Carnobacterium piscicola) and low concentrations of sulphite. Two pilot-scale trials showed that 0.6% chitosan combined with low sulphite (170 ppm) retarded the growth of spoilage organisms more effectively (3-4 log cfu/g) than high levels (340 ppm) of sulphite alone at 4xa0°C for up to 24 days. Microbial counts for frozen sausages showed that the preservative efficacy of the chitosan/sulphite combination was maintained following frozen storage. Carnocin did not protect sausages from spoilage but in a challenge trial, it reduced the numbers of Listeria innocua by up to 2.0 log cfu/g in the first 5 days of chill-storage. Sulphite was degraded rapidly within the first 3 days of storage in all the sausages that contained only this preservative but levels decreased less rapidly and persisted for longer in the presence of chitosan. Results of Quantitative Descriptive Analysis using 31 trained panellists reflected the gradual deterioration of all the sausages during storage. The batch containing chitosan and sulphite deteriorated less rapidly and was judged to be more acceptable for a longer period than all the other batches.

Chitosan potentiates the antimicrobial action of sodium benzoate on spoilage yeasts

Aims:u2002The objective of this study was to determine whether low concentrations of chitosan and benzoate in combination could be used to enhance the antimicrobial action of either compound alone against three spoilage yeasts in saline solutions.

Physiology of food spoilage organisms

A thorough understanding of the physiological responses of microorganisms to stresses imposed during food preservation is essential if novel combination systems based on mild food processing procedures are to be developed effectively. The influences of intrinsic characteristics as well as external factors such as water activity, temperature, preservatives, composition of the gaseous atmosphere, etc. on the stress response of microorganisms are discussed. The interaction of spoilage organisms with each other as well as with food pathogens and the ultimate consequences for food safety and quality are also explored in this review.

Solvent extraction of bacteriocins from model solutions and fermentation broths

A novel method for the extraction and purification of bacteriocins (small peptides with antimicrobial properties) from fermentation broths is described. Model solutions of nisin were prepared from Nisaplin®, a commercial whey-based powder containing about 2.5% nisin and sold as a food preservative. These model solutions and fermentation broths from a culture of nisin-producing Lactococcus lactis subsp lactis were cross-flow filtered and stirred with toluene to obtain a white suspension in the toluene layer, which was back extracted with 10u2009mmolu2005dm−3 HCl. Yields of the order of 90% for a single stage extraction were obtained followed by similar yields on back extraction. Yields dropped with decreasing pH, going through a minimum at pH 2. In larger scale experiments (11u2009dm3) at pH 6, a gel layer was formed which, on centrifugation and drying, gave a white solid containing about 50% nisin, the remainder being water and salt. Further process development would almost certainly bring this figure closer to the small-scale value. The phase transfer technique was also applied to solutions containing variacin and carnocin (produced by Micrococcus varians and Carnobacterium piscicola, respectively). The mechanism of bacteriocin extraction probably involves concentration of the peptides, which are surface active, at the interface until their solubility is exceeded and they form reverse submicelles, which aggregate into reverse micelles to give a gel. n n n n© 2000 Society of Chemical Industry