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.

Protein particulate retention and microorganism recovery for rapid detection of Salmonella.

The rapid detection of Salmonella in ground meat requires that living microorganisms be brought to levels detectable by PCR, immunoassays, or similar techniques within 8 h. Previously, we employed microfiltration using hollow fiber membranes to rapidly process and concentrate viable bacteria in food extracts through a combination of enzyme treatment and prefiltration in order to prevent blockage or fouling of the hollow fiber membranes. However, scanning electron microscopy and particle size analysis of enzyme hydrolysates showed that enzyme treatment followed by filtration caused submicron particles to form and be trapped within the prefiltration media, which in turn, retained about 80% of the bacteria. Filtering prior to enzyme treatment resulted in formation of a filter cake consisting of protein particles retained on the surface of the filter, while facilitating passage of the much smaller microorganisms through the filter, separating them from particulates. Subsequent enzyme treatment of the filtrate resulted in an extract that was microfiltered in less than an hour, while concentrating viable microorganisms in the extract by 500×. An inoculum of Salmonella enterica cells into turkey burger containing of 1-20 CFU/mL, consisting of spiked cells plus cells already present in the turkey burger sample, was rapidly brought to levels detectable by conventional PCR and BAX® PCR assays. The entire procedure from sample processing to detection of Salmonella enterica was achieved in less than 8 h. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:687-695, 2017.

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.

Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood.

Fundamental characterization of pretreated hardwood and its interactions with cellulolytic enzymes has confirmed that a pathway exists for dramatically reducing the loading of cellulase required for hydrolysis of pretreated biomass. We demonstrate that addition of protein effecting a seven-fold decrease in the specific activity of cellulases enables a ten-fold reduction in enzyme loading while maintaining a high level of cellulose hydrolysis in pretreated hardwood. While use of protein and other additives that adsorb on lignin have been reported previously, the current work demonstrates the effect in a dramatic manner and brings the rationale for this change into clear focus. The key to this result is recognizing and mitigating the pretreatment conundrum where increasingly severe pretreatment conditions enhance accessibility of the enzymes not only to cellulose, but also to lignin. The lignin adsorbs enzyme protein causing loss of cellulase activity. More enzyme, added to compensate for this lost activity, results in a higher cellulase loading. The addition of a different protein, such as BSA, prevents cellulase adsorption on lignin and enables the enzyme itself to better target its glucan substrate. This effect dramatically reduces the amount of cellulase for a given level of conversion with enzyme loadings of 15 FPU and 1.3 FPU/g solids both achieving 80% conversion. The understanding of this phenomenon reinvigorates motivation for the search for other approaches that prevent cellulase adsorption on lignin in order to achieve high glucose yields at low enzyme loadings for pretreated lignocellulose.

Severity factor coefficients for subcritical liquid hot water pretreatment of hardwood chips.

Single stage and multi-stage liquid hot water pretreatments of mixed hardwood pinchips were investigated at various severities (log R0 = 3.65-4.81) to assess the efficiencies of the pretreatments with respect to achieving high pentose sugar yields and improved enzymatic digestibility of pretreated cellulose. We investigate the effect of pretreatment parameters that is, temperature, and time, as expressed in the severity factor, on the recovery of sugars and hydrolyzability of pretreated cellulose. We find the severity factor, in its widely used form, is an incomplete measure for evaluating the pretreatment efficiencies and predicting overall sugar yields when pretreatment temperatures above 200°C are used. Corrections to the severity factor and its correlation to the measured pretreatment responses (% xylan solubilization, xylan recovery as fermentable sugars, cellulose enzymatic digestibility) indicate a greater influence of temperature on the pretreatment efficiencies than predicted by the commonly used severity factor. A low temperature, long residence time is preferred for hemicellulose dissolution during the pretreatment since the condition favors oligosaccharide and monomeric sugar formation over sugar degradation. On the contrary, high cellulose hydrolyzability is achieved with a high temperature (>200°C), high severity pretreatment when pretreatment is followed by enzyme hydrolysis. In multi-stage pretreatment, the first low-severity pretreatment is optimized for solubilizing fast-hydrolyzing hemicellulose while minimizing formation of furans. The subsequent pretreatment is carried out at over 200°C to recover the difficult-to-hydrolyze hemicellulose fraction as well as to increase susceptibility of pretreated cellulose to enzymes. High recovery (>92%) of hemicellulose-derived pentose sugars and enhanced enzymatic hydrolysis of pretreated cellulose (where >80% glucose yield results with 20 FPU = 32 mg protein/g glucan or 10-13 mg/g initial hardwood) are achieved by applying a multi-stage pretreatment. This work shows how the severity equation may be used to obtain a single characteristic curve that correlate xylan solubilization and enzymatic cellulose hydrolysis as a function of severity at pretreatment temperatures up to 230°C.

Fractionation of cellulase and fermentation inhibitors from steam pretreated mixed hardwood.

The purpose of liquid hot water and steam pretreatment of wood is to fractionate hemicelluloses, partially solubilize lignin, and enhance enzyme hydrolysis of cellulose. The pretreatment also solubilizes sugar oligomers, lignin-derived phenolic compounds, acetic acid, and furan derivatives that inhibit cellulase enzymes and/or impede fermentation of hydrolysates by yeasts. This work extends knowledge of the relative contribution of identified inhibitors, and the effect of temperature on their release when pretreated materials are washed and filtered with hot water. Dramatic yield improvements occur when polymeric or activated carbon adsorbs and removes inhibitors. By desorbing, recovering, and characterizing adsorbed molecules we found phenolic compounds were strong inhibitors of enzyme hydrolysis and fermentation of concentrated filtrates by Saccharomyces cerevisiae wine yeast NRRL Y-1536 or xylose fermenting yeast 424A (LNH-ST). These data show that separation of inhibitors from pretreatment liquid will be important in achieving maximal enzyme activity and efficient fermentations.