Characterization of antimicrobial properties on the growth of S. aureus of novel renewable blends of gliadins and chitosan of interest in food packaging and coating applications.

The biocide properties of chitosan-based materials have been known for many years. However, typical antimicrobial formulations of chitosan, mostly chitosonium salts, are known to be very water sensitive materials which may impair their use in many application fields such as food packaging or food coating applications. This first work reports on the development and characterization of the antimicrobial properties of novel fully renewable blends of chitosan with more water-resistant gliadin proteins isolated from wheat gluten. Chitosan release to the nutrient broth from a wide range of blends was studied making use of the ninhydrin method. The results indicated that both pure chitosan and its blends with gliadins presented significant antimicrobial activity, which increased with increasing the amount of chitosan in the composite formulation as expected. The gliadins-chitosan blends showed good transparency and film-forming properties and better water resistance than pure chitosan. The release tests revealed that dissolution of the biocide glucosamine groups, i.e. the chitosan water soluble fractions, also increased with the amount of chitosan present in the formulation. The release of these groups was for the first time directly correlated with the antimicrobial properties exhibited by the blends. Thus, incorporation of chitosan into an insoluble biopolymer matrix was revealed as a very feasible strategy to generate novel chitosan-based antimicrobial materials with potential advantages, for instance active food packaging applications.

Photoactivated chlorophyllin-based gelatin films and coatings to prevent microbial contamination of food products.

The aim of this work was to develop antimicrobial photosensitizer-containing edible films and coatings based on gelatin as the polymer matrix, incorporating sodium magnesium chlorophyllin (E-140) and sodium copper chlorophyllin (E-141). Chlorophyllins were incorporated into the gelatin film-forming solution and the inhibiting effect of the cast films was tested against Staphylococcus aureus and Listeria monocytogenes. The results demonstrated that water soluble sodium magnesium chlorophyllin and water soluble sodium copper chlorophyllin reduced the growth of S. aureus and L. monocytogenes by 5 log and 4 log respectively. Subsequently, the activity of self-standing films and coatings containing E-140 was assessed on cooked frankfurters inoculated with S. aureus and L. monocytogenes. These tests showed that it was possible to reduce microorganism growth in cooked frankfurters inoculated with S. aureus and L. monocytogenes by covering them with sodium magnesium chlorophyllin-gelatin films and coatings.

The effect of the growth environment on the lag phase of Listeria monocytogenes.

The duration of lag in Listeria monocytogenes was examined in relation to the physico-chemical properties of the growth environment. It was supposed that lag would be determined by two hypothetical quantities, the amount of work that a cell has to perform to adapt to new conditions and the rate at which it can perform that work. If the rate at which the cell can perform the necessary work is a function of the maximum specific growth rate in the new environment, the hypothesis predicts that lag time should be related in some way to growth rate, provided cells are initially in approximately the same physiological state. Literature data suggest this is true for many organisms when temperature is the sole growth limiting factor. However, lag times of L. monocytogenes displayed an unusual response to temperature in which lag times of cells precultured at 37 degrees C were shorter at 15 degrees C than at 20 degrees C or 25 degrees C. Analysis of data from the Food Micromodel in which growth of L. monocytogenes was controlled by combinations of pH, NaCl concentration and temperature, showed that there was a linear relationship between lag time and mean generation time although there was much scatter in the data. When the effects of pH, solute type and concentration were investigated individually in this work the correlation between lag time and mean generation time was often poor. It would thus appear that the relationship between growth environment and lag time is more complex than the corresponding relationship between growth environment and maximum specific growth rate.

Heat resistance of Bacillus stearothermophilus spores in alginate-mushroom puree mixture.

Thermal resistance of Bacillus stearothermophilus spores has been established inoculating spores in alginate-mushroom puree mixture (ungelled) and in alginate-mushroom puree mixture set in calcium chloride (gelled). Data are compared with those obtained suspending the spores in distilled water, mushroom extract and in calcium chloride. Results indicated that, in general, D values obtained in gelled mixture were higher than D values obtained in distilled water, mushroom extract or in ungelled mixture, while the D value in the ungelled mixture was similar to that obtained in distilled water. D121 value in gelled mixture was close to that obtained in 2% (w/v) calcium chloride.

Effect of heat activation and inactivation conditions on germination and thermal resistance parameters of Bacillus cereus spores.

The effect of isothermal and non-isothermal heat activation on germination and thermoresistance of two strains of Bacillus cereus spores was studied. Results indicated that the germination after isothermal activation was lower than after non-isothermal heating. The activation rate affected the z value, which increased with faster heating rates. For each temperature and inactivation rate, the non-isothermal activation at rate of 2 degrees C/min resulted in larger D values (D90 = 4.70 min) than isothermal activation (D90 = 4.04 min). The two mathematical equations used to analyse non-isothermal data produced similar predicted D and z values, nevertheless the Hayakawa equation modified in this work for non-linear regression analysis, requires less computational effort.

The effect of inoculum size on the lag phase of Listeria monocytogenes.

The effect of inoculum size on population lag times of Listeria monocytogenes was investigated using the Bioscreen automated microtitre plate incubator and reader. Under optimum conditions, lag times were little affected by inoculum size and there was little variation between replicate inocula even at very low cell numbers. However, in media containing inhibitory concentrations of NaCl, both the mean lag time and variation between replicate inocula increased as the inoculum size became smaller. The variation in lag time of cells within a population was investigated in more detail by measuring the distribution of detection times from 64 replicate inocula containing only one or two cells capable of initiating growth. The variance of the lag time distribution increased with increasing salt concentration and was greater in exponential than in stationary phase inocula. The number of cells required to initiate growth increased from one cell under optimum conditions to 10(5) cells in medium with 1.8 M NaCl. The addition of spent medium from a stationary phase culture reduced the variance and decreased lag times. The ability to initiate growth under severe salt stress appears to depend on the presence of a resistant sub-fraction of the population, although high cell densities assist adaptation of those resistant cells to the unfavourable growth conditions by some unspecified medium conditioning effect. These results are relevant to the prediction of lag times and probability of growth from low numbers of stressed cells in food.

Thermal resistance characteristics of PA 3679 in the temperature range of 110-121 degrees C as affected by pH, type of acidulant and substrate.

The influence of mushroom extract, acidification and type of acidulant (citric acid or glucono-delta-lactone) on the thermal resistance of PA 3679 spores has been studied. The pH values were 7 (phosphate buffer), 6.7 (non acidified mushroom extract), 6.22, 5.34 and 4.65 (acidified mushroom extract). Spores were less heat resistant in the non-acidified mushroom extract than in phosphate buffer. However, acidification of the extract had an effect on D values which was dependent on the specific pH. At pH 6.22, D values were higher than those in non-acidified extract, for both type of acidulants, but the difference between D values in both substrates was affected by temperature, the higher the temperature, the less the difference. Acidification at other pH levels did not significantly affect D values obtained, neither did the type of acidulant. The z value ranged from 9.79 to 12.09 degrees C for citric acid and from 8.18 to 10.18 degrees C for glucono-delta-lactone. When pH was decreased the z value increased for both acidulants.

Mathematical model for the combined effect of temperature and pH on the thermal resistance of Bacillus stearothermophilus and Clostridium sporogenes spores.

Two mathematical models have been studied to establish the relationship between the pH, treatment temperature and thermal destruction constant (k) of Bacillus stearothermophilus and Clostridium sporogenes spores. The study was carried out by heating the spores in mushroom extract acidified with two different acidulants (citric acid and glucono-delta-lactone). Among the models studied, the one that best described the inactivation was a second order polynomial equation, the precision of which depended on the microorganism studied.

Empirical model building based on Weibull distribution to describe the joint effect of pH and temperature on the thermal resistance of Bacillus cereus in vegetable substrate.

A mathematical model based on Weibull parameters was built to describe the joint effect of temperature and pH on thermal inactivation of Bacillus cereus spores (strain INRA TZ415). The effect of these factors on Weibull model parameters (beta, 1/alpha) was also studied. Heat inactivation tests were carried out in acidified carrot broth as vegetable substrate, following a full factorial design at four levels for temperature (80, 85, 90 and 95 degrees C) and pH (6.2, 5.8, 5.2 and 4.7). The Weibull distribution model provided good individual fits for the different combinations of temperature-pH tested, with discrepancy factors, Df, coming close to 25% for most cases. The temperature and pH did not have a significant effect on the shape parameter (beta), which yielded a mean value of 0.88. The scale parameter (alpha) decreased with pH, and its inverse (1/alpha) followed an Arrhenius-type relationship with temperature. A global model was built, including the dependence of the alpha parameter on temperature and pH, and the model parameters were estimated by using a one-step nonlinear least-squares regression to improve the precision of the estimates. Results indicated that the global model provides a satisfactory description of the thermal inactivation of B. cereus spores, with R2 equal to 0.983.

Improved model based on the Weibull distribution to describe the combined effect of pH and temperature on the heat resistance of Bacillus cereus in carrot juice.

The effect of pH and temperature on the thermal inactivation of different strains of Bacillus cereus was modeled. Inactivation tests were carried out in carrot broth, following a full factorial design at four levels for temperature (from 90 to 105 degrees C, depending on the strain) and pH (6.2, 5.8, 5.2, and 4.7). Individual inactivation curves were analyzed by applying the Weibull model function (with percent discrepancy close to 20% for most cases), and the effects of pH and temperature on the scale parameter (designated D(beta)) and the shape parameter (beta) were also studied. Temperature and pH did not have a significant effect on the shape parameter (beta). The effect of temperature on the scale parameter was modeled by the zeta concept. The scale parameter decreased with pH, although the behavior of the strains was not homogeneous. Two global models with a small number of parameters were developed, providing a satisfactory description of the thermal inactivation of B. cereus, with percent discrepancy ranging from 18 to 25%.

Evaluation of silver-infused polylactide films for inactivation of Salmonella and feline calicivirus in vitro and on fresh-cut vegetables.

There is a growing trend to develop packaging materials with an active role in guarantying that the quality and safety characteristics of packaged products will remain or improve from preparation throughout shelf-life. In the present study, 0.001-1.0 wt.% silver ions were satisfactorily incorporated into polylactide (PLA) films by a solvent casting technique. Silver migration from the films was measured by voltamperometry and then correlated with its antimicrobial efficacy against Salmonella enterica and feline calicivirus (FCV), a human norovirus surrogate, by using the Japanese industrial standard (JIS Z 2801). The PLA-silver films showed strong antibacterial and antiviral activity in vitro, with increasing effects at higher silver concentrations. Moreover, results show that FCV was less susceptible to silver than Salmonella. When films were applied on food samples, antibacterial and antiviral activity was reduced as compared to in vitro. Antimicrobial activity was very much dependent on the food type and temperature. In lettuce samples incubated at 4 °C during 6 days, 4 log CFU of Salmonella was inactivated for films with 1.0 wt.% and no infectious FCV was reported under the same conditions. On paprika samples, no antiviral effect was seen on FCV infectivity whereas films showed less antibacterial activity on Salmonella.

On the different growth conditions affecting silver antimicrobial efficacy on Listeria monocytogenes and Salmonella enterica.

Silver is known to inhibit microorganisms and therefore it is an ideal candidate for its incorporation in a wide variety of materials for food applications. However, there is still a need for understanding how silver prolonged exposure to bacterial contamination affects the bioavailability of the active silver species. In the present study, growth curves of Listeria monocytogenes and Salmonella enterica were performed for 3-5 days in Tryptic Soy Broth (TSB) and M9 minimal medium (M9) in the presence of silver ions and silver solutions previously in contact with the growth media. The cultivability of the bacteria under these conditions was correlated with the viability of the bacterial populations as measured by flow cytometry analysis (FC) using a LIVE/DEAD BacLight kit. It was found that, after a period where viable counts were not detected, bacterial populations recovered and were able to proliferate in most cases. The resuscitation of the cultures was explained by both the existence of a resilient fraction of bacteria in a compromised state and the parallel inactivation of the silver species. This inactivation was found to be highly influenced by time dependant chemical reactions taking place in the environment of exposure, producing differences of at least 3 fold between results for nutrient rich environments and results for limiting environments. This study points out the need for understanding these chemical interactions and bacterial mechanisms of adaptation and may have relevance in the design of silver-based antimicrobial systems for food-related applications.

Effects of chitosan films on the growth of Listeria monocytogenes, Staphylococcus aureus and Salmonella spp. in laboratory media and in fish soup.

The objective of this study was to assess the antimicrobial effectiveness of chitosonium acetate films on the growth of Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. The samples were tested in both laboratory conditions using Tryptone Soy Broth (TSB) and in a real food system using fish soup. The study was carried out at different temperatures (4, 12, and 37 degrees C) in order to discern the influence of such variables. Moreover, a sensory evaluation of the final product was performed as a parameter of consumer acceptance. The results showed a significant reduction of the bacterial growth, which greatly depended on the bacteria type, the temperature of incubation and the food substrate. Although the effectiveness of chitosan films decreased in the fish soup, neither the sensory properties nor the pH of the soup was affected upon their addition. The application of chitosonium acetate as an internal coating of the packaging material could be a very suitable means to assure safety of liquid food products such as fish soup at the range of temperatures studied.

Optimization of the film-forming and storage conditions of chitosan as an antimicrobial agent.

The aim of this work was to assess the antimicrobial capacity of chitosan-based films obtained by a dissolution and solvent evaporation (solvent casting) method at various temperatures (i.e., 37, 80, and 120 degrees C) on the growth of Staphylococcus aureus and Salmonella spp. bacteria. The effect of temperature (4, 23, 37 degrees C) and relative humidity (RH; 0, 75%) during storage on the biocide performance was also investigated. Color parameters and ATR-FTIR spectra were analyzed for each sample to investigate the relationship between structural and/or chemical alterations in the films during storage and biocide performance. The results indicated that films formed at 37 and 80 degrees C presented a significant inhibitory effect for both types of bacteria; however, when cast at 120 degrees C, the films ceased to exhibit antimicrobial properties. Curiously, chitosonium acetate films were seen to diminish to a large extent their biocide properties when stored at 23 degrees C and 75% RH for 2 months or alternatively when stored and 37 degrees C and 0% RH over the same period of time. In good agreement with this behavior the FTIR results indicated that under the previous conditions a significant fraction of the biocide carboxylate chemistry remained in the polymer after contact with the bacterial solution due to a strong reduction in cast film solubility. Because biopolymer active species migration from the film to the culture media is needed for the biomaterial to exhibit measurable antimicrobial effect, proper control of temperature and humidity during film formation and storage is necessary to design the optimum performance of chitosan as a biocide.

Film-forming process and biocide assessment of high-molecular-weight chitosan as determined by combined ATR-FTIR spectroscopy and antimicrobial assays.

This pioneering study reported about the film-forming properties of high-molecular-weight chitosan as followed in situ by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and has implications in fields such as biomedical, pharmaceutical, packaging, and coating applications. From the results, it was observed that immediately after dissolution in an acetic acid aqueous solution and subsequent casting over the ATR crystal, the formed carboxylate antimicrobial (-NH3+ -OOCH) species are not stable in the film formulation and become reduced over time; further assays confirmed previous research, which suggested that the presence and stability of these groups is strongly dependent, among other factors, on storage conditions. As-received chitosan and chitosan neutralized in NaOH films did not exhibit biocide performance towards Staphylococcus aureus. The antimicrobial tests were also found to strongly relate the presence of a sufficient quantity of these carboxylate groups to the chitosan activity as a biocide agent. Moreover, a novel methodology based on the use of a normalized infrared band centered at 1405 cm(-1) is proposed which can be correlated with the antimicrobial character of the biopolymer.

The use of ethidium bromide to assess a novel injury/recovery phenomenon in Listeria monocytogenes in inhibitory NaCl conditions.

An injury and recovery phenomenon was observed in Listeria monocytogenes inoculated into a medium containing 2.2 mmol l -1 NaCl, a concentration that was inhibitory to growth. The apparent loss then recovery of viability, as determined by plate counts, was compared with the uptake of ethidium bromide by the cells and found to be inversely related. Injury was caused not only by the initial osmotic up-shock but also by the subsequent down-shock involved in the spread plate protocol.

Thermal Resistance of Bacillus stearothermophilus Heated at High Temperatures in Different Substrates.

The effect of mushroom extract, with or without acidification with glucono-δ-lactone, and the overnight incubation of the spores in CaCl2, on the heat resistance of B. stearothermophilus ATCC 12980 spores was studied. The temperature range considered was 121 to 140°C for mushroom extract and CaCl2 and 121 to 145°C for double-distilled water as a reference substrate. The results indicated that mushroom extract without added acid significantly reduces the thermal resistance of the spores in comparison to the double-distilled water. Acidification of the mushroom extract reduces the heat resistance of spores of B. stearothermophilus at 121 °C. However, above 130°C lowering of the pH did not significantly reduce the thermal resistance of the spores, and so no generalizations should be made with regard to the effect of the pH when high temperature-short time (HTST) processes are being considered. Overnight incubation in CaCl2 and subsequent heat treatment lead to increased heat resistance at 121 °C compared to that observed in double-distilled water. However, at 130°C and above CaCl2 did not increase the apparent heat resistance of the spores.

Development and Validation of a Mathematical Model for HTST Processing of Foods Containing Large Particles.

A mathematical treatment for a heat penetration phenomenon with variable boundary conditions is presented. The system of differential equations for determining the unsteady-state temperature distribution inside a particle was solved by use of spectral methods as a new tool in food process development. A preliminary study was conducted on the use of a mathematical model to predict lethality in a sterilizing process. The model was validated using a calibrated time-temperature integrator (TTI) with immobilized Bacillus stearothermophilus spores, commonly used in TTIs for process validation. A comparison between the experimental data using Bacillus stearothermophilus and the predicted data obtained with the proposed model showed good agreement.

The effect of inoculum size and sublethal injury on the ability of Listeria monocytogenes to initiate growth under suboptimal conditions.

AIMS: To investigate the effect of inoculum size and physiological state on the ability of Listeria monocytogenes cells to initiate growth under suboptimal conditions of salt concentration and pH. METHODS AND RESULTS: Cell suspensions were serially diluted in media of different salt concentration or pH and replicate inocula distributed into 96-well microplates. The proportion of wells showing growth at each dilution level was determined after incubation for 6 weeks for each set of conditions. Growth occurred from single cells up to a concentration of 1.2 mol l-1 NaCl; above this threshold, the inoculum size needed to initiate growth became progressively larger. A similar effect was seen with decreasing pH but only very close to the growth/no growth boundary. The threshold for inoculum-dependent growth was lower in exponential phase cells than in stationary phase ones and sublethal injury greatly decreased the probability of growth from small inocula. CONCLUSIONS: The growth/no growth boundary for L. monocytogenes is not an absolute cut-off point but represents a region where the probability of growth rapidly decreases as conditions become more extreme. We interpret the requirement for a critical inoculum size for growth as being due to death of a proportion of cells in the inoculum rather than to co-operative population effects. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological heterogeneity within the cell population and inoculum size will affect the risk of L. monocytogenes growing in food.

Growth and Heat Resistance of Clostridium Sporogenes PA 3679 Spores Heated and Recovered in Acidified Media.

The effect of subculture medium composition (pH, type of acidulant, presence of EDTA) on the germination of spores of Clostridium sporogenes PA 3679, both with and without heat treatment in phosphate buffer (30 s at l25°C), was studied. Additionally, the effect of the heating medium substrate and pH and subculture medium pH and composition on the heat resistance of the spores was evaluated. The results indicate that the pH of the recovery medium and the type of acidulant have significant effects (P ≤ 0.05) on counts of PA 3679. Adding EDTA only increases inhibition of this microorganism when the pH is near neutral. For a given pH level, citric acid provided greater inhibiting power than glucono-δ-lactone, although the percentage of undissociated acid was less for citric acid. No growth was observed at pH 5.7, although in the case of glucono-δ-lactone sporadic colony development was seen in some plates. The pH of the heating medium acidified with glucono-δ-lactone did not affect the heat-resistance parameters (D and z) of spores heated to temperatures between 121 and 140°C in relation to the unacidified extract. Nor did we see any significant influence of the composition and pH of the recovery medium on these heat-resistance parameters when comparing the results with those obtained using the usual recovery medium.