Patent Grant
9651551
This application is a continuation of and claims the benefit of priority of International Application Serial No. PCT/US2013/034349, filed Mar. 28, 2013, which claims the benefit of priority of U.S. Provisional Patent Application Ser. No. 61/617,036 filed Mar. 28, 2012, each of which is hereby incorporated by reference in its entirety.
In certain aspects, the present invention relates to methods and systems that are useful for detecting the presence of pathogenic cells in a food sample, and in particular embodiments to methods and systems for processing solid food samples to concentrated, assayable liquid samples.
Pathogenic bacteria in foods are the cause of 90% of the cases of reported foodborne illnesses. The Centers for Disease Control and Prevention estimate that there 76 million cases of foodborne illnesses each year in the United States, resulting in hospitalization of 325,000 people, 5,500 deaths, and an annular cost of $7 to $23 billion. E. coli O157:H7, Salmonella and Listeria monocytogenes are the pathogens of major concern. Ground meat containing E. coli O157:H7 is now considered to be an adulterated food while Listeria monocytogenes has emerged as one of the most important food pathogens with a “zero tolerance” criterion for it in ready-to-eat processed (lunch) meats and dairy foods.
The genus Listeria is comprised of six species, L. monocytogenes, L. ivanovii, L. seeligeri, L. innocua, L. welshimeri, and L. grayi. Of these species, only L. monocytogenes is harmful to humans. Consumption of contaminated food may cause meningitis, encephalitis, liver abscess, headache, fever and gastroenteritis (diarrhea) in immunologically challenged individuals and abortion in pregnant women. L. monocytogenes is ubiquitous in nature and can be found in meat, poultry, seafood, and vegetables. Occurrence of this organism could be as high as 32%. In a food sample, L. monocytogenes is often present in close association with other nonpathogenic Listeria species, thereby complicating the specific detection procedures. A successful detection method ideally detects only L. monocytogenes in the presence of larger populations of nonpathogenic Listeria and other background resident bacteria.
The food processing industry annually carries out more than 144 million microbial tests costing $5 to $10 each. About 24 million of these tests are for detection of food pathogens based on biochemical profile analysis, immunogenic tests (such as enzyme linked immuno-sorbent assays or ELISA), and DNA/RNA probes. These tests are reliable but most require two to seven days to complete because of the steps that are needed to resuscitate cells, increase cell numbers or amplify genetic material needed for detection. This time period is too long for real-time detection of contamination in a food plant and is sufficiently long for contaminated food to be formulated, processed, packaged, shipped, and purchased and eaten by the consumer. Current tests require at least several days to confirm presence of Listeria monocytogenes. The number of annual tests is only expected to increase due to heightened consumer concerns about food safety and the requirement of compulsory testing.
In general, diagnostic tools used for detecting or quantitating biological analytes rely on ligand-specific binding between a ligand and a receptor. Ligand/receptor binding pairs used commonly in diagnostics include antigen-antibody, hormone-receptor, drug-receptor, cell surface antigen-lectin, biotin-avidin, substrate/enzyme, and complementary nucleic acid strands. The analyte to be detected may be either member of the binding pair; alternatively, the analyte may be a ligand analog that competes with the ligand for binding to the complement receptor.
A variety of devices for detecting ligand/receptor interactions are known. The most basic of these are purely chemical/enzymatic assays in which the presence or amount of analyte is detected by measuring or quantitating a detectable reaction product, such as a detectable marker or reporter molecule or ligand. Ligand/receptor interactions can also be detected and quantitated by radiolabel assays.
Quantitative binding assays of this type involve two separate components: a reaction substrate, e.g., a solid-phase test strip and a separate reader or detector device, such as a scintillation counter or spectrophotometer. The substrate is generally unsuited to multiple assays, or to miniaturization, for handling multiple analyte assays from a small amount of body-fluid sample.
In recent years, there has been a merger of microelectronics and biological sciences to develop what are called “biochips.” The term “biochip” has been used in various contexts but can be defined as a “micro fabricated device that is used for delivery, processing, and analysis of biological species (molecules, cells, etc.).” Such devices have been used, among other things, for the direct interrogation of the electric properties and behavior of cells (Borkholder et al. “Planar Electrode Array Systems for Neural Recording and Impedance Measurements”, IEEE Journal of Microelectromechanical Systems, vol 8(1), pp. 50-57, 1999); impedance-based detection of protein binding to surfaces, antigen-antibody binding, and DNA hybridization (DeSilva et al., “Impedance Based Sensing of the Specific Binding Reaction Staphylococcus Enterotoxin B and its Antibody on an Ultra-thin Platinum Film,” Biosensors & Bioelectronics, vol. B 44, pp 578-584, 1995); micro-scale capillary electrophoresis (Wooley et al., Ultra High Speed DNA Sequencing Using Capillary Electrophoresis Chips,” Analytical Chemistry, vol. 67(20), pp. 3676-3680, 1995); and optical detection of DNA hybridization using fluorescence signals in the commercially available “DNA-chips” (Fodor et al., “Light-directed Spatially Addressable Parallel Chemical Synthesis,” Science, vol. 251, pp. 767-773).
One of the most interesting uses of biochips is for the detection of small quantities of pathogenic bacteria or toxigenic substances in food, bodily fluids, tissue samples, soil, etc. In applications such as the screening of food products for the presence of pathogenic bacteria, it would be beneficial to detect between 100 and 1000 microorganisms per milliliter of sample, with a sample volume of a couple of milliliters. Not counting the fact that bacteria are substantially larger than single biomolecules (about 2 micrometers vs. about 10-100 Angstroms), 1000 cells are approximately equivalent to a 10−5 femto-moles of cells, which gives an idea of the difficulty in directly detecting such a small number suspended in a volume of 1 or 2 ml, along with large numbers of food debris, proteins, carbohydrates, oils, and other bacteria. Additionally, in many cases the screening technique must be able to discern between viable and dead cells. Many bacteria will not produce toxins when not viable and consequently will not be pathogenic in that state. DNA detection methods, which search for DNA sequences specific to the pathogen of interest, can be extremely sensitive because they rely on the very specific binding of complementary DNA strands, often coupled with Polymerase Chain Reaction (PCR) for amplification. But the detected DNA fragments cannot reveal whether the pathogen was viable or not. These are the main reasons why current methods of detection almost always involve a growth step, in which the bacteria are cultured to increase their numbers by several orders of magnitude. Once the bacteria are amplified to a large number, visual detection of colonies or Enzyme-Linked Immunosorbent Assays (ELISA) confirm their presence in the original sample. Even though bacteria can multiply very rapidly, this amplification by means of extended growth makes conventional detection methods extremely lengthy, taking anywhere from 2 to 7 days. Thus, goals in industry have been to reduce the time of sample processing and analysis, while also retaining or heightening the sensitivity of the analysis.
In one aspect, the present invention provides a method for processing a food sample. The method includes preparing a liquefied extract of a food sample, and processing the liquefied extract by hollow fiber membrane filtration to form a concentrate of cells from the liquefied extract. The step of preparing preferably includes enzymatically digesting the food sample. The hollow fiber membrane filtration can be conducted under cross flow conditions, with flow of a liquid on the permeate side of the hollow fiber(s). The method can also include assessing the concentrate of cells for the presence of pathogenic cells.
In another aspect, the present invention provides a method for preparing a liquefied extract of a food sample. The method includes homogenizing a solid food sample under controlled shear conditions to form a homogenized food sample substantially retaining a viable cellular content of the solid food sample. The method further includes enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample. Additionally, the digested food sample is processed to remove lipids from the digested food sample, to form a lipid-depleted food sample. The lipid-depleted food sample can be processed by hollow fiber membrane filtration, desirably under cross flow conditions, to concentrate cells in a retentate and generate a permeate. The method can also include assessing the concentrated cells for the presence of pathogenic cells.
In another aspect, the present invention provides a method for testing for pathogenic cells. The method includes contacting a viable cellular concentrate from a food sample with a fluorescent antibody dye that specifically binds to the pathogenic cells, to form a contacted sample. The method further includes processing the contacted sample under conditions to remove unbound amounts of the fluorescent antibody dye, to form a dye-depleted sample, and spectroscopically interrogating the dye-depleted sample for the fluorescent antibody dye. The viable cellular concentrate can be prepared by a process including: homogenizing a solid food sample under controlled shear conditions to form a homogenized food sample substantially retaining a viable cellular content of the solid food sample; enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample; processing the digested food sample to remove lipids from the digested food sample, to form a lipid-depleted food sample; and concentrating the lipid-depleted food sample in cells.
In another aspect, the invention provides a method for processing a hollow fiber membrane that has contacted a liquefied food sample. The method includes (i) passing a sodium hydroxide solution through the hollow fiber membrane; (ii) passing an aqueous medium through the hollow fiber membrane after step (i) to rinse sodium hydroxide residues from said membrane; (iii) passing a sterilant solution through the hollow fiber membrane after step (ii); and
(iv) passing an aqueous rinse medium through the hollow fiber membrane after step (iii).
In another aspect, the invention provides a method for concentrating and recovering cells from a food sample. The method includes (i) passing a liquefied extract of the food sample through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate of cells in the interior of the hollow fiber membrane; and (ii) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (i). The passing step (ii) can include passing the aqueous solution co-current to a flow of the liquefied extract through the interior of the hollow fiber membrane. The liquefied extract can be prepared by a process including enzymatic digestion of the food sample.
In another aspect, the invention provides a method for assessing a solid food sample for the presence of pathogenic cells. The method includes (i) suspending the solid food sample in an aqueous medium to form a suspended sample medium; (ii) homogenizing the suspended sample medium under controlled shear conditions to form a homogenized food sample, said homogenizing conducted under conditions effective to substantially retain any viable pathogenic cells from the solid food sample; (iii) enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample medium; (iv) processing the digested food sample medium to remove lipids and form a lipid-depleted food sample extract liquid; (v) passing the lipid-depleted food sample extract liquid through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate containing cells in the interior of the hollow fiber membrane; (vi) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (v); and (vii) assessing the liquid concentrate containing cells for the presence of viable pathogenic cells. The method can also include, after step (vii): (viii) passing a defouling liquid through the hollow fiber membrane; (ix) passing an aqueous rinse medium through the hollow fiber membrane to remove defouling solution residues from step (viii); (x) passing a sterilant liquid through the hollow fiber membrane; and (xi) passing an aqueous rinse medium through the hollow fiber membrane after step (x) to remove sterilant liquid residues from the hollow fiber membrane. The process can also include repeating steps (i) to (vii) after step (xi).
In additional embodiments, methods as described above or elsewhere herein can be conducted wherein any one, any combination, or all of the recited steps are conducted under the control of a controller, such as a computer.
Still further embodiments of the invention provide systems or instruments for processing food sample material, which are configured to carry out steps as in any method described herein.
Additional embodiments of the invention, as well as features and advantages thereof, will be apparent from the descriptions herein.
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments, some of which are illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates.
As disclosed above, in certain aspects the present invention relates to methods and systems for processing food materials that are useful for the detection of pathogenic cells in the materials. In the discussions that follow, a number of potential features of reagents, food samples, system equipment, steps, physical or chemical conditions, or other aspects, are disclosed. It is to be understood that each such disclosed feature or features can be combined with the features discussed in the Summary above or recited in the Specific Listed Embodiments below, to form a disclosed embodiment of the present invention. As well, it will be understood by those skilled in the art that methods and systems described herein will with preference be able to concentrate and detect viable pathogenic cells from food samples. Accordingly, liquid and other reagents or materials to contact the sample, e.g. feeds to systems described herein, are sterile as appropriate to avoid contaminating the sample with extraneous viable cells.
With reference to
With reference now to
System 30 also includes a second pump 54, such as a peristaltic pump as described above, and a permeate-side fluid reservoir 56 (for example distilled water) fluidly coupled to pump 54 by flow conduit 56a. Pump 54 feeds through feed conduit 54a to pressure transducer 58 (e.g. allowing a maximum pressure of 60 psi), which in turn feeds through flow conduit 60 to permeate-side inlet 32c of membrane module 32. Permeate-side opening 32c feeds into a space within a chamber of module 32 and external of the hollow fiber membrane(s) that extend through module generally from inlet 32a to outlet 32b. Permeate-side outlet 32d feeds from such chamber into a flow conduit 64 which in turn feeds to permeate collection reservoir 68. A first flow meter 62 is operably associated with flow conduit 60 and a second flow meter is operably associated with flow conduit 64. In this manner, the rate of flow into and out of the permeate side of module 32 can be measured and a visible readout provided to a user, for example using a computer-controlled system with a display (further described below).
System 30 includes a sample concentrate collection line 70 fluidly coupled to two-way valve 50, which in turn feeds to a sample concentration collection reservoir 72. System 30 also includes a liquid level sensor 74 operably associated with sample reservoir 36. Sensor 74 can be operable to sense when the liquid sample in reservoir 74 falls to or below a predetermined level. System 30 further includes a two-way valve 76 in retentate return flow conduit 52, fluidly coupled to flow conduit 78 which feeds into a sterilant reservoir 80 (e.g. a bleach solution such as a 10% bleach solution). A turbidity sensor 82 is operably associated with flow conduit 64 that receives flow from the permeate-side outlet 32d of module 32. Turbidity sensor 82 can be operable to sense turbidity in the permeate fluid exiting outlet 32d, which when sensed can provide a signal to actuate valve 76 to stop flow through retentate return flow conduit 53 back to sample reservoir 36 and instead direct liquid flow to flow conduit 78 and into sterilant reservoir 80. In this fashion, the turbidity sensed by sensor 82 can be indicative of a membrane breach in module 32, and the contaminated permeate can be redirected to the sterilant reservoir 80 to kill any pathogenic cells in the contaminated permeate. In addition to or as an alternative to providing permeate-side liquid from reservoir 56, a permeate return flow conduit 84 can be provided and fluidly coupled to flow conduit 56 in turn feeding to pump 54. In this manner, permeate liquid can be recirculated in a loop through the permeate side of module 32 under the power of pump 54.
In operation, a food sample is provided to sample reservoir 36. Such a food sample may be, and preferably is, a liquid sample that has been pre-processed as in steps 10, 12 and 14 discussed in conjunction with
After recovery of the concentrated retentate sample in reservoir 72, and prior to processing another food sample through system 30, a system regeneration cycle can be conducted. For these purposes, valve 44 can be actuated to direct flow from defoulant liquid (e.g. sodium hydroxide solution) from reservoir 40 through the hollow fiber membrane(s) of module 32, so as to remove cell membranes and/or other foulant materials from the surfaces of the hollow fiber membrane(s) of module 32. During processing-through of the defoulant liquid, flow of water or another suitable liquid, typically aqueous, on the permeate side may be maintained with a positive interior-to-exterior transmembrane pressure gradient also maintained. Amounts of the defoulant liquid passed through the membrane(s) of module 32 can be collected in reservoir 68.
After passage of the defoulant liquid, remaining residues of the defoulant liquid can be removed by passing an aqueous rinse medium, acceptably distilled water, through the hollow fiber membrane(s) of module 32 and the flow paths leading to and from such membrane(s). During processing-through of the aqueous rinse medium, flow of water or another suitable liquid, typically aqueous, on the permeate side may be maintained with a positive interior-to-exterior transmembrane pressure gradient also maintained. Amounts of the aqueous rinse medium and rinse residues passed through the membrane(s) of module 32 can be collected in reservoir 68.
After rinsing the module 32 as noted above, valve 44 can be actuated to direct a sterilant liquid from reservoir 42 through the interior of hollow fiber membrane(s) of module 32 and through the flow paths feeding to and from module 32, to kill any viable cells remaining therein. Again, during processing-through of the sterilant liquid medium, flow of water or another suitable liquid, typically aqueous, on the permeate side may be maintained with a positive interior-to-exterior transmembrane pressure gradient also maintained. Amounts of the sterilant liquid medium passed through the membrane(s) of module 32 can be collected in reservoir 68.
After passage of the sterilant liquid, remaining residues thereof can be removed by passing an aqueous rinse medium, acceptably distilled water, through the hollow fiber membranes of module 32. A flow of water or another suitable liquid, typically aqueous, on the permeate side may be maintained during processing-through of the aqueous rinse medium, with a positive interior-to-exterior transmembrane pressure gradient also maintained. Amounts of the rinse medium and rinsed sterilant liquid residues can be passed through the membrane(s) of module 32 can be collected in reservoir 68.
After the regeneration cycle including the above defoulant, rinse, sterilization, and rinse steps, the system 30 can be used to process another food sample charged to reservoir 36. For these purposes, the various other reservoirs of system 30 can be sufficiently large for processing of multiple samples, or these reservoirs can be recharged as well.
During processing, pressure on the retentate side can be measured using pressure transducer 46, and pressure on the permeate side can be monitored using pressure transducer 58. Flow into permeate-side inlet 32c can be monitored using flow meter 62, and flow out of permeate-side outlet can be monitored using flow meter 66. These pressure and flow readings can be used to maintain or adjust the speed of pumps 34 and 54 to maintain the flow and pressure conditions necessary for the filtration with module 34.
Should a membrane breach occur, turbidity monitor 82 will detect an increase in turbidity. This can provide a signal to actuate valve 76 to redirect flow of the retentate in flow conduit 52 from the sample reservoir 36 to instead the bleach, caustic or other sterilant reservoir 80 to kill any pathogenic cells in the permeate. The increase in turbidity may also provide a signal to turn off pumps 34 and 54, either immediately or after a predetermined period of time for shunting of contaminated permeate to reservoir 80.
The preferred system 30 also includes a controller 100, such as a computer, that automatically controls, and/or enables user inputs to control, some or all aspects of operation of system 30 as discussed above. For these purposes, controller 100 can be configured to electronically communicate with system 30 components so as to have the capacity to receive and/or send signals to the components (e.g. pumps, valves, sensors, or meters as discussed above) to achieve the noted operations, and potentially others. Controller 100 can also be communicably associated with a user input device, such as a keyboard 102, and/or a display device 104 which may also serve as a touch screen input in some embodiments. Computer or other controller 100 can operate software that enables display and control functions as noted herein for system 30. In one embodiment, software commercially available as LabVIEW 2009f3, available from National Instruments, is utilized. In this regard,
The membrane module 32 can contain any suitable number of hollow fiber membranes, and typically a plurality of membranes will be used in a bundle. For example, 3 or more, 5 or more, or 10 or more hollow fiber membranes can be used. In one embodiment, 12 hollow fiber membranes are used. The membranes can be made from any suitable material. Polysulfone hollow fiber membranes are suitable. The membranes can have a pore size sufficient to retain (prevent passage) of cells. A pore size of about 0.2 micrometers is suitable for this purpose. The inner diameter of the membranes may suitably be about 100-300 micrometers, for instance 280 micrometers, and an outer diameter may suitably be 150 to 400 micrometers, for instance about 360 micrometers. The membrane thickness may suitably be about 50 to 100 micrometers, and typically about 80 micrometers. In one module embodiment, about 0.2 micrometer pore size hollow fiber membranes having an inner diameter of 280 micrometers, an outer diameter of 360 micrometers, and a length of about 27 centimeters, are bundled together. 12 such hollow fibers can be used. The fiber bundle is packed into polymeric tubing, for example polyether ether ketone (PEEK) tubing, having an inner diameter of about 2 millimeters and an outer diameter of about 3.2 millimeters. Each end of the module is then sealed with two layers of epoxy to prevent leakage, and to separate the retentate side from the permeate side. “T” connectors are provided on the tubing and around the fiber bundle to provide a permeate-side inlet and a permeate-side outlet.
The concentrated sample provided by system 30 can be processed in any manner desired to assess for target pathogenic cells. Any of those methods, and systems or devices therefore, described hereinabove may be used. In further embodiments, such detection systems or devices can be operably associated with and part of system 30, receiving feed of the necessary sample for detection from reservoir 72 or potentially directly from concentrated sample collection conduit 70. Also in these embodiments, the feed to and operation of the detector may be controlled by controller 100.
In work to date, a working embodiment of system 30 has been used in several modes of testing, results of which are shown in
Provided in
In a further embodiment, system 30 can include an early detection feature for rapid detection of the presence of target pathogenic cells in the retentate. For these purposes, system 30 can include a dye reservoir 90, for example containing a fluorescent dye that specifically labels one or more target pathogenic cells to be detected. A suitable fluorescent marker for these purposes includes, for example, green fluorescent protein (GFP), although others are known and can also be used. System 30 can also include a pump 92 for pumping amounts of dye from reservoir 90 into retentate flow conduit 52. If needed or desired, a valve could also be provided between pump 92 and conduit 52 to selectively permit or prevent flow to conduit 52. The opening and closing of such a valve would be coordinated with the operation of pump 92 to feed amounts of the dye to conduit 52 at the appropriate time(s). System 30 can also include a detector 94 associated with retentate flow conduit 52 and operable to detect the presence of the dye in the retentate. In operation, an amount of the dye can be charged to the conduit 52 during a retentate circulation/concentration operation as described above. The dye will specifically bind to target pathogenic cell(s) in the retentate, and some amounts of the dye will remain unbound (free) in solution. Unbound amounts of dye will be continuously passed to the permeate in module 32, and thus the signal detected by detector 94 will diminish over time after input of a measured charge of dye into the retentate. A remaining reduced, but steady signal would indicate the presence of pathogenic cells in the retentate, and provide an early indication that the food sample was contaminated with the pathogenic cells. Several such pulses of dye can be added to the retentate over time as the sample continues to concentrate. Eventually, if the target pathogenic cells are present, the remaining signal from detector 94 (after removal of the unbound dye through the permeate) will be strong enough, due to the concentration of the pathogenic cells, to reliably indicate the presence of the target pathogenic cells in the retentate. Detector 94 is preferably a spectroscopic detector that spectroscopically interrogates the retentate in-line as it flows within system 30. The dye for these purposes is preferably a fluorescent dye, for example a fluorescent antibody dye, that binds specifically to a surface protein on the target pathogenic cell(s). As well, a mixture of such specific dyes could be used in the pulse(s) into the retentate, to enable the detection of different types of pathogenic cells by specifically binding of a dye to each of the cell types. In such cases the mixed dyes could each carry the same fluorescent marker and thus provide a signal additive of the different types of pathogenic cells, or could each carry a different fluorescent marker and thus provide a distinct signal relative to each of the different types of cells separately. The detector 94 could be configured to detect each such distinct signal, or one or more additional separate detectors could be incorporated into system 30 to detect the distinct signals. These and other variations will be apparent to those of ordinary skill in the art from the descriptions herein.
Target pathogenic cells for detection may be any pathogenic cells of interest. The pathogenic cells may in certain embodiments be E. coli O157:H7, Salmonella or Listeria monocytogenes pathogens.
The food sample processed may be any suitable food sample, preferably a solid material. It may be a meat product such as poultry or beef, for example a ground meat potentially containing E. coli O157:H7, or a ready-to-eat processed meat or dairy food that may contain Listeria monocytogenes, or a meat such as chicken that may contain a Salmonella pathogen. The food sample may also be a dry food such as a cereal or other starch, or a vegetable, in certain embodiments.
The following Examples describe various embodiments of the present invention, as well as features and further details that can be used in the above-described embodiments. Additionally, U.S. Pat. Nos. 7,306,924 and 7,547,526, which are incorporated herein by reference, describe additional details of pathogens, food products, processing conditions, and detection methods and devices that may be utilized in embodiments of the present invention.
Despite the development of rapid detection methods (such as ELISA and PCR), reduction and/or elimination of cultural enrichment remains an essential goal to attend the need for truly real time detection methods. While detection may be measured in hours, the steps leading up to detection add days to the whole process. Therefore, there is an essential role for a time effective bacterial concentration and purification from food sample matrices as a step preceding detection (known as pre-analytical sample processing) (Dwivedi, H. P. and Jaykus, L-A. 2011. Critical Reviews in Microbiology 37(1):40-63) (Kim, H.-Y., Park, H.-J., and Ko, G. 2010. Journal of Food Protection 72(12):2547-2552).
Microfiltration techniques represent one conceptually simple way to reduce large samples to a small volume and effectively increase cell concentration and recovery from food without lengthy culturing and enrichment steps. However, fouling of membrane is a challenging that needs to be addressed in order to make this process effective. Fouling is caused by the accumulation of fats, oils, particles, and proteins on the surface of the membrane, in a manner that causes a layer to form that blocks transmission of fluid through the membrane (Kimura, K., Hane, Y., Watanaeb, Y., Amy, G., Ohkuma, N. 2004. Water Research 38:3431-3441) (Ladisch, M. R. 2001. Bioseparations Engineering: Principles, Practice, and Economics, 17-19, 36-47, 53-106, Wiley-Interscience, NY) (Peterkin, P. I., Sharpe, A. N. 1980. Appl Environ Microbiol. 39(6):1138-1143).
In addition to the need of developing methods to reduce (or eliminate) membrane fouling, the understanding of bacterial surface adhesion to the substrate of interest (possibly contaminated food, in this case poultry meat) is also important. Goulter et al. (Goulter, R. M, Gentle, I. R, and Dykes, G. A. L. 2009. Appl. Microbiol. 49: 1-7) and Faille et al (Faille, C., Jullien, C, Fontanine, F., Bellon-Fontanine, M., Slomianny, C., and Benezech, T. 2002. Can. J. Microbiol. 48:728-738) have previously described hypothesis for bacterial binding to abiotic and metallic surfaces. Goulter suggested that bacterial bindings occurs via a two-step mechanism: (1) bacteria attaches to a surface with an initially reversible state which involves van der Waals forces, electrostatics, and hydrophobic/hydrophilic interactions; (2) irreversible attachment which can involve covalent bonding, hydrogen bonding, and very strong hydrophobic interactions. Faille suggested that cell adhesion exists through a three step process: (1) physiochemical interactions (similar as before); (2) adhesion reinforcement through polymer excretion; (3) cellular division. Faille also suggests a correlation between the micro-scale roughness of a sample and its adhesion ability.
An embodiment of the invention addresses both aspects through a method for food pretreatment prior filtration using hollow fiber membranes. In this method, the possibly contaminated sample (for example a piece of poultry meat) is mixed gently with water (e.g. in a weight/volume ratio of 1:9), bringing the pH to ˜6.0, and then homogenized in a STOMACHER bag, desirably at a relatively low speed (e.g. 100 rpm) for a suitable period of time, for example 30 seconds. The use of lower speed is to avoid additional stress to the microorganisms, as well as the release of more proteins, which has been found to be one of the major membrane foulants.
The resultant preparation is then mixed for 1 hour at 37° C. and 200 rpm with an enzyme (0.5% (v/v)) effective in hydrolyzing proteins at substantially neutral pH, for example the enzyme PROTEX™ 7 L, produced by Genencor International, Inc., Rochester, N.Y., USA. This is a bacterial endopeptidase preparation derived from a controlled fermentation of a non-genetically modified strain of Bacillus amyloliquefaciens. In order to remove lipids, another identified major foulant, the enzyme-treated preparation is then passed through a membrane (for example a 2.7 micrometer glass microfiber membrane (GF/D micro filter)), VWR, Batavia, Ill., USA) as a pre-filtration step. The sample is then ready to filtration by the use of hollow fiber membranes, e.g. in system 30 described in this application, for concentration and recovery of microorganisms.
The combination of pre-filtration using glass microfiber membranes and enzyme treatment prior concentration and recovery of microorganisms from the food sample (e.g. poultry meat) significantly reduces the necessary time (preferably 1-2 hours, compared to days when using standard methods) for the whole process and allows re-use of the hollow fiber membranes if appropriated sterilization procedures are taken.
Using the developed food pretreatment method disclosed herein, the results indicated that, although more challenging, efficient use of hollow fiber membranes targeting bacteria in food, previously homogenized before cell concentration and recovery, can be achieved in a significantly shorter time compared to standard methods (days) by the approach using few steps that can be integrated in one system for the whole process of microorganism concentration and recovery from contaminated food.
In the developed method described herein, initially a sample of bacterial cells is prepared from a liquid food extract using an automatically controlled microorganism concentration instrument. The operation of this instrument uses the simple principle of microfiltration in a continuous flowthrough type operation to decrease the volume of the sample to a small volume, having concentration of bacterial cells at sufficient number, so that they may be effectively probed for presence of pathogen on a “first alert” basis by the use of fluorescent antibodies, and detecting them using a spectrometer (for example a USB4000-UV-VIS Miniature Fiber Optic Spectrometer).
Preliminary tests have been done with E. coli GFP (sgGFP™: SuperGlo™ Green Fluorescent Protein: a single excitation peak at 474 nm, and emission at 509 nm). Further tests for optimization include the use of fluorescent micro-particles (7.-7.9 m, Spherotec, Inc., Lake Forest, Ill., USA) available with functional groups for covalent binding of antigens or antibodies. After these initial tests for optimization, the technology will be applied to test first the presence of Salmonella spp. artificially spiked in food at different concentration levels. The samples containing Salmonella will be incubated with fluorescent antibodies for 30-60 min at room temperature before applied to cell concentration and recovery in a system such as system 30 described herein (less than 10 min). The preparation and use of fluorescent antibodies for detection of Salmonella in food products was taught by Haglund et al. in 1964 (Haglund J. R., Ayres J. C., Paton A. L, Kraft A. A, and Quinn L. Y. 1964. Applied Microbiology, 12(5):447-450). The use of fluorescently-labeled monoclonal antibodies, with detection by multi-parameter flow cytometry for the rapid detection of Salmonellas in pure cultures has also been demonstrated even in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species is possible (McClelland R. G. and Pinder A. C. 1994. Journal of Applied Microbiology, 77 (4):440-447). The method of this and other embodiments of the invention can use anti-Salmonella fluorescent antibodies available commercially.
Increased interest has been developed for rapid detection of bacteria in food, biosafety and medicinal analysis, including the identification of infectious agents and the rapid detection of bio-warfare agents (Tauxe, R. V. Int. J. Food Microbiol. 78. 2002. PII S0168-1605(0102)00232-00235) (Love, T. E., Jones B., Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems, Springer, 2008, pp. 3-13) (Zhang, J. I., Talaty, N., Costa, A. B., Xia, Y., Tao, A., Bell, R., Callahan, J. H. and Cooks, R. G. 2011. International Journal of Mass Spectometry 301:37-44). Although traditional methods such as nucleic acid hybridization (McKillip, J. L. and Drake, M. 2004. J. Food Prot. 67:823-832), polymerase chain reaction (PCR) (McKillip, J. L. and Drake, M. 2004. J. Food Prot. 67:823-832) (Van Kessel, J. S, Karns, J. S. and Perdue, M. L. 2003. J. Food Prot. 66 1762-1767), enzyme linked immunosorbent assays (ELISA) (Mansfield, L. P. and Forsythe, S. J. 2000. Lett. Appl. Microbiol. 31:279-283) (Valdivieso-Garcia, A., Riche, E., Abubakar, O., Waddell, T. E. and Brooks, B. W. 2001. J. Food Prot. 64:1166-1171) immunofluorescence assays (IFAS) (Ng, S. P., Tsui, C. O., Roberts, D., Chau, P. Y., Ng, M. H. 1996. Appl. Environ. Microbiol. 62:2294-2302) or chemotaxonomic fingerprint by chromatographic (Huys, G., Altwegg, M., Hanninen, M. L., Vancanneyt, M., Vauterin, L., Coopman R., Torck, U., LuthyHottenstein, J., Janssen, P. and Kersters, K. 1996. Syst. Appl. Microbiol. 19:616-623) and methods (Busse, H. J., Denner, E. B. M. and Lubitz, W. 1996. J. Biotechnol. 47 (1996) 3-38) can differentiate bacteria at the species and sub-species level reliably, they may require hours to days to perform (Zhang, J. I., Talaty, N., Costa, A. B., Xia, Y., Tao, A., Bell, R., Callahan, J. H. and Cooks, R. G. 2011. International Journal of Mass Spectometry 301:37-44). The detection method of the invention differs from those methods, first by being integrated to an instrument such as system 30 disclosed herein for cell concentration and recovery, and second by probing targeted pathogens in real or near real time.
The technology integrated into a cell concentration and recovery (CCR) instrument providing system 30 herein can include easy to operate components: peristaltic pumps, low (or “zero”) dead volume tubing, automated valves, sterilizable fittings, sample and solvent reservoirs, and hollow fiber microfiltration membrane module packaged with reusable housings.
The food sample can be processed by microfiltration in a cross-flow operation mode to concentrate the sample to a small volume, increasing the number of microbial cells to a detectable level so that they may be effectively probed for the presence of pathogen. The running of this instrument includes two modes, i.e., the cell concentration or CC mode, and the cell recovery or CR mode. Under CC mode, the sample is feed to the membrane module with a pump. The permeate that passes the membrane goes to the permeate vessel, while the particles that are rejected by the membrane are concentrated in the retentate. The retentate is circulated back to the sample reservoir and further concentrated until no or very little sample is left in the sample reservoir. A second pump is used to pump a liquid (e.g. sterile water) flow against the hollow fiber membranes from the permeate side, e.g. to achieve a substantially constant transmembrane pressure. Under the CR mode, the retentate sample is pumped to the collection tube which can be facilitated by a two-valve that can selectively circulate retentate back to the sample reservoir or to the collection tube.
A simplified software interface compatible with the most recent version of LabVIEW with graphical programming for measurement and automation has been developed using simulation software and an interface. The program and interface are installed in a lab laptop for displaying the pressure and flow data and controlling the unit's operation. A cycle of sample concentration, sample recovery, and chemical asepsis (e.g as discussed in conjunction with
This integrated instrument can be used to concentrate and recovery microbial cells from large volume of food suspensions/extracts in a time-efficient manner. The concentration and recovery of microbial cells from food matrices is the basis of rapid food borne pathogen detection.
In the developed instrument, cell concentration and recovery are integrated. The microbial cells are not only separated from the permeate but also recovered for further analysis. This embodiment is highly automated and the manual handling can be minimized. The recovered cell-containing retentate sample can be processed for subsequent pathogen detection.
Hollow fiber (HF) ultrafiltration has been used as a potential approach to simultaneously concentrate different microorganisms in water, and reduce clogging problems compared to conventional ultrafilters (Morales-Morales H. A., Vidal G., Olszewski J., Rock C. M., Dasgupta D., Oshima K. H. and Smith G. B. 2003. Appl. Environ. Microbiol. 69(7): 4098-4102) (Hill V. R., Polaczyk A. L., Hahn D., Narayanan J., Cromeans T. L., Roberts, J. M., and Amburgey J. E. 2005. Appl. Environ. Microbiol. 71(11): 6878-6884) (Smith C. M. and Hill V. R. 2009. Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl. Environ. Microbiol. 75(16): 5284-5289) (Kim H.-Y., Park H.-J., and Ko G. 2010. Journal of Food Protection 72(12): 2547-2552). Its use may offer advantages, such as facilitating the detection of multiple bacterial strains, removal of matrix-associated reaction inhibitors and provision of adequate sample size reductions to allow for the use of representative food sample sizes (Stevens, K. A. Jaykus, L. A. 2004. Critical Reviews Microbiology. 30: 7-24). Recent literature has indicated the potential efficient use of HF membranes for pathogen concentration and recovery from water samples (Morales-Morales H. A., Vidal G., Olszewski J., Rock C. M., Dasgupta D., Oshima K. H. and Smith G. B. 2003. Appl. Environ. Microbiol. 69(7): 4098-4102) (Hill V. R., Polaczyk A. L., Hahn D., Narayanan J., Cromeans T. L., Roberts, J. M., and Amburgey J. E. 2005. Appl. Environ. Microbiol. 71(11): 6878-6884) (Smith C. M. and Hill V. R. 2009. Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl. Environ. Microbiol. 75(16): 5284-5289) (Kim H.-Y., Park H.-J., and Ko G. 2010. Journal of Food Protection 72(12): 2547-2552) (Stevens, K. A. Jaykus, L. A. 2004. Critical Reviews Microbiology. 30: 7-24) (Park H., Kim M. and Ko G. 2010. Journal of Food Protection 73(9): 1651-1657). Recent results indicated that, although more challenging, by using a developed instrument based on the use of hollow fiber membrane modules, targeted pathogens in food can be effectively concentrated and recovered for further identification.
In order to allow instrument re-use, and consequently enhance the economics for the whole process, we developed a method for cleaning and sterilization of a hollow fiber membrane instrument (e.g. providing system 30 described herein) for cell concentration and recovery.
Positive results has been systematically achieved by sequential use of NaOH (0.2 M, 10 min), H2O (5 min) to clean any trace of caustics in the system, 70% (v/v) ethanol (10 min) to return the system back to a sterile state, and finally water one more time at the same pressure and flow rate for 5 min to clean any trace of ethanol and rehydrate the membrane. This method has been consistently used without any negative impact on any of the instrument parts.
The effectiveness of cleaning and sterilization has been accessed through microscopy analysis and plating in non- and selective media for monitoring. Results indicate the possible re-use of the hollow fiber membranes for cells concentration from food for at least about 10-20 times (depending on the volume applied, and still under optimization). These tests have been done with different new hollow fiber membrane modules.
The disinfection of the whole instrument for concentration and recovery of microorganisms before and after each filtration is important taking in consideration that bacteria have the potential for rapid reproduction under diverse environmental conditions. The disinfection method developed and described herein allows for multiple re-use of the hollow fiber membrane modules, which will also enhances economics associated to the whole process.
The rapid detection of microorganisms against a background of nonpathogenic bacteria in contaminated food samples requires recovering the bacteria from colloidal matter, fats, proteins, and other components which rapidly foul membranes and stop flow in a matter of minutes. Microfiltration techniques represent a conceptually simple way to reduce large samples to a small volume and effectively increase cell concentration without lengthy culturing and enrichment steps. Dead-end filtration techniques using a flat-sheet membrane have proven to be effective for concentrating microbial cells, including L. monocytogenes and Gram-negative pathogens including Salmonella for microbiological analysis of water, dairy, and food products (Carroll, S. A., Carr, L. E., Mallinson, E. T., Lamichanne, C., Rice, B. E., Rollins, D. M., Joseph, S. W. 2000. J. Food Prot. 63(3):347-353) (Chen, W. T., Hendrickson, R. L., Huang, C. P., Sherman, D., Geng, T., Bhunia, A. K., Ladisch, M. R. 2005. Biotechnol Bioeng. 89:263-273) (Chen, W. T., Ladisch, M. R., Geng, T., Bhunia, A. K. 2005. AIChE. J. 51:3305-3308) (Entis, P., Lerner, I. 2000. J Food Prot. 63(3):354-363) (Ladisch, M. R. 2001. Bioseparations Engineering: Principles, Practice, and Economics, 17-19, 36-47, 53-106, Wiley-Interscience, NY) (Peterkin, P. I., Sharpe, A. N. 1980. Membrane filtration of dairy products for microbiological analysis. Appl. Environ. Microbiol. 39(6):1138-1143) (Hill, V. R., Polaczyk, A. L., Hahn, D., Narayanan, J., Cromeans, T. L., Roberts, J. M., and Amburgey, J. E. 2005. Appl. Environ. Microbiol. 71(11):6878-6884). It has been shown that sequential filtration through a depth filter followed by a screen filter could concentrate 100 ml samples having as few as 20 CFU/ml to a volume of 50 l with 2,000 CFU/ml (Chen, W. T., Ladisch, M. R., Geng, T., Bhunia, A. K. 2005. AIChE. J. 51:3305-3308) (Entis, P., Lerner, I. 2000. J Food Prot. 63(3):354-363). However, due to fouling of the membrane and the need for removing and handling it, an alternate method was needed. Hollow fiber (HF) membranes offer several advantages over the traditional flat-sheet membranes: a high surface area to volume ratio, a higher flux per unit volume of the membrane module, and continuous operation that avoids manual handling of the membrane and sample. The membrane may be back-flushed to recover concentrated cells in an aqueous buffer or flushed with an elution buffer to recover cells from the device platforms for detection and identification. Recent literature has taught certain uses of HF membranes for pathogen concentration and recovery from water samples (Kim, H.-Y., Park, H.-J., and Ko, G. 2010. Hollow fiber ultrafiltration for the concentration and simultaneous recovery of multiple pathogens in contaminated foods. Journal of Food Protection 72(12):2547-2552) (Morales-Morales, H. A., Vidal, G., Olszewski, J., Rock, C. M., Dasgupta, D., Oshima, K. H. and Smith, G. B. 2003. Appl. Environ. Microbiol. 69(7):4098-4102) (Smith, C. M. and Hill, V. R. 2009. Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl. Environ. Microbiol. 75(16):5284-5289) (Fukushima, H, Katsube, K, Hata, Y, Kishi, R, Fujiwara, S. 2007. Appl. Environ. Microbiol 73(1):92-100).
In aspects of the present invention a hollow fiber ultrafiltration membrane module (HFM) was assembled for use in a continuous cell concentration device (CCC), for example providing system 30 described herein. This HFM was constructed from twelve 0.22 μm commercial polysulfone hollow fibers available from Minntech Corporation, IDEX PEEK tubing, and Loctite Medical Device Epoxy Adhesive. Hollow fiber membranes are inserted through an assembled PEEK tubing skin and glued at the ends with medical device epoxy. HFMs, are based size-exclusion ultrafiltration. Specifically for this purpose, the HFM was designed to operate under creep flow. The HFM also uses a second water circulation line to increase the transmembrane flux through the device by maintaining a positive pressure on the outside of the ultrafiltration membrane. This ultrafiltration membrane module allows for the ultrafiltration and concentration of particles (including pathogens and food stuffs) which are larger than 0.22 micrometers in diameter. Rapid concentration of food samples can be used for the early detection and identification of contaminants and pathogens present in food samples.
A combination of rapid concentration of cells from washate or extracted food matrices will be accompanied by the addition of GFP (Green Fluorescent Protein) linked to antibodies specific for proteins displayed by pathogenic cells. The principle of detection is based on the binding of a polyclonal or monoclonal antibody to the surface of a cell, causing the cell to anchor a chromophore (GFP) to the surface. Consequently, as the cell becomes more concentrated the bound antibody will become visible as specks of light, associated with specific particles (i.e. cells). In order to achieve detection, however, the concentration of pathogenic cells would needs to be relatively high (e.g. beneficially 1,000 to 10,000 cells per ml), and therefore an indirect measurement will be needed for this approach to be practical and economical.
The method of this embodiment concentrates the cells, and then injects an amount (typically small) of monoclonal antibody with the GFP attached so that it binds to the pathogenic cells, which are in the concentrate being re-circulated through the hollow fiber membrane system (e.g. system 30 described herein). Initially, the GFP would cause a very strong response (such as would be obtained using the spectrometer from Ocean Optics for detection, when applying green fluorescent protein to it), and consequently, reliable detection of the pathogenic cells would not be possible. However, as the liquid from the sample being processed is washed through the membrane of the hollow fibers (becoming permeate), cells to which the GFP would be attached (pathogenic cells) would be retained. Consequently, as a background, due to the presence of soluble GFP-bound monoclonal antibodies, is reduced through membrane microfiltration, the signal from cells attached to the monoclonal antibodies would then become more prevalent.
Since the number of pathogenic cells is small, a measure that can be beneficially used for their detection is an increase in baseline noise. The detection is noisy, particularly at higher sensitivities. Thus, if cells are present to which the monoclonal antibody with GFP attached are concentrated, the signal would become more noisy as GFP bound to cells passes through the detector. Consequently, an online spectral analysis of this phenomenon would indicate rather random noise is becoming less random, and thereby indicate the potential presence of pathogenic cells against a background of a small amount of GFP, and a large number of non-pathogenic cells. Thus, a combination of cell concentration, measured and timed addition of amounts of monoclonal antibodies to which GFP is attached been reacted, binding of the antibody to pathogenic cells, coupled with simultaneous washing out of monoclonal antibody with GFP (in free solution) through the action of the hollow fiber membrane device may be used to enable rapid and on-line detection of pathogenic cells. Since the number of pathogenic cells, if present, would likely be small, a change in the signal-to-noise-ratio and the frequency of spikes in the noise, would be used as a first indicator of the presence of pathogenic cells.
Mathematically, this would be achieved in real time using spectral analysis, coupled with a mini-GFP detector, such as the Ocean Optics System, that is being used on the concentration device. An embodiment of the invention thus includes: 1) Use of a concentration device to rapidly increase the total number of living microbial cells; 2) measured and timed addition of small amounts of monoclonal antibody, to which GFP is attached, during times during which cell concentration is occurring; 3) simultaneous removal of soluble monoclonal antibody/GFP through the washing action of the cell concentration step (additional buffer may be added) with specific binding of the antibodies (green fluorescing or other fluorescing to pathogens) occurring at the same time; and 4) detection of cells to which the fluorescent antibody has bound through changes in baseline noise. This can give a first indicator of the potential presence of pathogenic cells, i.e. first alert. If pathogenic cells are found, the sample could then be further processed in order to give a more thorough analysis, using ELISA, or PCR-type analysis, or any other suitable analysis. In addition, the pattern of subsequent growth in culture may be measured using a laser light optical system (such as Bardot), or using Raman spectroscopy for detecting large amounts of pathogenic cells, as a second analysis.
An interface for hands-off concentration, recovery, and aseptic clean-up of a rapid system for concentrating and recovering living cells in food matrices is described. The interface enables hands-off control and monitoring of a system in which fluid is re-circulated through a membrane, which allows some of the fluid to pass. The resulting solids include microbial cells, which are concentrated, and then re-circulated until the desired concentration is attained. The system (e.g. system 30 as described herein) has various monitors including pressure, flow rate, and mass, as well as an on-line spectrophotometer for detecting fluorescent protein, which may be attached to pathogenic cells. In addition, the system includes a controller with executable software which communicates with the pumps and the valves on the instrument (e.g. system 30), is capable of detecting leaks so that fluid, which may contain pathogens, upon which the fluid is automatically diverted into caustic or bleach or another sterilization agent to kill the microorganisms. In addition, the system can have programmed into it an automated sequence for cleaning of the membranes, aseptic processing of the system so that ethanol or other fluids for killing microorganisms between samples is circulated, followed by a clean-up step, in which cleaning solutions are removed from the system so that it may be used again. This hands-off system enables the re-use of the membranes, for example for up to 10-20 times and thereby enables a more economic operation of a cell concentration and recovery system.
This embodiment involves the manner and sequence in which the concentration, recovery, wash, cleaning, and equilibration steps are carried out; and the controller (e.g. computer) with executable software or other programming, which enables operation of the system in a hands-off and safe manner, and so that detection of leaks would result in diversion of the fluid into a solution which would kill any microorganisms, whether they are pathogenic or not. This particular system embodiment also enables the cell concentration recovery instrument to be operated such that rapid concentration and recovery, as well as first alert detection of the cells, is possible.
Membrane filtration of microorganisms from extracts from food matrices are hindered by fouling. This must be overcome in order to achieve rapid recovery and concentration of microorganisms derived from extracts of food matrices, so that the concentrated microorganisms may be probed for the presence of pathogens. Reliable detection of foodborne pathogens is an integral part of food safety and that current detection of food pathogens is slow in large part due to the time required to amplify microbial cultures derived from foods, followed by selective culture to increase the numbers of pathogenic microorganisms if they are present.
A faster detection scheme is desirable, since the detection of pathogens takes place before foods are distributed to the consumers. A lab-on-a-chip is used for rapid detection using either capture by selective antibodies, and/or on-chip PCR to identify cells that may have been captured (e.g. in a retentate sample produced by system 30) with respect to whether they are pathogens. In order to achieve rapid detection, concentrated cells must be presented to the chip so that detection can proceed with reasonable accuracy. The volumes on such chips are typically on the order of 100 nL, with 1 cell/100 nL's being equivalent to 10,000 cells/mL, or 107 cells/L. Consequently, for the presence of pathogens to be detected at concentrations from one cell to 1,000 cells a volume of 100 mL's or more must be processed into a final volume of 1 mL or less. Consequently, large volumes of fluid containing potential pathogens, together with a large background of non-pathogenic microorganisms, must be amplified. The preferred approach is presented herein, in which the cells are concentrated preferably to a volume of less than 500 microliters, giving cell concentrations which may for example be 100-1,000 cells/ml. When preceded by or followed by growth of the microbial cells for a period of several hours, detectable concentrations can result.
This embodiment can use simultaneous flow on both retentate and permeate side of a hollow fiber (e.g. system 30) or equivalent membrane system. The flow of fluid through the membrane into a moving fluid on the permeate side keeps the pressure drop across the membrane at a constant or substantially constant difference, while enabling a rapid flow rate across the inner side of the membrane (retentate side) to help minimize fouling. Fouling by cake formation will be negligible when shear induced diffusion is greater than actual convection. Small particles can foul the membrane, because shear gradients affect them less (these particles are larger than smaller colloidal matter or large molecules, and are less mobile).
Fouling may be non-uniform along the length of the membrane. Since actual convection and shear diffusion are directly coupled under normal operation, the flux, and therefore tangential convection, is proportional to transmembrane pressure. As fluid flows along the length of the membrane, there will be pressure loss per unit length of the membrane. The actual convection will start at a high rate and then decrease along the axis of the membrane. At the same time, the magnitude of shear-induced diffusion remains constant. Ideally, the shear gradient will be high and the tangential connection will remain low. Under normal operations, this is not achieved because tangential convection and shear-induced diffusion are coupled.
To address these issues, this embodiment can include pumping a permeate-side liquid (e.g. water or a buffer solution) across the permeate side of the hollow fiber or other membrane such that the transmembrane pressure will remain constant along the length of the membrane. This will uncouple the forward and backward transport mechanisms, thereby allowing for increased membrane performance. A flow of buffer, which has a much lower concentration of dissolved solutes than typically occurs on the retentate side, as well as a lower viscosity, when pumped on the permeate side of the membrane, has been found to enable enhanced flux and reduced fouling for a solution containing extracted molecules and particles, as well as microorganisms. Operating according to this embodiment of the invention has been found to maintain flux and enables rapid concentration even when infant food formula (which has many fat molecules and other species that rapidly foul the membrane) is used on the retentate side of the membrane.
Various materials that are used in the production of foods, and the preparation of baked goods, may contain microbial pathogens. These foods might include flour, condiments, spices, chocolate, and related dry goods. These materials may contain microorganisms, and in order for them to be probed for possible presence of pathogens, the food material must somehow be altered so that any microorganisms present would be released into a liquid solution. Once the liquid solution is obtained, the microorganisms may be rapidly concentrated and recovered using a membrane-based concentration instrument. Foods that may harbor bacteria or pathogens include dry cereals (such as corn flakes, Cheerios, and other dry breakfast cereals), as well as numerous confectionary products such as cookies, cakes, and crackers. The embodiment herein includes the utilization of a combination of cellulases, hemicellulases, xylanases, amylases, glucosidases, proteases, lipases, nucleases, and/or esterases, formulated in a manner that they can be added to relatively small samples of food and liquefy these foods by hydrolyzing carbohydrates, proteins, and lipids into their monomeric components, thereby allowing the food material to dissolve. Once this occurs, microorganisms that are contained in these foods are released, and may be filtered and concentrated using the membrane-based device described in the present application and embodiments thereof.
The types of enzymes, and their formulation will depend on the type of food being processed into a sample for probing for the presence of pathogenic microorganisms and therefore will lead to a number of different enzyme formulations. It is also possible that additives, including small amounts of detergents, salts, buffer, and proteins (in addition to enzymes) will enhance the rate and extent of the release of microorganism that may be present in the food.
The invention, coupled with methods for rapid concentration and detection of cells (for example as described with
Consequently, combinations of enzymes are used which will break down these food matrices for the purpose of generating either soluble solutions, or suspensions of very fine particles of these food materials so that they might be rapidly filtered and ultimately concentrated using a novel rapid concentration and recovery system, e.g. system 30 described herein. The use of enzymes, including at high concentrations, is within the realm of economic viability for the processing of food samples so that presence of pathogens might be detected.
This approach may also be applied to vegetables (in which microorganisms are taken up by the vascular tissue, and therefore, can only be probed or detected if the vascular tissue is dissolved (i.e. hydrolyzed away). At the same time, it has been shown that a subset of these enzymes may be used in order to assist in the processing of extracts from chicken and that these microorganisms do not have negative effect on the microorganisms present in these food samples. At a high level, this embodiment involves a formulation of enzymes that may be added to food materials preferably at room conditions in order to quickly dissolve these materials within 30 minutes so that they may be filtered, and the liquid passed through a membrane-based filtration and concentration system (e.g. system 30 described herein). This method embodiment may have possible applications to other approaches to rapid pathogen detection in which the microorganisms are suspended would be either plated out, subjected to imaging, for ELISA assays, PCR, labeling with biomarkers, and use of microscopy. Since the food particles would represent a background which must be removed for analyses to be carried out, and since the food particles may, in fact, encapsulate or enclose the microorganisms, the use of enzyme will greatly aid the release of microorganisms so they may be tested for the presence of pathogens.
After cells are concentrated using an instrument to process food sample by microfiltration in a cross-flow operation to a small volume (e.g. using system 30 described herein), and consequently increasing the number of microbial cells to a detectable level, the cells need to be efficiently recovered in order to be effectively probed for the presence of possible pathogens in food.
This embodiment provides a method to recovery the microbial cells that have been concentrated in the retentate after microfiltration. A recovery buffer is used. It is prepared for example by dissolving a nonionic surfactant such as tween 20 in sterile deionized water at the dilution of 0.001% (v/v). Tween 20, also known generically as Polysorbate 20, is a surfactant and spreading agent that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid, and thus can be used to remove the microbial cells attached to the membrane surface. Other suitable surfactants for these purposes may also be utilized. The stability and relative non-toxicity of tween 20 allows it to be beneficially used in the recovery of microbial cells. The dosage of tween 20 is as low as 0.001% (v/v) for the reason that high concentrations of tween may produce a large amount of foam which is undesirable for the performance of microfiltration (e.g. in system 30). The dose of the recovery buffer used in the cell recovery step can be lower than the initial food sample volume, for example 10 ml of buffer per 250 ml of initial sample loading. After the microfiltration of the sample containing microbial cells, the recovery buffer is processed through the microfiltration system (e.g. system 30) under the same or different conditions until it is collected with the prior-collected cell retentate sample.
The method can be applied in the recovery/harvest of microbial cells concentrated in the retentate after microfiltration, such as described in connection with
After cells are concentrated using an instrument to process food sample by microfiltration in a cross-flow operation to a small volume, and consequently increasing the number of microbial cells to a detectable level, the cells need to be efficiently recovered in order to be effectively probed for the presence of possible pathogens in food.
Developed is a method to recovery the microbial cells that have been concentrated in the retentate after microfiltration. The recovery buffer is prepared by dissolving tween 20 in sterile deionized water at the dilution of 0.001% (v/v). Tween 20, also known generically as Polysorbate 20 is a surfactant and spreading agent that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid, and thus can be used to remove the microbial cells attached to the membrane surface. Its stability and relative non-toxicity allows it to be used in the recovery of microbial cells. The dosage of tween 20 is as low as 0.001% (v/v) for the reason that results indicate that higher concentrations of tween may produce large amount of foam which is undesirable for the performance of microfiltration. The dose of the recovery buffer is 10 ml per 250 ml initial sample loading. After the microfiltration of the sample containing microbial cells, the recovery buffer is processed by the microfiltration system under the same conditions until finished.
The method can be applied in the recovery/harvest of microbial cells concentrated in the retentate after microfiltration. This method is essential to get significant and consistent results for concentration and recovery of microbial cells (and therefore provide enough cells for probing to the presence of pathogens) from many types of food matrices.
The results in literature for pathogens recovered from water indicated recoveries varying from ˜40-95% depending on the experimental conditions used samples (Kim, H.-Y., Park, H.-J., and Ko, G. 2010. Hollow fiber ultrafiltration for the concentration and simultaneous recovery of multiple pathogens in contaminated foods. Journal of Food Protection 72(12):2547-2552) (Smith, C. M. and Hill, V. R. 2009. Dead-end hollow-fiber ultrafiltration for recovery of diverse microbes from water. Appl. Environ. Microbiol. 75(16):5284-5289) (Park H., Kim M. and Ko, G. 2010. Journal of Food Protection 73(9):1651-1657). Cells from natural flora and artificially spiked Salmonella (102-103 CFU/mL) cells present in a more complex sample matrix (chicken rinse) varying from 60-80%, depending on the experimental conditions used have been recovered cells.
The disclosure herein includes the following embodiments:
A method for processing a food sample, comprising:
The method of Embodiment 1, wherein said preparing includes enzymatically digesting the food sample.
The method of Embodiment 1 or 2, wherein the hollow fiber membrane filtration is conducted with a cross flow pattern.
The method of any preceding Embodiment, wherein the concentrate of cells includes pathogenic cells.
The method of any preceding Embodiment, also comprising assessing the concentrate of cells for the presence of pathogenic cells.
A method for preparing a liquefied extract of a food sample, comprising:
The method of Embodiment 6, wherein said processing includes filtering.
The method of Embodiment 7, wherein the filtering includes filtering with a glass fiber membrane
The method of any one of Embodiments 6 to 8, also comprising concentrating the lipid-depleted food sample in cells.
The method of Embodiment 9, wherein said concentrating comprises filtering the lipid-depleted food sample with a hollow fiber membrane filter.
The method of Embodiment 10, wherein said filtering is conducted under cross flow conditions.
A method for testing for pathogenic cells, comprising:
processing the contacted sample under conditions to remove unbound amounts of the fluorescent antibody dye, to form a dye-depleted sample; and
The method of Embodiment 12, wherein the viable cellular concentrate is prepared by a process including:
homogenizing a solid food sample under controlled shear conditions to form a homogenized food sample substantially retaining a viable cellular content of the solid food sample;
concentrating the lipid-depleted food sample in cells.
The method of Embodiment 13, wherein said concentrating comprises filtering the lipid-depleted food sample with a hollow fiber membrane filter.
The method of Embodiment 14, wherein said filtering is conducted under cross flow conditions.
The method of any of Embodiments 12 to 15, wherein said spectroscopically interrogating is conducted while the lipid-depleted food sample is flowing in a conduit.
The method of any one of Embodiments 12 to 16, wherein said contacting, processing, and spectroscopically interrogating are each conducted multiple times.
A method for concentrating and recovering cells from a food sample, comprising:
recovering the liquid concentrate of cells.
The method of Embodiment 18, wherein said recovering comprises passing an aqueous surfactant solution through the hollow fiber membrane.
The method of Embodiment 19, wherein the aqueous surfactant solution has a surfactant concentration of less than about 0.01% volume/volume.
The method of Embodiment 19 or 20, wherein the surfactant is a polysorbate.
The method of Embodiment 21, wherein the surfactant is polysorbate 20.
A method for processing a hollow fiber membrane that has contacted a liquefied food sample, comprising:
(i) passing a sodium hydroxide solution through the hollow fiber membrane;
(ii) passing an aqueous medium through the hollow fiber membrane after step (i) to rinse sodium hydroxide residues from said membrane;
(iii) passing a sterilant solution through the hollow fiber membrane after step (ii); and
(iv) passing an aqueous rinse medium through the hollow fiber membrane after step (iii).
The method of Embodiment 23, wherein the aqueous medium in step (i) and/or step (iv) is water.
The method of Embodiment 23 or 24, wherein the sterilant solution comprises an alcohol.
The method of Embodiment 25, wherein the alcohol is ethanol.
A method for concentrating and recovering cells from a food sample, comprising:
(i) passing a liquefied extract of the food sample through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate of cells in the interior of the hollow fiber membrane; and
(ii) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (i).
The method of Embodiment 27, wherein said passing step (ii) comprises passing the aqueous solution co-current to a flow of the liquefied extract through the interior of the hollow fiber membrane.
The method of Embodiment 27 or 28, also comprising preparing the liquefied extract by a process including enzymatic digestion of the food sample.
A method for assessing a solid food sample for the presence of pathogenic cells, comprising:
(i) suspending the solid food sample in an aqueous medium to form a suspended sample medium;
(ii) homogenizing the suspended sample medium under controlled shear conditions to form a homogenized food sample, said homogenizing conducted under conditions effective to substantially retain any viable pathogenic cells from the solid food sample;
(iii) enzymatically digesting the homogenized food sample to hydrolyze proteins in the homogenized food sample and form a digested food sample medium;
(iv) processing the digested food sample medium to remove lipids and form a lipid-depleted food sample extract liquid;
(v) passing the lipid-depleted food sample extract liquid through the interior of a hollow fiber membrane to form a permeate exterior of the hollow fiber membrane and a liquid concentrate containing cells in the interior of the hollow fiber membrane; and
(vi) passing an aqueous solution against an exterior surface of the hollow fiber membrane during said passing step (v); and
(vii) assessing the liquid concentrate containing cells for the presence of viable pathogenic cells.
The method of Embodiment 30, also comprising, after step (vii):
(viii) passing a defouling liquid through the hollow fiber membrane;
(ix) passing an aqueous rinse medium through the hollow fiber membrane to remove defouling solution residues from step (viii);
(x) passing a sterilant liquid through the hollow fiber membrane; and
(xi) passing an aqueous rinse medium through the hollow fiber membrane after step (x) to remove sterilant liquid residues from the hollow fiber membrane.
The method of Embodiment 31, also comprising repeating steps (i) to (vii) after step (xii).
The method of any one of Embodiments 1 to 32, wherein any one, any combination, or all of the recited steps are conducted under the control of a controller.
The method of Embodiment 33, wherein the controller is a computer.
A processing system, configured to be capable of carrying out a method according to any one of Embodiments 1 to 34.
The system of Embodiment 35, comprising respective reservoirs for any one or all recited liquids or mediums used in the method according to any one of Embodiments 1 to 34.
The system of Embodiment 35 or 36, comprising a hollow fiber membrane filtration module.
The system of Embodiment 37, comprising a first pump for powering circulation of liquid on a retentate side of said module, and a second pump for powering circulation of liquid on a permeate side of said module.
The system of Embodiment 38, comprising a detector configured to detect fluorescence in-line in a liquid flowing in a path travelling through the retentate side of said module.
The system of Embodiment 39, comprising a source of fluorescent dye fluidly coupled to said path.
The system of Embodiment 40, wherein said fluorescent dye binds specifically to a target pathogenic cell.
The system of any of Embodiments 35 to 41, comprising:
a hollow fiber membrane filtration module having a retentate side and a permeate side;
a reservoir for holding an aqueous recovery buffer for removing cells from membrane surfaces fluidly coupled to the retentate side;
a reservoir for holding a liquefied food sample fluidly coupled to the retentate side;
a reservoir for holding a defoulant liquid fluidly coupled to the retentate side; and
a reservoir for holding a sterilant fluid fluidly coupled to the retentate side.
The system of Embodiment 42, also comprising a reservoir for holding a liquid medium fluidly coupled to the permeate side of the filtration module.
The system of any of Embodiments 37 to 43, also comprising a detector for detecting pathogenic cells, said detector positioned to receive a concentrated cell sample formed on the retentate side of a hollow fiber filtration module.
The uses of the terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.
This invention was made with government support under contract number 59-1935-8-850 awarded by the United States Department of Agriculture. The government has certain rights in the invention.
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