Rapid Sample Preparation of Foodborne Pathogens by Membrane Filt
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Rapid Sample Preparation of Foodborne Pathogens by Membrane Filtration Wan-Tzu Chen 1,3, Rick Hendrickson 3, Michael Ladisch 1,2,3 Wan- 1 Department of Biomedical Engineering 2 Department of Agricultural and Biological Engineering 3 Laboratory of Renewable Resource Engineering Purdue University ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS Detection of foodborne pathogens requires that food samples The experimental set-up is shown below. TWEEN 20 EFFECT FOR 0.45 µm MEMBRANE FILTERS Hotdog juice made by massaging the hotdog package in the stomacher have to be processed in order to remove interfering factors, including bag mimic the environment of where L. monocytogenes really lives food particles, proteins and lipids, and concentration microorganisms inside food package and simplify the difficulty of filtering food that are to be probed for the presence of pathogens. Conventional Since by membrane filtration, large amount of bacteria can be particles. involving culture steps may require up to 7 days, which is not enough concentrated into about 15 µl, resulting in 10000-fold more concentrated before food is distributed throughout the nation. sample. The problem is to wash them off. We choose Tween 20 as Simplified sample preparation procedures for food samples have been In current study, membrane filtration is able to concentrate the washing solution and converted our results to a concentration factors, a established by membrane filtration. foodborne pathogen, Listeria monocytogenes by a factor of 10000X, ratio of cell count in the retentates to that in the original hotdog juice. Polycarbonate membrane filters are proved to be the most effective with 90% recovery of microorganisms by filtering 50 mL of food Higher concentration factors mean that we made more concentrated ones to retain L. monocytogenes and concentrate them into small sample inoculated with Listeria monocytogenes using a syringe filter. bacteria solutions. volumes. Tween 20 was required to prevent irreversible adsorption of the Nucleopore Recovery percentage polycarbonate (%) MicronSep mixed Recovery percentage (%) microorganism to the membrane, due to hydrophobic interactions. 0.4 um cellulose 10000-fold more concentrated L. monocytogenes samples can be made 0.45 um Polycarbonate, mixed cellulose, nylon and PVDF membranes were Without With Without With Tween from large amount of starting inoculated hotdog juice. To quantify the Tween Tween Tween tested for their ability to retain Listeria monocytogenes and to separate concentration, larger volume (0.5 ml) of washing solution is needed. 1 ml 5.27 65.36 1 ml 3.44 70.41 proteins from microorganisms. The polycarbonate membrane filters with Tween 20, a non-ionic surfactant can successfully wash off the 5 ml 5.27 58.44 5 ml 5.05 86.28 straight through, mono-radial pores was proved to be the most bacteria from the membrane filters without having any detrimental effect 10 ml 8.44 67.68 10 ml 4.61 38.03 successful one. The results show that Listeria monocytogenes 25 ml 8.99 48.35 25 ml 1.74 13.94 on bacteria numbers. concentrated in this manner gives sufficient volume of sample for 50 ml 10.78 72.24 50 ml 4.82 58.48 processing on a protein biochip where as little as 1 µL of sample is Figure 1 Experimental set-up Proteins inside the hotdog juice can be separated from bacteria by needed. membrane filtration and almost 100% of proteins were recovered in the Table 1 Comparison of concentration factors with and without Tween 20 Hotdog juice was made by mixing a pack of hotdog with 250 ml filtrates, meaning that no proteins would appear in the retentates, The visual evidence of how Tween 20 work on removing Listeria of PBS in the stomacher bag. By massaging in the 10 minutes intervals resulting in the blocking of active sites on the Biochips. monocytogenes from the membrane is shown below on the fluorescent for 2 hours, hotdog juice was obtained. Filter it through the filter unit images. Significant amount of bacteria were washed off. A relative quick sample preparation with filtration of 5 minutes would containing 0.22 µm cellulose nitrate membrane filter. After that, hotdog be done within 2 hours compared to the conventional ones which need juice is ready to be inoculated. a b c days for enrichment and culture Listeria monocytogenes strain V7 is obtained from isolating it from real contaminated hotdog samples. Before each experiment, fresh culture is made beforehand. Inoculate hotdog juice with it and serially dilute the hotdog juice to make the final concentration in the order of 102 cells/ml. After that, filter different volumes of inoculated hotdog juice Figure 4 Fluorescent images of ( a ) before washing and ( b ), ( c ) after through syringe and collect the membrane filters and the filtrates. For the retentates, membrane filters (four different materials: washing with PBST REFERENCES INTRODUCTION nylon, mixed cellulose, PVDF and polycarbonate) were soaked into Eppendof tube containing 0.5 ml PBS-1% Tween (PBST). Subsequent The filtrates were tested on their protein contents by Bradford protein assay. (Figure 4) Almost all the proteins go through the pores Sharpe AN, Peterkin PI, Dudas I, 1979, Membrane filtration of food shaking and incubation of 30 minutes was carried out. The washing and appear in the filtrates. suspensions, Appl Environ Microbiol, 37(1): 21-35 Listeria monocytogenes is one of the most commonly occurred solution was enumerated on the Oxford agar by plating out 100 µl of it. 0.3 bacteria in food industry. It has caused about 2500 cases of listeriosis Polycarbonate 0.4 um Ladisch MR. 2001, Bioseparations Engineering. New York: John Wiley Protein Conc(mg/ml) For the filtrates, both protein assay and bacteria enumeration were 0.25 & Sons. p 85-90 annually in the U.S. and 500 of them died. There were many of the L. carried out. 0.2 Mixed Cellulose 0.45 um monocytogenes outbreaks related to the contaminated hotdogs happening 0.15 Control-blank Hotdog juice Hale KA, Doores S, Walsh RA, 1990, An Enzyme/surfactant treatment in recently years. This has caused thousands of people sick and tons of a b c 0.1 and filtration technique for the retrieval of Listeria monocytogenes from ice products being recalled. (Sara Lee recall) It is needed to establish a quick 0.05 cream mix, Food Structure, 9: 61-67 and accurate detection technique for it. 0 Besse NG, Lafarge V, 2001, Development of a membrane filtration Biochips designed for foodborne pathogens have the specificity 10 ml Filtered volumes(ml) 50 ml method for enumeration of Listeria monocytogenes from soft cheese, Food to L. monocytogenes due to a layer of antibodies on top of it. Because of Figure 2 SEM of ( a ) nylon ( b ) PVDF and ( c ) polycarbonate Figure 5 Protein contents in filtrates compared to control Microbiol. 18: 669-676 this layer of specific antibodies, successful separation of food samples would be required in order to remove all the other food proteins and BEST RESULTS 0.2 µ m POLYCARBONATE lipids so that the bio-active sites on the Biochips will not be blocked. RESULTS AND DISCUSSION Finally, the pore size effect on the concentrating was examinated. 100 Preliminary tests without Tween 20 was done. This is carried out Membrane filtration is a good method to carry this out. Due to the by not using any Tween 20 in the washing solution. According to the 90 ACKNOWLEDGEMENTS size difference between L. monocytogenes and proteins inside the food 80 Concentrated factors Nucleopore 0.2 um results, (data not shown) polycarbonate and mixed cellulose gave much 70 samples, a certain pore size can be decided to separate these two better recovery than the rest filters. We thought it is probably due to the 60 Nucleopore 0.4 um • Thisresearch was supported through a cooperative components. At the same time, membrane filter will retain these bacteria structure difference of them. Polycarbonate (below) has a more defined 50 agreement with the Agricultural Research Service of the in the retentate and keep them alive and viable for further detection and pore sizes and straight pathway while nylon and PVDF have a tortuous 40 identification since no harmful chemical was used. pathway and non-uniform pore size distribution. 30 United States Department of Agriculture project number The objective of this study was to separate L. monocytogenes 20 1935-42000-035 from other interfering substances like proteins, lipids and large food 10 particles in hotdog samples. The goal of this experiment is to find a 0 •We appreciate the efforts Dr. Richard Linton has done 1 mL 5 mL 10 mL 25 mL 50 mL simplified procedure to reduce the labor-extensive and time-consuming Filtered volumes (ml) to initially start this project culture steps. Various materials of membrane filters were tested for their Figure 6 Concentration factors for different pore sizes ability to recover L. monocytogenes and the best one can make Figure 3 L. monocytogenes on polycarbonate membrane filter Smaller pore sizes would more easily retain the L. monocytogenes on the •Helpful suggestions from Dr. Arun Bhunia and Nate microorganisms 100-fold more concentrated and pass through almost all membrane due to the size restriction. Mosier the proteins.