Amanda J. Deering

Melamine detection in infant formula powder using near- and mid-infrared spectroscopy.

Near- and mid-infrared spectroscopy methods (NIR, FTIR-ATR, FTIR-DRIFT) were evaluated for the detection and quantification of melamine in infant formula powder. Partial least-squares (PLS) models were established for correlating spectral data to melamine concentration: R(2) > 0.99, RMSECV ≤ 0.9, and RPD ≥ 12. Factorization analysis of spectra was able to differentiate unadulterated infant formula powder from samples containing 1 ppm melamine with no misclassifications, a confidence level of 99.99%, and selectivity > 2. These nondestructive methods require little or no sample preparation. The NIR method has an assay time of 1 min, and a 2 min total time to detection. The FTIR methods require up to 5 min for melamine detection. Therefore, NIR and FTIR methods enable rapid detection of 1 ppm melamine in infant formula powder.

Detection and differentiation of live and heat-treated Salmonella enterica serovars inoculated onto chicken breast using Fourier transform infrared (FT-IR) spectroscopy.

Aims:  To evaluate Fourier transform infrared (FT‐IR) techniques for detecting, quantifying, and differentiating viable and heat‐treated cells of Salmonella enterica serovars from chicken breast.

Identification of the Cellular Location of Internalized Escherichia coli O157:H7 in Mung Bean, Vigna radiata, by Immunocytochemical Techniques

Escherichia coli O157:H7 has been associated with numerous outbreaks involving fresh produce. Previous studies have shown that bacteria can be internalized within plant tissue and that this can be a source of protection from antimicrobial chemicals and environmental conditions. However, the types of tissue and cellular locations the bacteria occupy in the plant following internalization have not been addressed. In this study, immunocytochemical techniques were used to localize internalized E. coli O157:H7 expressing green fluorescent protein in germinated mung bean (Vigna radiata) hypocotyl tissue following contamination of intact seeds. An average of 13 bacteria per mm(3) were localized within the sampled tissue. The bacteria were found to be associated with every major tissue and corresponding cell type (cortex, phloem, xylem, epidermis, and pith). The cortical cells located on the outside of the vascular bundles contained the majority of the internalized bacteria (61%). In addition, the bacteria were localized primarily to the spaces between the cells (apoplast) and not within the cells. Growth experiments were also performed and demonstrated that mung bean plants could support the replication of bacteria to high levels (10(7) CFU per plant) following seed contamination and that these levels could be sustained over a 12-day period. Therefore, E. coli O157:H7 can be internalized in many different plant tissue types after a brief seed contamination event, and the bacteria are able to grow and persist within the plant.

Accelerating Sample Preparation Through Enzyme-Assisted Microfiltration of Salmonella in Chicken Extract

Microfiltration of chicken extracts has the potential to significantly decrease the time required to detect Salmonella, as long as the extract can be efficiently filtered and the pathogenic microorganisms kept in a viable state during this process. We present conditions that enable microfiltration by adding endopeptidase from Bacillus amyloliquefaciens to chicken extracts or chicken rinse, prior to microfiltration with fluid flow on both retentate and permeate sides of 0.2 μm cutoff polysulfone and polyethersulfone hollow fiber membranes. After treatment with this protease, the distribution of micron, submicron, and nanometer particles in chicken extracts changes so that the size of the remaining particles corresponds to 0.4–1 μm. Together with alteration of dissolved proteins, this change helps to explain how membrane fouling might be minimized because the potential foulants are significantly smaller or larger than the membrane pore size. At the same time, we found that the presence of protein protects Salmonella from protease action, thus maintaining cell viability. Concentration and recovery of 1–10 CFU Salmonella/mL from 400 mL chicken rinse is possible in less than 4 h, with the microfiltration step requiring less than 25 min at fluxes of 0.028–0.32 mL/cm2 min. The entire procedure—from sample processing to detection by polymerase chain reaction—is completed in 8 h.

Differentiation of live, dead and treated cells of Escherichia coli O157:H7 using FT-IR spectroscopy

Aims:  To apply specific collection techniques and spectroscopy to differentiate between live and dead Escherichia coli O157:H7 cells, as well as cells subjected to various inactivation treatments, including heat, salt, UV, antibiotics and alcohol.

Thymol nanoemulsions formed via spontaneous emulsification: Physical and antimicrobial properties.

In this work, we prepared various sub-micron thymol emulsions with high hydrophilic-lipophilic balance (HLB) surfactants via spontaneous emulsification. Emulsion properties, such as size, polydispersity and charge, were assessed for each surfactant type and mass fraction. Emulsion stability was characterized by monitoring droplet size following exposure to physical (centrifugation) and thermal stressors (freeze, thaw cycling). Emulsions were subsequently screened against several challenge pathogens to evaluate antimicrobial efficacy. Based on these time-kill assays, exemplary formulations were further tested as sanitizing washes on lettuce and blueberries inoculated with food-borne bacterial biofilms. Antimicrobial data elucidate both surfactant and formulation specific antagonisms between thymol and the emulsifying agents. However, the best emulsion compositions were capable of reducing planktonic bacteria by >4 logs and biofilm bacteria by 1.5-2.5 logs in 60 s. These results are comparable to the efficacy of chlorine at ∼50-200ppm. The experimental results have implications in emulsion formulations involving thymol and other terpenoids.

Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella.

We report detection of <13 CFU of Salmonella per 25 g egg white within 7 h by concentrating the bacteria using microfiltration through 0.2‐μm cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross‐flow on both sides of the hollow fibers, and media selection were key to controlling membrane fouling so that rapid concentration and the subsequent detection of low numbers of microbial cells were achieved. We leveraged the protective effect of egg white proteins and peptone so that the proteolytic enzymes did not attack the living cells while hydrolyzing the egg white proteins responsible for fouling. The molecular weight of egg white proteins was reduced from about 70 kDa to 15 kDa during hydrolysis. This enabled a 50‐fold concentration of the cells when a volume of 525 mL of peptone and egg white, containing 13 CFU of Salmonella, was decreased to a 10 mL volume in 50 min. A 10‐min microcentrifugation step further concentrated the viable Salmonella cells by 10×. The final cell recovery exceeded 100%, indicating that microbial growth occurred during the 3‐h processing time. The experiments leading to rapid concentration, recovery, and detection provided further insights on the nature of membrane fouling enabling fouling effects to be mitigated. Unlike most membrane processes where protein recovery is the goal, recovery of viable microorganisms for pathogen detection is the key measure of success, with modification of cell‐free proteins being both acceptable and required to achieve rapid microfiltration of viable microorganisms.

Listeria monocytogenes Internalizes in Romaine Lettuce Grown in Greenhouse Conditions

Listeria monocytogenes has been implicated in a number of outbreaks involving fresh produce, including an outbreak in 2016 resulting from contaminated packaged salads. The persistence and internalization potential of L. monocytogenes in romaine lettuce was evaluated, and the persistence of two L. monocytogenes strains was assessed on three romaine lettuce cultivars. Seeds were germinated, and plants grown in three soil types (i.e., standard potting mix, autoclaved potting mix, and top soil) and sterile soft-top agar for up to 21 days. Average CFU per gram of L. monocytogenes on seeds and plants was calculated from five replicates per harvest day. Up to 8.2 log CFU/g L. monocytogenes persisted on romaine lettuce plants (Braveheart cultivar) grown in soft-top agar, while those grown in commercial potting mix (initial soil aerobic plate count of 4.0 × 104 CFU/g) had a final concentration of 5.4 log CFU/g, and autoclaved commercial potting mix had a final concentration of 3.8 ± 0.2 log CFU/g after a 21-day period. Pathogen levels dropped below the limit of detection (2 log CFU/g) by day 18 in 75% topsoil (initial soil aerobic plate count of 4.0 × 101 CFU/g); this did not occur in sterile media. Although L. monocytogenes strain differences and presence of a clay coating on seeds did not affect persistence, differences were observed in L. monocytogenes growth and survival among cultivars. To assess internalization, seeds were inoculated with L. monocytogenes expressing green fluorescent protein. Three plants were fixed, paraffin embedded, and sectioned; localization was studied by using standard immunohistochemistry techniques. A total of 539 internalized L. monocytogenes cells were visualized among three 20-day seedlings. L. monocytogenes cells were located in all major tissue types (pith followed by cortex, xylem, phloem, and epidermis). The presence of L. monocytogenes in the plant vasculature suggests potential for transport throughout the plant into edible tissue.

Movement of Salmonella serovar Typhimurium and E. coli O157:H7 to Ripe Tomato Fruit Following Various Routes of Contamination

Salmonella serovars have been associated with the majority of foodborne illness outbreaks involving tomatoes, and E. coli O157:H7 has caused outbreaks involving other fresh produce. Contamination by both pathogens has been thought to originate from all points of the growing and distribution process. To determine if Salmonella serovar Typhimurium and E. coli O157:H7 could move to the mature tomato fruit of different tomato cultivars following contamination, three different contamination scenarios (seed, leaf, and soil) were examined. Following contamination, each cultivar appeared to respond differently to the presence of the pathogens, with most producing few fruit and having overall poor health. The Micro-Tom cultivar, however, produced relatively more fruit and E. coli O157:H7 was detected in the ripe tomatoes for both the seed- and leaf- contaminated plants, but not following soil contamination. The Roma cultivar produced fewer fruit, but was the only cultivar in which E. coli O157:H7 was detected via all three routes of contamination. Only two of the five cultivars produced tomatoes following seed-, leaf-, and soil- contamination with Salmonella Typhimurium, and no Salmonella was found in any of the tomatoes. Together these results show that different tomato cultivars respond differently to the presence of a human pathogen, and for E. coli O157:H7, in particular, tomato plants that are either contaminated as seeds or have a natural opening or a wound, that allows bacteria to enter the leaves can result in plants that have the potential to produce tomatoes that harbor internalized pathogenic bacteria.

Quality and safety attributes of afghan raisins before and after processing

Raisins are an important export commodity for Afghanistan; however, Afghan packers are unable to export to markets seeking high-quality products due to limited knowledge regarding their quality and safety. To evaluate this, Afghan raisin samples from pre-, semi-, and postprocessed raisins were obtained from a raisin packer in Kabul, Afghanistan. The raisins were analyzed and compared to U.S. standards for processed raisins. The samples tested did not meet U.S. industry standards for embedded sand and pieces of stem, total soluble solids, and titratable acidity. The Afghan raisins did meet or exceed U.S. Grade A standard for the number of cap-stems, percent damaged, crystallization levels, moisture content, and color. Following processing, the number of total aerobic bacteria, yeasts, molds, and total coliforms were within the acceptable limits. Although quality issues are present in the Afghan raisins, the process used to clean the raisins is suitable to maintain food safety standards.