Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices.

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

Effect of microorganism characteristics on leak size critical to predicting package sterility.

The effects of microorganism size and motility on the leak size critical to the sterility of a package, along with the imposed pressure required to initiate liquid flow for the critical leak size, were measured. Pseudomonas fragi Lacy-1052, Bacillus atrophaeus ATCC 49337, and Enterobacter aerogenes ATCC 29007 were employed to assess package sterility. One hundred twenty-six 7-mm-long microtubes with interior diameters of 5, 10, and 20 microm were used to simulate package defects. Forty-two solid microtubes were used as controls. No significant differences were found between sizes or motility statuses of test organisms with respect to loss of sterility as a result of microbial ingress into test cells with microtube interior diameters of 5, 10, and 20 microm (P > 0.05). Interactions between the initiation of liquid flow as a result of applied threshold pressures and sterility loss for test cells were significant (P < 0.05).

Comparative study of a semisynthetic derivative of natamycin and the parent antibiotic on the spoilage of shredded cheddar cheese.

A water-soluble N-alkyl semisynthetic derivative of natamycin was synthesized by the Michael addition reaction of the parent with an N-substituted malemide. A comparative study was carried out to investigate the effectiveness of the semisynthetic derivative and the parent antibiotic in suppressing mold growth on shredded Cheddar cheese stored in modified atmosphere packaging (MAP). The effects of 0-, 10-, and 20-ppm antimycotic treatments were examined. A 20-ppm natamycin treatment effectively suppressed visible mold growth (< 10(4) CFU/g) in MAP samples for up to 30 days after packages were opened. The performance of the 20-ppm semisynthetic derivative was similar to that of the 10-ppm natamycin treatment. For these treatments, visible mold growth did not occur in MAP samples until 20 days after packages were opened. These results indicate that the semisynthetic derivative of natamycin is less effective than the parent compound in suppressing mold growth on shredded Cheddar cheese.

Application of fluid modeling to determine threshold leak size for liquid foods.

In this study, the mechanism by which a package defect converts to a leaker was examined in an effort to develop a relationship between threshold leak size and loss of package sterility. The threshold leak size is the hole size at which the onset of leakage occurs. The threshold pressure is the pressure required to initiate a leak. Leak initiation was studied in terms of the interaction between three components: liquid attributes of liquid food products, defect size, and pressures required to initiate liquid flow. Liquid surface tension, viscosity, and density values were obtained for 16 liquids. The imposed pressures required to initiate flow through microtubes with interior diameters of 0, 2, 5, 7, 10, 20, and 50 microm were measured with the use of 63 test cells filled with safranin red dye, tryptic soy broth, and distilled water with surface tensions of 18.69, 44.09, and 64.67 mN/m, respectively. Significant differences (P<0.05) between threshold pressures observed for safranin red dye, tryptic soy broth, and distilled water were found. Liquids with low surface tensions, such as safranin red dye, required significantly lower threshold imposed pressures than did liquids with high surface tensions, such as distilled water (P<0.05). An equation to quantify the relationship between liquid surface tension, threshold imposed pressure, and defect size was developed. Threshold pressures observed were not significantly different (P>0.05) from those predicted by the equation. Imposed pressures and vacuums generated within packages during random vibration and sweep resonance tests were measured for brick-style aseptic packages (250 ml), metal cans (76.2 by 114.3 mm [425 ml]), 1-qt gable-top packages (946 ml), 0.5-gal gable-top packages (1.89 liters), and 1-gal milk jugs (4.25 liters). Significant differences between packages were found with respect to observed generated pressures during vibration testing (P<0.05). An equation to calculate threshold size on the basis of liquid surface tension and imposed pressure was established.

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries.

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.

Predictive Model with Improved Statistical Analysis of Interactive Factors Affecting the Growth of Staphylococcus aureus 196E.

The growth of pathogenic bacteria in foods is affected by several factors which may interact to enhance or inhibit microbial growth. A model to predict the growth of Staphylococcus aureus 196E in microbiological media was developed using a modified Gompertz function and response-surface methodology. The predictive equation required the estimation of 23 parameters which describe singular and interactive effects of the growth factors studied. S. aureus 196E was inoculated into brain heart infusion broth formulated with either 0.5, 4.5, or 8.5% NaCl, adjusted to pH 5.0, 6.0, or 7.0, and incubated aerobically at 12, 20, or 28°C. Several interactive relationships between time, temperature, pH, and NaCl concentration were significant. The model adequately predicted the growth of S. aureus 196E. Predicted responses to multiple-factor interactions were displayed with three-dimensional and contour plots. A second model developed from a smaller subset of the growth data demonstrated that models could be produced with much less data collection. This methodology can provide important information to food scientists about the growth kinetics of microorganisms and prediction ranges or confidence intervals for growth parameters. Consequently, the effects of food formulations and storage conditions on the growth kinetics of foodborne pathogens or spoilage microorganisms could be predicted.

Bioaerosol Exposure Method for Package Integrity Testing †.

Test organism motility, concentration, aerosol exposure time, hole diameter and length were evaluated to determine their influence on microbial ingress into a flexible plastic pouch. Microtubes with 10- and 20-µm hole diameters and of 5- and 10-mm lengths were used as defects in 128 flexible pouches. A bioaerosol with a 2.68-µm mean particle size comprised of 102 or 106 CFU/ml source concentrations of motile or nonmotile Pseudomonas fragi TM 849 was introduced into a 119,911-cm3 chamber for exposures of 15 or 30 minutes. Six pouches showed test organism growth after a 72-h incubation period. Microbial ingress was significant (P < .05) for motile test organisms with source concentrations of 106 CFU/ml.

Contamination of Flexible Pouches Challenged by Immersion Biotesting †.

Immersion biotesting has long been used to challenge packages, particularly cans, for pinholes and channel leaks. Such testing for all types of plastic packaging may not be appropriate because some packages (e.g., aseptic, hot fill) are not exposed to water. As the food-packaging industry develops alternative environmental biotests there is a need to benchmark them against traditional immersion testing. The purpose of this research was to examine the threshold of critical-defect dimensions using artifically created channel leaks of 10 and 20 µm and 5- and 10-mm lengths sealed into plastic pouches which were subsequently tested by immersion at 102 and 106 CFU of motile and nonmotile Pseudomonas fragi TM849 per ml. Forty-four percent (44%) of the pouches tested became contaminated, indicating the threshold defect value is below 10 µm. Microbial ingress was significant (P < .05) for motile test organisms with a concentration of 106 CFU/ml. The interaction of concentration and time was also significant at 102 CFU/ml at 30 min exposure and 106 CFU/ml at 15 min. Channel length was not statistically significant. The markedly greater contamination rate using immersion testing versus that of aerosol testing highlights the importance of using test methods that reflect environmental exposure conditions of the packages.

Improved Recovery of Stressed Bifidobacterium from Water and Frozen Yogurt.

A roll-tube repair-detection procedure was developed to enumerate injured and noninjured cells of Bifidobacterium species from water and food samples. This procedure combined the Virginia Polytechnic Institute and State University's anaerobic roll-tube procedure and the repair-detection technique for detecting stressed cells. Mara and Oragui's human bifid sorbitol agar medium was modified for use in the roll-tube procedure by replacing the indicator bromocresol purple with phenyl red (0.027 g/l), adding 0.0006 g of methylene blue per 1, increasing the agar content to 25 g/l and adjusting the pH of the medium to 7.1 ± 0.1. The repair-detection roll-tube technique was shown to recover Bifidobacterium cells significantly (P < 0.01) better than pour plates, using the same medium incubated in anaerobe jars, even when a repair-detection system was used. Most repair in the roll tubes occurred in the first hour. B. adolescentis had a poor survival rate after 96 hours in water. Glucose was substituted for sorbitol in the medium used for enumeration of B. longum added to frozen yogurt, because this organism cannot utilize sorbitol. This medium, when used as part of a repair detection system, significantly (P < 0.01) recovered more cells than anaerobic pour plate techniques and was able to separate Bifidobacterium species and Lactobacillus acidophilus by colony morphology. Bifidobacterium cells were 1mm or larger, round and yellow, while the L. acidophilus colonies were so small (< 1/4 mm) their detection and enumeration was difficult.

Low Temperature Growth and Thermal Inactivation of Listeria monocytogenes in Precooked Crawfish Tail Meat 1.

Growth of Listeria monocytogenes in precooked crawfish tail meat at 0, 6, and 12°C was determined. Thermal death times were also determined. Growth curves for L. monocytogenes revealed that little multiplication was observable for the entire storage time of 20 d at 0°C. At 6 and 12°C, exponential growth began immediately with no observed lag phase. Generation times of 72.2, 17.0, and 6.9 h were calculated at 0, 6, and 12°C, respectively. Observed D values at 55, 60, and 65°C were 10.23, 1.98, and 0.19 min, respectively. The z value for L. monocytogenes in precooked crawfish tail meat was calculated to be 5.5°C. Results from this study indicate that a refrigeration temperature of 6°C (42.8°F) will support growth of L. monocytogenes and short-term temperature abuse at 12°C will induce very rapid growth of the organism on crawfish tail meat. Thermal treatment values from this study can be used to establish postpicking heat treatments that would eliminate L. monocytogenes from packaged crawfish tail meat prior to retail sale.

Seasonal Variation in the Fecal Coliform Population of Louisiana Oysters and its Relationship to Microbiological Quality.

Seasonal variation was observed in the type of bacteria which comprised the fecal coliform population of oysters. Escherichia coli was the principal fecal coliform when water temperatures were below 22°C. Conversely, Klebsiella sp. predominated during the summer months. No significant relationship was observed between levels of E. coli and enterococci and non- E. coli fecal coliforms in oysters. Fecal coliform and E. coli levels were significantly (p >0.001) related in water. Klebsiella sp. isolated from oysters demonstrated considerably less multiple antimicrobial agent resistance than clinical isolates of K. pneumoniae . Fecal coliform-positive Klebsiella species had characteristics of environmental organisms. Results of this study suggest that high levels of non- E coli fecal coliforms in oysters harvested in the summer from beds meeting the fecal coliform water standard are not indicative of sewage pollution. Furthermore, it is suggested that the safety indicator in the guideline for oyster meats should be changed form fecal coliforms to E. coli .

Evaluation of Methods for Extraction of Enteric Virus from Louisiana Oysters.

Six techniques were evaluated for recovery of poliovirus from Louisiana oysters. The methods were compared for percent recovery rates, toxicity, ease of extraction, bacterial contamination, and final volume of oyster concentrate. Oyster samples were contaminated with 30-40 plaque forming units of Poliovirus type 1 and processed by six variations of adsorption-elution-precipitation and elution-precipitation methods. The method developed by Ellender et al. (Natural enterovirus and fecal coliform contamination of gulf coast oysters. J. Food Prot. 43:105-110) was judged to be the preferred method for gulf coast oysters.

Detection of Enteroviruses and Bacterial Indicators and Pathogens in Louisiana Oysters and Their Overlying Waters.

Field studies were conducted for 1 year to determine levels of enteroviruses in Louisiana Gulf Coast oysters and their overlying waters. Levels of human enteric viruses were compared with bacterial pathogens ( Salmonella and Vibrio parahaemolyticus ), fecal coliform levels, and physicochemical water parameters (pH, salinity, temperature, and conductivity). Samples of 20-30 oysters and 380 L of overlying water were collected monthly from both "open" and "closed" oyster growing areas. Enteric viruses were found predominantly in January and February. Viruses were isolated only from areas which exceeded the 14 fecal coliforms/100 ml standard for shellfish harvesting waters.

Enteric Bacterial and Viral Pathogens and Indicator Bacteria in Hard Shell Clams.

Enteric bacteria and virus levels were determined in hard shell clams, Mercenaria mercenaria , harvested from areas open or closed for commercial shellfishing on the basis of total coliform levels in water. Four pairs of open and closed stations were sampled seasonally over a 1-year period. Enteric viruses were isolated from 3 of 13 100-g clam samples from open beds and 6 of 15 samples from closed beds. Salmonella was found in 1 of 15 samples from closed areas, but not in any samples from open areas. No Shigella or Yersinia were isolated from clams taken from either open or closed beds. Levels of Vibrio parahaemolyticus , an indigenous estuarine microorganism, were similar in clams from open and closed areas. No statistically significant difference was found in the occurrence of enteric viruses in clams from open and closed areas. Product-moment correlations between concentrations of enteric viruses and bacteria in clams or water demonstrated no statistically significant correlations between virus concentrations in clams and total coliforms or fecal coliforms in water or total coliforms, fecal coliforms, fecal streptococci or aerobic plate counts in clams.

Occurrence of Enteric Bacteria and Viruses in Oysters.

Enteric bacteria and virus levels were determined in oysters from paired stations that were opened or closed for commercial shellfishing on the basis of total coliform levels in the water. Six pairs of stations were sampled quarterly over a 1-year period. Enteric viruses were found in 3 of 24 50-g oyster samples from closed areas and in none of 23 samples from open areas. Salmonella was found in 2 of 47 samples of 40 g each, one from an open and the other from a closed area. Although enteric pathogens of fecal origin were found only in oysters that exceeded the recommended market limit of 230 fecal coliforms per 100 g of meat, the fecal coliform levels in some virus-positive samples were much lower than those in Salmonella -positive samples. Vibrio parahemolyticus levels were similar in oysters from both open and closed beds, indicating no particular association with fecal pollution. However, there was a marked seasonal variation in V. parahemolyticus levels. Total but not fecal coliform levels in oysters from open beds correlated with the occurrence of rainfall 1 or 2 days before sample collection. Neither total nor fecal coliform levels in oysters from closed beds correlated with rainfall. These findings suggest that fecal coliforms levels in oysters are less influenced by rainfall than are total coliforms, and therefore may be a more specific indicator of recent fecal pollution.