Cationic peptides produced by multicellular organisms are an evolutionarily ancient and rapidly mobilized primary defense against infections caused by a broad range of microbes (8). The cationic antimicrobial peptides are ribosomally synthesized, proteolytically processed species of 12 to ϳ50 amino acids that comprise about 50% hydrophobic residues and that have a net excess of positive charge (9). They are usually found on epithelial cell surfaces and in phagocytic cells at sites of microbial invasion, and only a few instances of constitutive or induced resistance to cationic peptides have been detected (for a review, see reference 44). Although the cationic peptides are subdivided into several structural classes (10), they are, in general, amphipathic molecules that preferentially bind to acidic phospholipids, acidic polysaccharides, and lipopolysaccharides on the exterior of the lipid bilayer of invading microbes rather than to the cholesterol-rich and neutral plasma membrane surfaces of mammalian host cells. The bound cationic peptides are then thought to kill target microbes, including fungi (4), by forming assemblies that alter the lipid bilayer structure and disrupt the functional properties of the microbial membrane. A few cationic peptides may affect intracellular targets, including mitochondria and DNA and RNA metabolism; but apart from the binding of the salivary histatin 5 to a cell surface receptor in Candida albicans (16), there is no evidence of direct effects on fungal cell surface proteins. We hypothesized that the incorporation of a cationic peptide-like motif into an antifungal would enhance its potency by concentrating the compound at fungal cell surfaces. This idea has been validated in the present study by obtaining a membraneimpermeant and surface-active cationic peptide inhibitor of the fungal plasma membrane proton-pumping ATPase (Pma1p), an essential enzyme involved in fungal energy transduction (36).Pma1p is an ϳ100-kDa electrogenic, polytopic integral membrane protein of the P-type ATPase class which contributes 10 to 20% of the yeast plasma membrane protein. It generates the plasma membrane electrochemical gradient that is required for the maintenance of intracellular pH, cellular ion balance, and the uptake of numerous nutrients (36). The amount of functional Pma1p is tightly regulated (5), and yeast growth requires at least 25% of normal Pma1p activity (35). Pma1p was postulated to be a target for surface-mediated, broad-spectrum antifungal intervention because of the structural similarity between cell surface loops in Pma1ps from fungal cells and their dissimilarity to the comparable loops in P-type ATPases from other organisms, as well as the specificity achieved with therapies targeting mammalian P-type ATPases (28). Pma1p was validated as an antifungal target by demonstrating that acid-activated omeprazole is a fungicidal Pma1p inhibitor that acts from outside the cell (25,37). This paper describes a drug discovery strategy that targets Pma1p. Screen-* Corresponding author. Mai...
The antimicrobial impact of purified and natural sources of both nitrite and ascorbate were evaluated against Clostridium perfringens during the postthermal processing cooling period of deli-style turkey breast. The objective of phase I was to assess comparable concentrations of nitrite (0 or 100 ppm) and ascorbate (0 or 547 ppm) from both purified and natural sources. Phase II was conducted to investigate concentrations of nitrite (50, 75, or 100 ppm) from cultured celery juice powder and ascorbate (0, 250, or 500 ppm) from cherry powder to simulate alternative curing formulations. Ground turkey breast (75% moisture, 1.2% salt, pH 6.2) treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in 10 additional hours. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar; experiments were replicated three times. In phase I, uncured, purified nitrite, and natural nitrite treatments without ascorbate had 5.3-, 4.2-, and 4.4-log increases in C. perfringens, respectively, at 15 h, but <1-log increase was observed at the end of chilling in treatments containing 100 ppm of nitrite and 547 ppm of ascorbate from either source. In phase II, 0, 50, 75, and 100 ppm of nitrite and 50 ppm of nitrite plus 250 ppm of ascorbate supported 4.5-, 3.9-, 3.5-, 2.2-, and 1.5-log increases in C. perfringens, respectively. In contrast, <1-log increase was observed after 15 h in the remaining phase II treatments supplemented with 50 ppm of nitrite and 500 ppm of ascorbate or ≥75 ppm of nitrite and ≥250 ppm of ascorbate. These results confirm that equivalent concentrations of nitrite, regardless of the source, provide similar inhibition of C. perfringens during chilling and that ascorbate enhances the antimicrobial effect of nitrite on C. perfringens at concentrations commonly used in alternative cured meats.
Sodium nitrite has been identified as a key antimicrobial ingredient to control pathogens in ready-to-eat (RTE) meat and poultry products, including Listeria monocytogenes. This study was designed to more clearly elucidate the relationship between chemical factors (ingoing nitrite, ascorbate, and residual nitrite) and L. monocytogenes growth in RTE meats. Treatments of cooked, cured pork sausage (65% moisture, 1.8% salt, pH 6.6, and water activity 0.98) were based on response surface methodology with ingoing nitrite and ascorbate concentrations as the two main factors. Concentrations of nitrite and ascorbate, including star points, ranged from 0 to 352 and 0 to 643 ppm, respectively. At one of two time points after manufacturing (days 0 and 28), half of each treatment was surface inoculated to target 3 log CFU/g of a five-strain L. monocytogenes cocktail, vacuum packaged, and stored at 7°C for up to 4 weeks. Growth of L. monocytogenes was measured twice per week, and enumerations were used to estimate lag time and growth rates for each treatment. Residual nitrite concentrations were measured on days 0, 4, 7, 14, 21, and 28, and nitrite depletion rate was estimated by using first-order kinetics. The response surface methodology was used to model L. monocytogenes lag time and growth rate based on ingoing nitrite, ascorbate, and the residual nitrite remaining at the point of inoculation. Modeling results showed that lag time was impacted by residual nitrite concentration remaining at inoculation, as well as the squared term of ingoing nitrite, whereas growth rate was affected by ingoing nitrite concentration but not by the remaining residual nitrite at the point of inoculation. Residual nitrite depletion rate was dependent upon ingoing nitrite concentration and was only slightly affected by ascorbate concentration. This study confirmed that ingoing nitrite concentration influences L. monocytogenes growth in RTE products, yet residual nitrite concentration contributes to the antimicrobial impact of nitrite as well.
Organic acids and sodium nitrite have long been shown to provide antimicrobial activity during chilling of cured meat products. However, neither purified organic acids nor NaNO2 is permitted in products labeled natural and both are generally avoided in clean-label formulations; efficacy of their replacement is not well understood. Natural and clean-label antimicrobial alternatives were evaluated in both uncured and in alternative cured (a process that uses natural sources of nitrite) deli-style turkey breast to determine inhibition of Clostridium perfringens outgrowth during 15 h of chilling. Ten treatments of ground turkey breast (76% moisture, 1.2% salt) included a control and four antimicrobials: 1.0% tropical fruit extract, 0.7% dried vinegar, 1.0% cultured sugar-vinegar blend, and 2.0% lemon-vinegar blend. Each treatment was formulated without (uncured) and with nitrite (PCN; 50 ppm of NaNO2 from cultured celery juice powder). Treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in an additional 10 h. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar. Uncured control and PCN-only treatments allowed for 4.6- and 4.2-log increases at 15 h, respectively, and although all antimicrobial treatments allowed less outgrowth than uncured and PCN, the degree of inhibition varied. The 1.0% fruit extract and 1.0% cultured sugar-vinegar blend were effective at controlling populations at or below initial levels, whether or not PCN was included. Without PCN, 0.7% dried vinegar and 2.0% lemon-vinegar blend allowed for 2.0- and 2.5-log increases, respectively, and ∼1.5-log increases with PCN. Results suggest using clean-label antimicrobials can provide for safe cooling following the study parameters, and greater inhibition of C. perfringens may exist when antimicrobials are used with nitrite.
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