Comparing the effectiveness of delivery style in produce safety training for growers.

The Produce Safety Alliance grower training has been offered since 2016. Prior to the pandemic, the course was offered exclusively in-person. During the pandemic, trainers were allowed to offer the course remotely. The effectiveness of in-person and remote delivery options was compared utilizing four methods: course evaluations completed at the training; a pre- and post-training knowledge assessment; a 1-year follow-up survey; and focus groups with course trainers. All methods, except the focus groups, were used as evaluation tools starting before and continuing during the pandemic. On the course evaluations, remote delivery and in-person participants rated their satisfaction with the training and their confidence in their ability to make changes at the same high rate. The knowledge assessment found remote delivery participants scored higher on the posttest than in-person participants when controlling for pretest score (p < 0.001); the effect size was between low and medium (ηp 2 = 0.025). On the follow-up survey, remote delivery participants reported making changes to food safety practices or infrastructure at a higher rate than in-person participants (68% vs. 53%, respectively, Χ2 (1, N = 700) = 6.372, p = 0.012, Cramer's V = 0.012 (very low)). There were demographic differences in educational level, job description, and number of years farming between the two populations. The focus group revealed advantages and disadvantages of both delivery methods, including internet availability, engagement activity, and course logistics and planning. Because no practical differences in outcome were measured between delivery methods and each had unique strengths, researchers recommend that educators should utilize both methods in the future. PRACTICAL APPLICATION: 1. When deciding between offering in-person or synchronous virtual training, trainers can feel confident that both delivery methods result in positive experiences from participants, learning, and behavior change. 2. PSA trainers may choose to offer the training remotely to increase accessibility for people who live in areas where there may not be enough growers to warrant holding an in-person training nearby, but should also consider that reliable high-speed internet access may not be available to all. 3. Remote delivery trainings can be smoother by hosting remote delivery participants at local extension or other partner offices where high-speed internet is available.

Minimizing Escherichia coli O157:H7 contamination in indoor farming: effects of cultivar type and ultra-violet light quality.

BACKGROUND: Bacterial contamination of produce is a concern in indoor farming due to close plant spacing, recycling irrigation, warm temperatures, and high relative humidity during production. Cultivars that inherently resist contamination and photo-sanitization using ultraviolet (UV) radiation during the production phase can reduce bacterial contamination. However, there is limited information to support their use in indoor farming. RESULTS: Lettuce (Lactuca sativa) cultivars with varying plant architectures grown in a custom-built indoor farm exhibited differences in E. coli O157:H7 survival after inoculation. The survival of E. coli O157:H7 was lowest in the leaf cultivar (open architecture) and highest in the romaine and oakleaf cultivars (compact architecture). Of the different UV wavelengths that were tested (UV-A, UV-A + B, UV-A + C), UV A + C at an intensity of 54.5 µmol m-2 s-1 (with 3.5 µmol m-2 s-1 of UV-C), provided for 15 min every day, was found to be most efficacious in reducing the E. coli O157:H7 survival on romaine lettuce with no negative effects on plant growth and quality. CONCLUSION: Contamination of E. coli O157:H7 on lettuce plants can be reduced and the food safety levels in indoor farms can be increased by selecting cultivars with an open leaf architecture coupled with photo-sanitization using low and frequent exposure to UV A + C radiation. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Detection of Salmonella Typhimurium with Gold Nanoparticles Using Quartz Crystal Microbalance Biosensor.

Demonstration of the Salmonella Typhimurium detection system was shown utilizing a quartz crystal microbalance (QCM) biosensor and signal enhancement by gold nanoparticles. In this study, a benchtop system of a QCM biosensor was utilized for the detection of Salmonella Typhimurium. It was designed with a peristaltic pump system to achieve immobilization of antibodies, detection of Salmonella, and the addition of gold nanoparticles to the sensor. As a series of biochemical solutions were introduced to the surface, the proposed system was able to track the changes in the resonant frequency which were proportional to the variations of mass on the sensor. For antibody immobilization, polyclonal antibodies were immobilized via self-assembled monolayers to detect Salmonella O-antigen. Subsequently, Salmonella Typhimurium was detected by antibodies and the average frequency before and after detecting Salmonella was compared. The highest frequency shifts were −26.91 Hz for 109 CFU/mL while the smallest frequency shift was −3.65 Hz corresponding to 103 CFU/mL. For the specificity tests, non-Salmonella samples such as E. coli, Listeria, and Staphylococcus resulted in low cross-reactivity. For signal amplification, biotinylated antibodies reacted to Salmonella followed by streptavidin­100 nm AuNPs through biotin-avidin interaction. The frequency shifts of 103 CFU/mL showed −28.04 Hz, and consequently improved the limit of detection.

Designing a Computer-Vision Application: A Case Study for Hand-Hygiene Assessment in an Open-Room Environment.

Hand-hygiene is a critical component for safe food handling. In this paper, we apply an iterative engineering process to design a hand-hygiene action detection system to improve food-handling safety. We demonstrate the feasibility of a baseline RGB-only convolutional neural network (CNN) in the restricted case of a single scenario; however, since this baseline system performs poorly across scenarios, we also demonstrate the application of two methods to explore potential reasons for its poor performance. This leads to the development of our hierarchical system that incorporates a variety of modalities (RGB, optical flow, hand masks, and human skeleton joints) for recognizing subsets of hand-hygiene actions. Using hand-washing video recorded from several locations in a commercial kitchen, we demonstrate the effectiveness of our system for detecting hand hygiene actions in untrimmed videos. In addition, we discuss recommendations for designing a computer vision system for a real application.

Development of a smartphone-based lateral-flow imaging system using machine-learning classifiers for detection of Salmonella spp.

Salmonella spp. are a foodborne pathogen frequently found in raw meat, egg products, and milk. Salmonella is responsible for numerous outbreaks, becoming a frequent major public-health concern. Many studies have recently reported handheld and rapid devices for microbial detection. This study explored a smartphone-based lateral-flow assay analyzer which employed machine-learning algorithms to detect various concentrations of Salmonella spp. from the test line images. When cell numbers are low, a faint test line is difficult to detect, leading to misleading results. Hence, this study focused on the development of a smartphone-based lateral-flow assay (SLFA) to distinguish ambiguous concentrations of test line with higher confidence. A smartphone cradle was designed with an angled slot to maximize the intensity, and the optimal direction of the optimal incident light was found. Furthermore, the combination of color spaces and the machine-learning algorithms were applied to the SLFA for classifications. It was found that the combination of L*a*b and RGB color space with SVM and KNN classifiers achieved the high accuracy (95.56%). A blind test was conducted to evaluate the performance of devices; the results by machine-learning techniques reported less error than visual inspection. The smartphone-based lateral-flow assay provided accurate interpretation with a detection limit of 5 × 104 CFU/mL commercially available lateral-flow assays.

Occurrence of Chemical Contaminants in Peruvian Produce: A Food-Safety Perspective.

The presence of chemical contaminants in agricultural products is a continued food-safety challenge in Peru. This country has robust agriculture potential, but its output of fruits and vegetables is severely impacted by massive mining activities, as well as poor farming practices, including the use of polluted irrigation water, misuse of pesticides, and inadequate postharvest conditions. This review examines the current scientific knowledge on the levels of pesticide residues, heavy metals, and mycotoxins on crops produced in Peru. The available data shows that several crop varieties are contaminated with these classes of chemical contaminants, and at levels that exceed the national and international permissible limits. The abundance of chemical contaminants in produce indicates a relevant food-safety issue, which increases the risks of chronic human diseases, like cancer-a leading cause of death in Peru. Finally, this review presents recommendations to address these contamination problems in produce grown in the Andean country.

Food safety in Peru: A review of fresh produce production and challenges in the public health system.

Peru has a commodities-based economy where agriculture plays an essential role in the nation's development. Among agricultural products, fruits and vegetables are foundational to Peruvian culture and a healthy and nutritious diet. Produce is also the primary income source for thousands of small-scale farmers and producers throughout the country. Peru has significant potential to export agricultural and value-added products. Nevertheless, the Peruvian food chain has weak food safety and quality standards, limiting access to international markets. The inherent lack of food safety surveillance and management systems negatively affects public health. In the past decade, fresh and raw produce has been associated with several foodborne outbreaks worldwide, resulting in significant health and economic losses. This alarming situation for public health officials and regulators has called for the strengthening of produce safety standards and food safety risk management for safer food and to reduce the incidence of foodborne illnesses. This review summarizes the current status of produce safety in Peru and explores opportunities (e.g., policy, university capacity development) toward a safer food system.

Microwave pasteurization of apple juice: Modeling the inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium at 80-90 °C.

Although due to their acidity some fruit juices are considered safe, several outbreaks have been reported. For processing fruit juices, microwave heating offers advantages such as shorter come-up time, faster and uniform heating, and energy efficiency. Thus, it could be a beneficial alternative to conventional pasteurization. The objective of this study was to study the inactivation kinetics of Escherichia coli O157:H7 and Salmonella Typhimurium under microwave pasteurization at temperatures between 80 and 90 °C, i.e., at conditions that are employed in conventional pasteurization. Inoculated juices were treated at different power levels (600 W, 720 W) and treatment times (5s, 10s, 15s, 20s, 25s). Time-temperature profiles were obtained by fiber-optic sensors in contact with the samples allowing continuous data collection. The log-logistic and Arrhenius equations were used to account for the influence of the temperature history; thus, resulting in two different modeling approaches that were compared in terms of their prediction abilities. Survival kinetics including non-isothermal conditions were described by a non-linear ordinary differential equation that was numerically solved by the Runge-Kutta method (ode45 in MATLAB ®). The lsqcurvefit function (MATLAB®) was employed to estimate the corresponding survival parameters, which were obtained from freshly made apple juice, whereas the prediction ability of these parameters was evaluated on commercial apple juices. Results indicated that inactivation increased with power level, temperature, and treatment time reaching a microbial reduction up to 7 Log10 cycles. The study is relevant to the food industry because it provides a quantitative tool to predict survival characteristics of pathogens at other non-isothermal processing conditions.

Multi-View Hand-Hygiene Recognition for Food Safety.

A majority of foodborne illnesses result from inappropriate food handling practices. One proven practice to reduce pathogens is to perform effective hand-hygiene before all stages of food handling. In this paper, we design a multi-camera system that uses video analytics to recognize hand-hygiene actions, with the goal of improving hand-hygiene effectiveness. Our proposed two-stage system processes untrimmed video from both egocentric and third-person cameras. In the first stage, a low-cost coarse classifier efficiently localizes the hand-hygiene period; in the second stage, more complex refinement classifiers recognize seven specific actions within the hand-hygiene period. We demonstrate that our two-stage system has significantly lower computational requirements without a loss of recognition accuracy. Specifically, the computationally complex refinement classifiers process less than 68% of the untrimmed videos, and we anticipate further computational gains in videos that contain a larger fraction of non-hygiene actions. Our results demonstrate that a carefully designed video action recognition system can play an important role in improving hand hygiene for food safety.

Smartphone-based lateral flow imaging system for detection of food-borne bacteria E.coli O157:H7.

We report an application for the smartphone as an accurate and unbiased reading platform of a lateral flow immunoassays for food safety application. In particular, this report focuses on detection of food-borne bacteria in samples extracted from food matrices such as ground beef and spinach. The lateral flow assay is a widely accepted methodology owing to its on-site results, low-cost analysis, and ease of use with minimum user inputs, even though sensitivity is not quite equivalent to that of standard laboratory equipment. An antibody-antigen relationship is transduced into a color change on a nitrocellulose pad while visual interpretation of this color change can result in uncertainty, particularly near the detection limit of the assay. Employing the high resolution integrated camera, constant illumination from light source, and computing power of a smartphone, we provide an objective and accurate method to determine the bacterial cell concentration in a food matrix based on the regression model from the color intensity of test lines. A 3D-printed sample holder was designed for representative commercial lateral flow assays and an in-house application was developed in Android Studio to solve the inverse problem to provide cell concentration information from the color intensity. Test results with E.coli O157:H7 as a model organism suggests that smartphone-based reader can detect 104-105 CFU/ml from ground beef and spinach food matrices.

Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7.

Infectious outbreaks caused by foodborne pathogens such as E. coli O157:H7 are still imposing a heavy burden for global food safety, causing acute illnesses and significant industrial impact worldwide. Despite the growth of biosensors as a research field, continuous innovation on detection strategies, novel materials and enhanced limits of detection, most of the platforms developed at the laboratory scale never will get to meet the market. The use of aptamers as capture biomolecules has been proposed as a promising alternative to overcome the harsh environmental conditions of industrial manufacturing processes, and to enhance the performance under real, complex, conditions. In this work, we present the feasibility of using aptameric DNA sequences, covalently conjugated to 4-aminothiophenol-gold nanoparticle complexes for the sensitive and highly specific detection of E. coli O157:H7 via surface enhanced Raman spectroscopy (SERS) analysis. Low concentrations of E. coli O157:H7 were detected and quantified within 20 min in both pure culture (∼101 CFU mL-1) and ground beef samples (∼102 CFU mL-1). The SERS intensity response showed a strong negative linear correlation (r2 = 0.995) with increasing concentrations of E. coli O157:H7 (ranging from 102 to 106 CFU mL-1). High specificity was achieved at genus (L. monocytogenes, S. aureus S. typhimurium) species (E. coli B1201) and serotype (E. coli O55:H7) level, demonstrating with 95% of confidence that the interferent microorganisms tested generated a Raman signal response not significantly different from the background (p = 0.786). This work evaluates the incorporation of aptameric DNA sequences as bio capture molecules exclusively. The successful performance presented using non-modified citrate reduced GNPs, is promising for potential low-cost, high-throughput applications. The findings might be applied simultaneously to the detection of a wide variety of foodborne pathogens in a multiplexed fashion employing unique Raman probes and strain-specific aptamer sequences.

Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach.

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.

Inkjet Printed Nanopatterned Aptamer-Based Sensors for Improved Optical Detection of Foodborne Pathogens.

The increasing incidence of infectious outbreaks from contaminated food and water supply continues imposing a global burden for food safety, creating a market demand for on-site, disposable, easy-to-use, and cost-efficient devices. Despite of the rapid growth of biosensors field and the generation of breakthrough technologies, more than 80% of the platforms developed at lab-scale never will get to meet the market. This work aims to provide a cost-efficient, reliable, and repeatable approach for the detection of foodborne pathogens in real samples. For the first time an optimized inkjet printing platform is proposed taking advantage of a carefully controlled nanopatterning of novel carboxyl-functionalized aptameric ink on a nitrocellulose substrate for the highly efficient detection of E. coli O157:H7 (25 colony forming units (CFU) mL-1 in pure culture and 233 CFU mL-1 in ground beef) demonstrating the ability to control the variation within ±1 SD for at least 75% of the data collected even at very low concentrations. From the best of the knowledge this work reports the lowest limit of detection of the state of the art for paper-based optical detection of E. coli O157:H7, with enough evidence (p > 0.05) to prove its high specificity at genus, species, strain, and serotype level.

Capacity Building through Water Quality and Safety Analyses in Herat, Afghanistan.

Contaminated water is a leading cause of approximately 600 million annual cases of foodborne disease globally. Twenty percent of all child mortality in Afghanistan is attributed to diarrheal diseases. There are limited data on water quality and safety in Afghanistan as well as limited laboratory capacity for food and water analyses. The purpose of this study was to conduct a regional water assessment study and, in doing so, train the first class of food technology undergraduate students at Herat University (Herat City, Afghanistan) in basic water quality and safety laboratory techniques. In total, 235 water samples from private wells ( n = 128) and municipal water system taps ( n = 107) were collected from Herat Province, Afghanistan. Samples were aseptically collected, transferred, and analyzed at the Herat University Food Technology Laboratory for nitrate, nitrite, lead, phosphate, and arsenic concentrations; hardness; total coliforms; and Escherichia coli. We did not detect arsenic in any sample tested ( n = 234), and lead levels in samples tested ( n = 28) were below the U.S. Environmental Protection Agency maximum contaminant level (15 µg/L). In contrast, 38 of 232 samples had nitrate (NO3) levels greater than the maximum contaminant level (10 mg/L) and 15 of 232 samples had nitrite (NO2) levels > 0.3 mg/L. On average, well water was harder than municipal water; there were no differences in phosphate (PO4) levels. Furthermore, 93 (43.9%) of 212 samples had detectable coliforms (average CFU/100 mL) and 52 (24.3%) of 214 samples had detectable E. coli (average 28.6 CFU/100 mL). E. coli was detected in 21.4 and 26.7% of municipal and well water samples, respectively. This study indicates a clear need for systematic analyses of Herat City water to develop plans for water quality and safety improvement and management. The students engaged in the research project now have the basic research and analytical skills needed to address water and foodborne disease issues endemic in the area.

Biofilm of Escherichia coli O157:H7 on cantaloupe surface is resistant to lauroyl arginate ethyl and sodium hypochlorite.

Biofilms formed by Escherichia coli O157:H7 on cantaloupe rind were characterized in this study. Cantaloupe rind pieces inoculated with E. coli O157:H7 B6-914 was sampled after 2, 12, and 24h incubation for imaging with cryo-scanning electron microscopy (Cryo-SEM) or treating with lauroyl arginate ethyl (LAE) or sodium hypochlorite (SHC). Cryo-SEM images showed that E. coli O157:H7 formed a biofilm within 12h on the rind surface. For rind samples treated with LAE or SHC, the residual cell counts were significantly different (p<0.05) between 2 and 12h incubation, and between 2 and 24h of incubation. For the 2h incubation samples, E. coli O157:H7 was undetectable (>5-log reduction) after treatment with 2000µg/mL of LAE or SHC. In contrast, for 12h incubation samples, 2000µg/mL of LAE or SHC could only achieve 1.74 or 1.86-log reduction, respectively. The study showed the low efficacy of LAE and SHC on cantaloupe rind surface to reduce the E. coli biofilm, suggesting the needs for cantaloupe cleaning methods beyond washing with conventional antimicrobial agents.

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.

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.

Pathogen biofilm formation on cantaloupe surface and its impact on the antibacterial effect of lauroyl arginate ethyl.

Pathogen biofilm at fruit surface may pose a particular risk to food safety. In this study, the biofilms of Listeria monocytogenes V7 and Salmonella enterica serovar Typhimurium ATCC 13311 on cantaloupe fruit surface were visualized, and the resistance of biofilms against lauroyl arginate ethyl (LAE, an antibacterial compound) was evaluated. Each bacterium was inoculated on isolated cantaloupe rind surfaces at 105-106 CFU/cm2 and after incubation for 2, 12, 24, and 48 h, the surfaces were imaged using cryo-scanning electron microscopy (Cryo-SEM). The images showed that both pathogens formed biofilms on rind surfaces, with S. Typhimurium forming biofilm in 12 h and L. monocytogenes cells starting to aggregate in 2 h. For the inoculated rind surfaces treated with LAE, the cell counts were affected by both the incubation time and LAE concentration. For rind surface with 2 h incubation of S. Typhimurium, 400 and 800 µg/mL LAE was able to achieve >2.00 log reduction; however, 12 h incubation required 1600 and 2000 µg/mL LAE for >2.00 log reduction. In contrast, even the highest LAE concentration (2000 µg/mL) was unable to cause 1.00 log reduction for L. monocytogenes regardless the incubation time applied. The results showed that the biofilms of both bacteria substantially reduced LAE efficacy, and that the biofilm of L. monocytogenes was more resistant than that of S. Typhimurium.

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. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1464-1471, 2016.

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/cm(2) min. The entire procedure-from sample processing to detection by polymerase chain reaction-is completed in 8 h.