Influence of high voltage atmospheric cold plasma process parameters and role of relative humidity on inactivation of Bacillus atrophaeus spores inside a sealed package.

BACKGROUND: Non-thermal plasma has received much attention for elimination of microbial contamination from a range of surfaces. AIM: This study aimed to determine the effect of a range of dielectric barrier discharge high voltage atmospheric cold plasma (HVACP) parameters for inactivation of Bacillus atrophaeus spores inside a sealed package. METHODS: A sterile polystyrene Petri dish containing B. atrophaeus spore strip (spore population 2.3 × 10(6)/strip i.e. 6.36 log10/strip) was placed in a sealed polypropylene container and was subjected to HVACP treatment. The HVACP discharge was generated between two aluminium plate electrodes using a high voltage of 70kVRMS. The effects of process parameters, including treatment time, mode of exposure (direct/indirect), and working gas types, were evaluated. The influence of relative humidity on HVACP inactivation efficacy was also assessed. The inactivation efficacy was evaluated using colony counts. Optical absorption spectroscopy (OAS) was used to assess gas composition following HVACP exposure. FINDINGS: A strong effect of process parameters on inactivation was observed. Direct plasma exposure for 60s resulted in ≥6 log10 cycle reduction of spores in all gas types tested. However, indirect exposure for 60s resulted in either 2.1 or 6.3 log10 cycle reduction of spores depending on gas types used for HVACP generation. The relative humidity (RH) was a critical factor in bacterial spore inactivation by HVACP, where a major role of plasma-generated species other than ozone was noted. Direct and indirect HVACP exposure for 60s at 70% RH recorded 6.3 and 5.7 log10 cycle reduction of spores, respectively. CONCLUSION: In summary, a strong influence of process parameters on spore inactivation was noted. Rapid in-package HVACP inactivation of bacterial spores within 30-60s demonstrates the promising potential application for reduction of spores on medical devices and heat-sensitive materials.

Atmospheric cold plasma inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes inoculated on fresh produce.

Atmospheric cold plasma (ACP) represents a potential alternative to traditional methods for non-thermal decontamination of foods. In this study, the antimicrobial efficacy of a novel dielectric barrier discharge ACP device against Escherichia coli, Salmonella enterica Typhimurium and Listeria monocytogenes inoculated on cherry tomatoes and strawberries, was examined. Bacteria were spot inoculated on the produce surface, air dried and sealed inside a rigid polypropylene container. Samples were indirectly exposed (i.e. placed outside plasma discharge) to a high voltage (70 kVRMS) air ACP and subsequently stored at room temperature for 24 h. ACP treatment for 10, 60 and 120 s resulted in reduction of Salmonella, E. coli and L. monocytogenes populations on tomato to undetectable levels from initial populations of 3.1, 6.3, and 6.7 log10 CFU/sample, respectively. However, an extended ACP treatment time was necessary to reduce bacterial populations attached on the more complex surface of strawberries. Treatment time for 300 s resulted in reduction of E. coli, Salmonella and L. monocytogenes populations by 3.5, 3.8 and 4.2 log10 CFU/sample, respectively, and also effectively reduced the background microflora of tomatoes.

Bacterial inactivation by high-voltage atmospheric cold plasma: influence of process parameters and effects on cell leakage and DNA.

AIMS: This study investigated a range of atmospheric cold plasma (ACP) process parameters for bacterial inactivation with further investigation of selected parameters on cell membrane integrity and DNA damage. The effects of high voltage levels, mode of exposure, gas mixture and treatment time against Escherichia coli and Listeria monocytogenes were examined. METHODS AND RESULTS: 10(8) CFU ml(-1) E. coli ATCC 25922, E. coli NCTC 12900 and L. monocytogenes NCTC11994 were ACP-treated in 10 ml phosphate-buffered saline (PBS). Working gas mixtures used were air (gas mix 1), 90% N2 + 10% O2 (gas mix 2) and 65% O2 + 30% CO2 + 5% N2 (gas mix 3). Greater reduction of viability was observed for all strains using higher voltage of 70 kVRMS and with working gas mixtures with higher oxygen content in combination with direct exposure. Indirect ACP exposure for 30 s inactivated below detection level both E. coli strains. L. monocytogenes inactivation within 30 s was irrespective of the mode of exposure. Leakage was assessed using A260 absorbance, and DNA damage was monitored using PCR and gel electrophoresis. Membrane integrity was compromised after 5 s, with noticeable DNA damage also dependent on the target cell after 30 s. CONCLUSIONS: Plasma treatment was effective for inactivation of challenge micro-organisms, with a greater sensitivity of L. monocytogenes noted. Different damage patterns were observed for the different bacterial strains attributed to the membrane structure and potential resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Using atmospheric air as working gas resulted in useful inactivation by comparison with high nitrogen or high oxygen mix. The mechanism of inactivation was a function of treatment duration and cell membrane characteristics, thus offering potential for optimized process parameters specific to the microbial challenge.

Atmospheric cold plasma inactivation of Escherichia coli in liquid media inside a sealed package.

AIMS: The main objective of this study was to determine the inactivation efficacy of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) generated inside a sealed package for Escherichia coli ATCC 25922. METHODS AND RESULTS: A plasma discharge was generated between two circular aluminium electrodes at 40 kV. E. coli suspensions (10(7) CFU ml(-1)) in either maximum recovery diluent (MRD) or phosphate buffered saline (PBS) were treated in a 96-well microtitre plate inside a sealed package. The effects of treatment time, post-treatment storage time, either direct or indirect samples exposure to the plasma discharge and suspension media were studied. Regardless of the media tested, 20 s of direct and 45 s of indirect plasma treatment resulted in complete bacterial inactivation (7 log CFU ml(-1)). At the lower plasma treatment times (10-30 s) investigated, the effects of suspension media and mode of exposure on the inactivation efficacy were evident. The inactivation efficacy was also influenced by the post-treatment storage time. CONCLUSIONS: It was demonstrated that the novel DBD-ACP can inactivate high concentrations of E. coli suspended in liquids within sealed packages in seconds. SIGNIFICANCE AND IMPACT OF THE STUDY: A key advantage of this in-package nonthermal novel disinfection approach is the elimination of post-processing contamination.

Maximizing carbon dioxide content of shell eggs by rapid cooling treatment and its effect on shell egg quality.

Rapid cooling of shell eggs using liquid CO2 has been shown to cool eggs to 7°C within minutes, as opposed to days required by traditional cooling treatments. This quick-cooling technique is component in the maintenance of egg quality and extended shelf life beyond the current 30- to 45-d period. The hypothesis for the current study was that maximizing CO2 content of the eggs during cooling may increase Haugh units and thus extend shelf life (physical quality factors). The objective of this study was to maximize CO2 content of shell eggs during rapid cooling with liquid CO2 and determine its effect on egg quality during 12 wk of refrigerated storage. Three cooling conditions selected for the study were -45°C for 18 min (treatment A), -60°C for 15 min (treatment B), and -75°C for 12 min (treatment C). After rapid-cooling treatment, it took approximately 25 min for the internal temperature of eggs to equilibrate to 7°C. The Haugh units of the rapidly cooled eggs were significantly higher than the traditionally cooled (control) eggs. After 12 wk of refrigerated (5-7°C) storage, control eggs were only 37% AA-grade, 57% A-grade, and 6% B-grade. In comparison, all the rapidly cooled eggs averaged to 80% AA-grade and 20% A-grade. After 6 wk, the average quality of control eggs reduced to grade A, whereas rapid cooling treatment was able to maintain AA quality up to 12 wk. The CO2 content of the rapidly cooled eggs (1.8 mg of CO2/g of albumen) showed no difference between treatments A, B, and C, but it was significantly higher than the control (1.3 mg of CO2/g of albumen). In addition, the vitelline membrane strength of the control decreased 65% during storage and was between 30 and 50% of the vitelline membrane strength of CO2-cooled eggs at 12 wk. Rapid cooling with liquid CO2 extends shelf life of shell eggs.

Influence of carbon dioxide on the activity of chicken egg white lysozyme.

Rapid cooling of shell eggs by using liquid CO(2) has shown increased bactericidal effects along with saturation of the egg albumen with CO(2). Lysozyme is a bactericidal enzyme present in chicken eggs, and it lyses gram-positive bacteria. Newly laid chicken eggs have an initial pH of 7.6 to 8.5 and are saturated with CO(2). During storage, the pH gradually increases to 9.7, accompanied by a loss of CO(2). It is hypothesized that the lysozyme activity is influenced by either CO(2) concentration or pH changes resulting from CO(2) loss. The objective of this study was to determine the lytic activity of purified lysozyme and chicken egg white (unpurified lysozyme) under varying conditions of temperature, pH, and CO(2) gas concentration. Lytic activity was determined by a standard microbial assay using lyophilized Micrococcus lysodeikticus. A 2 × 4 × 2 × 2 × 3 factorial design consisting of 2 temperatures (5 and 22°C), 4 pH (4.5, 6.5, 8.0, and 9.5), 2 treatments (with and without CO(2)), 2 types of lysozyme (purified and unpurified egg white), and 3 replicates was used. The highest lytic activity was found at pH 6.5 and 22°C. At pH 4.5 and 8.0, the addition of CO(2) increased lytic activity by more than 50% at both temperatures. At pH 6.5, lytic activity was maintained with CO(2) addition at both temperatures. At pH 9.5, lytic activity without CO(2) addition was high; however, adding CO(2) reduced lytic activity to zero. In conclusion, both pH and CO(2) treatment influence lysozyme activity.

The effects of edible coatings on chicken egg quality under refrigerated storage.

Seventy-three billion chicken eggs are produced annually in the United States. However, less than 0.1% of these eggs are exported. Increasing the shelf-life of eggs may increase export sales. The goal of this research was to determine whether food-grade coatings on eggs may extend shelf-life under refrigerated storage. Four food-grade coatings were selected: paraffin wax, mineral oil, soy protein isolate, and whey protein isolate (WPI). These coatings were applied to fresh chicken eggs. The eggs were stored for 12 wk in refrigerated storage at 7 degrees C. Two replicates of the 12-wk study were conducted. Egg properties measured included Haugh units, albumen pH, yolk pH, albumen CO(2) content, vitelline membrane strength, water loss, shell strength, and shell color. Egg functionality measurements included foam volume, angel food cake volume, and emulsion stability. Statistical analysis was performed using the SAS PROC GLIMMIX method (P < 0.05). Results found that coated eggs maintained higher Haugh units beyond 6 wk compared with the uncoated eggs. Also, coated eggs maintained a higher CO(2) content and lower albumen pH than the uncoated eggs over the storage period. Vitelline membrane strength slightly decreased over time in uncoated eggs, but did not change in coated eggs. Overall, oil-, wax-, and WPI-coated eggs maintained higher vitelline membrane strength (14%) than the uncoated eggs. Coating of chicken eggs with a food-grade film (oil, wax, WPI) will extend shelf-life beyond 6 wk.

Effect of testing temperature on internal egg quality measurements.

The objective of this study was to determine the effect of egg testing temperature on quality measurements of shell eggs. The quality measurements compared included 3 Haugh unit (HU) devices (electronic Haugh, tripod Haugh, and Haugh meter), egg weight, albumen height, albumen width, albumen index, yolk width, yolk height, yolk index, percentage of thin albumen, and vitelline membrane strength at 3 temperatures of 5, 13, and 23 degrees C from 2 strains of laying hens (Hyline W36 and Bovans White) at 2 storage times. The HU measurements averaged 72.44 at time zero and 59.99 at 7 wk. At 7 wk for all devices, HU values decreased 6 units with increased temperature (P < 0.05). The electronic Haugh and tripod Haugh devices gave equal measurements for all testing conditions. The Haugh meter gave equal values at 5 degrees C for fresh eggs but lower HU at higher temperatures and 7 wk storage. Thus, it is recommended that egg testing temperature be reported when HU are measured. Coefficient of variation generally increased for all HU methods with increasing temperature. Although there was a proportionately different amount of thin albumen detected between the strains of laying hens, no significant difference was seen in HU. From the evaluated methods for measuring quality, the electronic Haugh, which electronically measures albumen height and calculates HU, provided the lowest coefficient of variation, was sensitive to quality loss, and gave the highest quality measurement (5 degrees C).

Effects of carcass washers on Campylobacter contamination in large broiler processing plants.

Campylobacter, a major foodborne pathogen found in poultry products, remains a serious problem facing poultry processors. Campylobacter research has primarily focused on detection methods, prevalence, and detection on carcasses; limited research has been conducted on intervention. The aim of this study was to assess the effectiveness of carcass washing systems in 4 large broiler-processing plants in removing Campylobacter species. Washing systems evaluated included combinations of inside/outside carcass washers and homemade cabinet washers. Processing aids evaluated were trisodium phosphate (TSP) and acidified sodium chlorite (ASC). The washer systems consisted of 1 to 3 carcass washers and used from 2.16 to 9.73 L of water per carcass. The washer systems used chlorinated water with 25 to 35 ppm of total chlorine. These washer systems on average reduced Campylobacter populations by log 0.5 cfu/mL from log 4.8 cfu/mL to log 4.3 cfu/mL. Washer systems with TSP or ASC reduced Campylobacter populations on average by an additional log 1.03 to log 1.26, respectively. Total average reductions in Campylobacter populations across the washer system and chill tank were log 0.76 cfu/mL. Washer systems that included antimicrobial systems had total average reductions in Campylobacter populations of log 1.53 cfu/mL. These results suggest that carcass washer systems consisting of multiple washers provide minimal reductions in Campylobacter populations found on poultry in processing plants. A more effective treatment of reducing Campylobacter populations is ASC or TSP treatment; however, these reductions, although significant, will not eliminate the organism from raw poultry.

Determination of cooling rates and carbon dioxide uptake in commercially processed shell eggs using cryogenic carbon dioxide gas.

The ability to rapidly cool shell eggs to 7 degrees C is important in the prevention of Salmonella Enteritidis (SE) growth. In addition, quality may also be maintained longer from rapid cooling of shell eggs. A commercial cryogenic CO2 egg cooling unit was designed and installed in a commercial egg processing facility. This unit was installed on a packer head to rapidly cool eggs individually prior to packaging. The objective of this study was to determine cooling rates and CO2 gas changes that result from rapidly cooling eggs using this commercial cryogenic egg cooling system and subsequent storage for 15 wk. Results indicated that cryogenic CO2 cooling quickly cooled shell eggs in approximately 45 min, whereas traditional cooling required from 19 to 116 h. CO2 uptake into the albumen was greater in cryogenically cooled eggs (2.11 mg/g) than in traditionally cooled eggs (1.81 mg/g) immediately after processing. No differences were observed in albumen CO2 content after 2 wk of storage; at 10 wk statistically greater CO2 content remained in the cryogenically cooled eggs (1.75 mg/g) compared with the traditionally cooled eggs (1.60 mg/g). These results suggest that a large amount of CO2 enters the egg during the cryogenic cooling process but is quickly lost during storage. Beyond 10 wk of storage, the albumen CO2 content in cryogenically cooled eggs was higher than in the traditionally cooled eggs suggesting chemical changes may have occurred in the albumen.

Comprehensive Review of Campylobacter and Poultry Processing.

Campylobacter has been recognized as a leading bacterial cause of human gastroenteritis in the United States, with 40000 documented cases annually. Epidemiological data suggest that contaminated products of animal origin, especially poultry, contribute significantly to campylobacteriosis. Thus, reduction of contamination of raw poultry would have a large impact in reducing incidence of illness. Contamination occurs both on the farm and in poultry slaughter plants. Routine procedures on the farm such as feed withdrawal, poultry handling, and transportation practices have a documented effect on Campylobacter levels at the processing plant. At the plant, defeathering, evisceration, and carcass chillers have been documented to cross-contaminate poultry carcasses. Carcass washings and the application of processing aids have been shown to reduce populations of Campylobacter in the carcasses by log10 0.5 log10 1.5; however, populations of Campylobacter have been shown to enter a poultry processing plant at levels between log10 5 colony-forming units (CFU)/mL and log10 8 CFU/mL of carcass rinse. The purpose of this article is to review Campylobacter, the infection that it causes, its association with poultry, contamination sources during processing, and intervention methods.

Characterization of radiant emitters used in food processing.

Radiant emissions from short, medium, and long wavelength thermal radiant emitter systems typically used for food processing applications were quantified. Measurements included heat flux intensity, emitter surface temperature, and spectral wavelength distribution. Heat flux measurements were found highly dependent on the incident angle and the distance from the emitter facing. The maximum flux measured was 5.4 W/cm2. Emitter surface temperature measurements showed that short wavelength radiant systems had the highest surface temperature and greatest thermal efficiency. The emitter spectral distributions showed that radiant emitter systems had large amounts of far infrared energy emission greater than 3 microm when compared to theoretical blackbody curves. The longer wavelength energy would likely cause increased surface heating for most high moisture content food materials.

Parametric analysis of cryogenic carbon dioxide cooling of shell eggs.

Parametric analysis of cryogenic cooling of shell eggs was performed using finite element analysis. Two cooling temperatures (-50 and -70 C), three cooling convective heat transfer coefficients (20, 50, and 100 W/ m2K), two equilibration temperatures (7 and 25 C), and two equilibration heat transfer coefficients (0 and 20 W/ m2K) were considered in the analysis. Lower temperatures and higher cooling convective heat transfer coefficients resulted in higher cooling rates and lower final egg temperatures. A chart and equation were developed to identify combinations of processing parameters to yield the desired egg temperature (7 C) at the end of adiabatic equilibration. Results show that a cooling time of 8.2 min was required to reach a final egg temperature of 7 C for a cooling temperature of -50 C and a convective heat transfer coefficient of 20 W/m2K. The cooling time decreased to 2 min when the convective heat transfer coefficient increased to 100 W/m2K, at a cooling temperature of -50 C. Processing at -70 C and 20 W/m2K, required 5.3 min to reach a final temperature of 7 C. At a higher convective heat transfer coefficient (100 W/m2K) and -70 C, a processing time of 1.3 min was sufficient to reach the target temperature of 7 C. The results may be used as a reference in process or equipment design for shell egg cooling in cryogenic CO2.

Effects of cryogenic cooling of shell eggs on egg quality.

This study was conducted to investigate the effects of cryogenic cooling on shell egg quality. Gaseous nitrogen (GN), liquid nitrogen (LN), and gaseous carbon dioxide (GC) were utilized to rapidly cool eggs in a commercial egg processing facility and were compared to traditional cooling (TC). A modified food freezer was attached to existing egg processing equipment in order to expose eggs to the selected cryogen. In Experiment 1, eggs were treated with GN, LN, and TC then stored and tested over 10 wk. Experiment 2 eggs were treated (GC and TC) and evaluated for 12 wk. Quality factors that were measured included Haugh units, vitelline membrane strength and deformation at rupture, and USDA shell egg grades for quality defects. Haugh unit values were greater for cryogenically treated eggs as compared to traditionally cooled eggs (Experiment 1: 73.27, GN; 72.03, LN; and 71.4, TC and Experiment 2: 74.42, GC and 70.18, TC). The percentage of loss eggs in the GN treatment was significantly (P < 0.01) greater than those of the LN and TC treatments. Vitelline membrane strength was greater for the cryogenically cooled eggs versus traditional processing. Vitelline membrane breaking strength decreased over storage time. Vitelline membrane deformation at rupture was significantly (P < 0.05) greater for the cryogenically cooled eggs compared to the traditional eggs in each experiment. Use of the technology could allow for egg quality to be maintained for a longer time, which could increase international markets and potentially lead to extended shelf lives.

Chemical method for determination of carbon dioxide content in egg yolk and egg albumen.

The safety, quality, and shelf life of shell eggs is a function of carbon dioxide content. A commercial process was recently developed for rapidly cooling shell eggs by using cryogenic CO2. The benefit of this new process over existing cooling processes is that the CO2 addition during cryogenic cooling provides additional safety and quality enhancements. In order for these benefits to be fully developed into a process that can be adopted by the egg industry, and thus realized by the consumer, the amount of CO2 absorbed by the egg during this process needs to be quantified. Because the albumen pH of rapidly cooled eggs was reduced to pH <6.5, existing titrametric methods were not adequate for determining CO2 content. They did not prevent CO2 loss during neutralization. A simple and accurate method for determining CO2 content in acidified egg albumen and yolk samples was developed. This method involves the liberation of CO2 from an acidified egg sample into a standardized, dilute sodium hydroxide solution inside a sealed jar. The egg sample and a small beaker containing the standardized sodium hydroxide solution are placed in a glass jar and sealed. Next, a concentrated acid phosphate solution is injected through a rubber septum in the cap of the jar onto the egg sample, while avoiding contact with the sodium hydroxide solution. The sample is then stored at 37 C for 24 h. During this storage period, the carbon dioxide is released from the egg sample and is absorbed into the sodium hydroxide solution. Afterwards, the dilute sodium hydroxide solution is removed and titrated to the phenolphthalein endpoint using a dilute, standardized hydrochloric acid solution. The amount of hydrochloric acid solution required for neutralization can be directly related to CO2 content in the sample.

The influence of rapid air cooling and carbon dioxided cooling and subsequent storage in air and carbon dioxide on shell egg quality.

This study examined the effect of rapid cooling with air and CO2 on shell egg quality over 14 wk. The 240 fresh eggs were initially heated to 47 C for 24 h in an incubator, cooled using rapid air cooling or CO2 cooling, and then stored in air or CO2 in 250-mL jars for 14 wk. The CO2 levels were recorded of the jar atmosphere, of the egg air cell, and of the egg albumen. The Haugh units of each egg, pH, and of albumen from five eggs per group were also recorded. Haugh units are a logarithmic, empirical relationship between albumen height and egg weight (Stadelman, 1995). Haugh units for the control eggs averaged 70.8 over 10 wk of the study. The control eggs were of such poor quality that they could not be sampled after 10 wk. The air-cooled and CO2-stored eggs averaged 70.3 Haugh units over the 14-wk storage period; however, the egg quality significantly deteriorated after 10 wk. The CO2-cooled and CO2-stored eggs averaged 75.9 Haugh units over the 14 wk study, with no observable decrease in quality. Rapid air-cooling produces a lower quality egg than rapid cooling with CO2. Subsequent storage of rapidly air-cooled eggs in C02 may increase shelf life, but Haugh units were not statistically different from rapid air-cooled eggs. CO2-cooling and subsequent storage in CO2 increased Haugh units. The shelf life of shell eggs could be extended to greater than 14 wk when the eggs were CO2-cooled and CO2-stored.

Gas exchange into shell eggs from cryogenic cooling.

The gas composition of the air cell in a shell egg is influenced by heating from egg washing and candling and the method of cooling and storage. This study found that N2 gas (-122 C), CO2 gas (-45 C), and cold air (-15 C) could be used to rapidly cool shell eggs from 47.7 C to 7 C in 30 min or less. These results suggest that the gas composition of the air cell in shell eggs can be significantly modified using N2 cooling and CO2 cooling. Commercial field studies have shown that these modifications, which take place during cryogenic cooling, can significantly reduce microbial levels and increase shelf life of shell eggs. Storage in a modified atmosphere environment further enhanced these changes. It was found that the CO2 concentration in the air cell of a shell egg can be increased from 0.04 to 48% by CO2 cooling and storage in a CO2 environment.