Source: AGRICULTURAL RESEARCH SERVICE submitted to
OPTICAL DETECTION OF FOOD SAFETY AND FOOD DEFENSE HAZARDS
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0421113
Grant No.
(N/A)
Project No.
6040-42000-041-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 1, 2011
Project End Date
Mar 31, 2016
Grant Year
(N/A)
Project Director
PARK B
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
ATHENS,GA 30613
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
33%
Applied
33%
Developmental
34%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7113260202068%
7123270202010%
7117410202012%
7123599202010%
Goals / Objectives
1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents in poultry and swine with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. The funds are to be used to enhance current research conducted by the project to develop and validate methods and instruments to detect and identify microbiological and toxicological hazards, and biothreat agents throughout food processing systems. Expansion of this research within the current project. objectives is considered a high priority.
Project Methods
Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry and swine products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research.

Progress 04/01/11 to 03/31/16

Outputs
Progress Report Objectives (from AD-416): 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents in poultry and swine with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. The funds are to be used to enhance current research conducted by the project to develop and validate methods and instruments to detect and identify microbiological and toxicological hazards, and biothreat agents throughout food processing systems. Expansion of this research within the current project. objectives is considered a high priority. Approach (from AD-416): Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry and swine products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Hyperspectral imaging has the potential for early and rapid detection of pathogenic foodborne bacteria through identification of their unique spectral and spatial information, particularly when combined with microscopy. However, multiple lighting sources are available for use with a hyperspectral microscope system. ARS researchers in Athens, Georgia, compared multiple lighting sources and selected the optimal lighting system for research purposes which was found to be a tungsten halogen light source. For use in a commercial/processing setting, where a rapid data processing is needed (reduced datasets), the most significant wavelengths from a metal halide light were also identified. For tungsten halogen lights, the full range of 89 wavelengths should be used, whereas the metal halide light can be reduced to about 10 wavelengths. Both lighting options resulted in high classification accuracies for five Salmonella serotypes at a cellular level resulting in a highly sensitive detection method requiring only a few cells. Detection of Salmonella Typhimurium with surface enhanced Raman spectroscopy (SERS) and DNA aptamer. Salmonella Typhimurium (ST) is one of the major causes of gastrointestinal infections in human and animals. Conventional detection methods are time consuming and inefficient for outbreak intervention. Therefore, biosensors that can detect Salmonella rapidly and with both high sensitivity and specificity are required for modern food industry. ARS researchers in Athens, Georgia, in collaboration with researchers at University of Georgia, have developed rapid detection methods based on SERS. This method uses low cost and stable DNA aptamers for Salmonella binding and a material, known as a nanosubstrate, with the capacity to handle 40 samples simultaneously. With different surface modification techniques, surface enhanced Raman spectroscopy (SERS) has the ability to detect ST with or without an external Raman labels. The methods can facilitate further development of versatile biosensors for the detection of various pathogens and toxins in foods. Salmonellae are the leading causes of foodborne outbreaks and animal infections in the U.S. Timely detection and characterization of Salmonella serotypes are important for disease treatment and outbreak investigation. ARS researchers in Athens, Georgia, developed methods, based on surface enhanced Raman spectroscopy (SERS) and immunomagnetic separation (IMS), for label-free detection and characterization of Salmonella spp. Salmonella cells are captured (isolated) from a food matrix with a material known as polyclonal antibody functionalized paramagnetic beads. Then SERS �fingerprints� of the captured bacteria can be compared with a spectral library of various pathogens and serotypes. The method can be used to detect and serotype Salmonella within 24 hours with high sensitivity and between-species specificity. Specificity between serotypes is still challenging due to highly similar spectral features of closely related Salmonella serotypes. Accomplishments 01 Development of a neutralizing solution for use by USDA-FSIS in regulatory monitoring. Previously, it was demonstrated that the current FSIS protocol for Salmonella testing of whole chicken carcasses may potentially lead to carry-over of intervention solutions, used to reduce pathogens in poultry processing, into the collection broth tested by FSIS inspectors. This carry-over could result in under estimating Salmonella levels in poultry processing operations. ARS scientists in Athens, Georgia, developed a modified collection broth that is capable of neutralizing a wide range of sanitizers, resulting in a more accurate reporting of Salmonella in poultry processing. FSIS implemented this modified collection broth on July 1, 2016 and FSIS field inspectors are currently utilizing it in the collection of samples for Salmonella testing. 02 Table egg candling lights for official grading. The official graders in the Agricultural Marketing Service (AMS) needed a new system for inspecting (candling) table eggs. ARS scientists from Athens, Georgia, designed, developed, and prototyped a high-intensity light emitting diode (LED) white light for candling eggs. Both a portable battery- powered model and a stationary model were created. A material transfer agreement was used to complete the design and transfer the technology to a U.S. commercial partner. The company has now completed the second prototype revision and has just started selling the lights to the public. AMS has plans to purchase several hundred of these lights to replace all of their existing obsolete candling lights. 03 Label-free biosensing of Salmonella enterica serovars at the single- cell level. The food industry is constantly facing challenges in detecting various toxic pathogens that can contaminate food. The detection of Salmonella enterica serovars from food sources is critical for the prevention and control of the outbreak of salmonellosis and emerging nanotechnologies have greatly facilitated the development of label-free biosensors for detection. A technique, known as Atomic force microscopy (AFM), has been used to study the reaction mechanisms between proteins and aptamers at the molecular level. Another technique, known as surface plasmon resonance (SPR), has been used in rapid detection of bacteria. Scientists in Athens, Georgia, used both AFM and SPR to study the complex reactions between aptamers and outer membrane proteins (OMPs) on the surface of Salmonella Typhimurium (ST) and two DNA aptamers, which have specific binding affinities to the OMPs, were used for label-free detections of ST. At the single-molecule level, high-resolution AFM topography and recognition images distinguished the OMPs on the bacteria surface, which is the first time the location of individual outer membrane protein have been rapidly determined on Salmonella surface with these methods. 04 Protocol development of fluorescence in-situ hybridization (FISH) probes with hyperspectral microscopic imaging to target the main pathogens associated with poultry and poultry processing environments. FISH probes, while beneficial for rapid detection of pathogens know to contaminate poultry products, are sometimes hard to detect in mixtures where multiple probes are needed. Furthermore, there is a need to increase the intensity and lifespan of the FISH fluorescent signals by optimizing the protocols used for analysis which include formamide concentration, antifade reagents, washing, and hybridization time. A wide-field hyperspectral microscope and spectral confocal laser scanning microscope were both used to demonstrate the concept of separating the combined responses of numerous probes to ultimately identify specific individual pathogens. This is known as spectrally- unmixing image analysis and is used to separate overlapping signals compounded by signal interaction or crosstalk.

Impacts
(N/A)

Publications

  • Gamble, G.R., Berrang, M.E., Buhr, R.J., Hinton Jr, A., Bourassa, D.V., Johnston, J.J., Ingram, K.D., Adams, E.S., Feldner, P.W. 2016. Effect of simulated sanitizer carryover on recovery of salmonella from broiler carcass rinsates. Journal of Food Protection. 79(5):710-714.
  • Park, B., Yoon, S.C. 2015. Real-time hyperspectral imaging for food safety applications. Book Chapter. Chapter 13: pp 305-329.
  • Park, B. 2015. AOTF hyperspectral microscope imaging for foodborne bacteria detection. In: Park, B., Lu, R., editors. Hyperspectral Imaging Technology in Food and Agriculture. New York, NY: Springer Science and Business Media. p. 359-390.
  • Yoon, S.C., Lawrence, K.C., Park, B. 2015. Automatic counting and classification of bacterial colonies using hyperspectral imaging. Food and Bioprocess Technology. 8:2047-2065.
  • Yoon, S.C., Shin, T., Lawrence, K.C., Heitschmidt, G.W., Park, B., Gamble, G.R. 2015. Hyperspectral imaging using RGB color for foodborne pathogen detection. Journal of Electronic Imaging. 24(4):043008.


Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents in poultry and swine with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. The funds are to be used to enhance current research conducted by the project to develop and validate methods and instruments to detect and identify microbiological and toxicological hazards, and biothreat agents throughout food processing systems. Expansion of this research within the current project. objectives is considered a high priority. Approach (from AD-416): Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry and swine products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Discrimination of poultry pathogens based on laser-induced breakdown spectroscopy (LIBS). Four common pathogen genotypes found in processed poultry (Listeria, Salmonella, Pseudomonas, and Staphylococcus) can be discriminated by LIBS when cultured in liquid media and isolated by repeated steps of rinsing and centrifugation. The source of the rinse solution (e.g. deionized water, phosphate-buffered saline) was determined to cause significant changes in the proportions of minerals bound to the bacteria due to pH and mineral exchange effects, thus affecting the repeatability of the LIBS measurement and discrimination. In order to mitigate these effects, a rinse solution has been developed that is buffered to minimize pH effects, contains no mineral constituents that will compete for binding sites on the bacteria cell wall, and is non- complexing. Use of this rinse solution enabled the four bacteria genera to be reliably and repeatedly discriminated by LIBS upon multiple replicates. The resulting stabilization of the LIBS spectra is expected to allow for the more sensitive discrimination of groups of bacteria that are inherently very similar, such as various Salmonella serotypes. The ability to discriminate between serotypes will potentially lead to a more rapid assessment of serotypes responsible for foodborne outbreaks. Hairline egg crack detection with improved sixty egg modified-pressure imaging system. Official graders with the USDA Agricultural Marketing Service (AMS) are having difficulty in accurately grading eggs with fresh hairline cracks and have asked for a system to help in the task. Scientists at ARS Athens, Georgia have developed a system that uses a short quick negative pressure change (vacuum gradient) along with a digitial camera system, associated hardware and software to control and analyze the data. Modifications and updates to the system include a higher resolution camera, full and independent LED light-intensity adjustment for each of the sixty eggs, improved software analysis, upgraded computer, automated opening and preloaded closing of the enclosure, and a stand-alone cart. These updates consolidated and simplified the operation of the system without affecting the accuracy of the system. Detection of Salmonella Typhimurium (ST) with atomic force microscopy (AFM) and surface Plasmon resonance (SPR). Foodborne pathogen Salmonella enterica is one of the major causes of gastrointestinal infections in human and animals. The conventional detection methods are time consuming and not effective enough under emergency circumstances to immediately control outbreaks. Therefore, biosensors that can detect Salmonella rapidly with high sensitivity and specificity are needed. ARS researchers in Athens, Georgia, in collaboration with researchers at University of Georgia, have developed label-free detection methods with DNA aptamers. This method can improve detection efficiency by increasing sensitivity at the single cell level with low-cost and stable binding reagents. AFM has the ability to detect single ST bacterium in samples and SPR can detect ST in the bulk solution with surface modification using nanotechnology. The methods can facilitate further development of label-free biosensor for the detection of various pathogens and toxins in foods. Hyperspectral microscopic image analysis for foodborne pathogenic bacteria identification. Hyperspectral imaging is an imaging technique that combines aspects of conventional imaging with spectral information. However, the exploration with spatial information including physical and geometric observations of size, orientation, shape, color, and texture has been limited for hyperspectral imaging research in food safety and quality. ARS researchers in Athens, Georgia developed image processing techniques for segmenting and extracting shape and texture features, known as morphological features, from five different foodborne bacteria. Using extracted morphological features, bacteria can be differentiated and classified. However, due to the limitation of morphological differentiation within serotypes, hyperspectral image data containing both spatial and spectral information could improve classification accuracy to identify and classify foodborne pathogenic bacteria at the serotype level. Development of a hyperspectral image reconstruction method from RGB color: A spectral recovery technique, converting color images with three RGB channels of broad spectra to hyperspectral images with hundreds of narrow spectra was developed for safety inspection of poultry carcasses. ARS researchers in Athens, Georgia, developed a multispectral image classification method using a spectral wavelength ratio algorithm (565 nm / 517 nm) that was originally used on hyperspectral images that were used to detect fecal materials on poultry carcasses. The study demonstrated the feasibility of an advanced statistical method for spectral image recovery from a simpler and less expensive color or multispectral imaging systems for fecal detection. Accomplishments 01 Classification of Salmonella enterica serotypes with visible/near- infrared hyperspectral microscope imaging (HMI). Optical method with HMI has the potential for rapid detection and classification for foodborne bacteria by analyzing spectral signatures of bacterial species and serotypes. ARS researchers in Athens, Georgia, developed HMI methods to classify five Salmonella serotypes. To improve performance of processing time and classification accuracy, optimizing selective spectral bands and testing different classification algorithms have been carried out. HMI method with selected classification algorithms can be employed for rapid detection of bacteria and further applied for presumptive screening foodborne pathogen in foods with minimum sample preparation.

Impacts
(N/A)

Publications

  • Park, B., Seo, Y., Yoon, S.C., Hinton Jr, A., Windham, W.R., Lawrence, K.C. 2015. Hyperspectral microscope imaging methods to classify gram-positive and gram-negative foodborne pathogenic bacteria. Transactions of the ASABE. 58(1):5-16.
  • Wu, X., Huang, Y., Park, B., Tripp, R., Zhao, Y. 2015. Differentiation and classification of bacteria using vancomycin functionalized silver nanorods array based surface-enhanced raman spectroscopy an chemometric analysis. Talanta. 139:96-103.
  • Eady, M.B., Park, B., Choi, S. 2015. Rapid and early detection of salmonella serotypes with hyperspectral microscope and multivariate data analysis. Journal of Food Protection. 78(4):668-674.
  • Wang, B., Zhichao, L., Park, B., Kwon, Y., Xu, B. 2014. Surface conformations of anti-ricin aptamer and its affinity to ricin determined by atomic force microscopy and surface plasmon resonance. Physical Chemistry Chemical Physics. 17:307-14. doi: 10.1039/c4cp03190c.


Progress 10/01/13 to 09/30/14

Outputs
Progress Report Objectives (from AD-416): 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents in poultry and swine with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. The funds are to be used to enhance current research conducted by the project to develop and validate methods and instruments to detect and identify microbiological and toxicological hazards, and biothreat agents throughout food processing systems. Expansion of this research within the current project. objectives is considered a high priority. Approach (from AD-416): Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry and swine products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Discrimination of poultry pathogens based on laser induced breakdown spectroscopy (LIBS). LIBS is an atomic spectroscopy technique useful for discrimination of materials based on elemental composition. LIBS was used to discriminate between four pathogen genotypes (Listeria, Salmonella, Pseudomonas, Staphylococcus) cultured in Tryptic Soy Broth using three different water sources. Each of the 12 cultures was discriminated with 100% accuracy. This provides a basis for continuing studies using LIBS to discriminate between serotypes of Salmonella, Escherichia coli and other pathogens, using LIBS to identify colonies directly on agar plates, and using LIBS as a diagnostic test for repeatability of other optical detection methods for foodborne pathogens. Repeatability improvements of hyperspectral imaging (HSI) in the differentiation of Non-O157 Shiga Toxin E. coli (STEC) colonies. Reliable discrimination of non-O157 STECs with HSI was shown to be hampered by spectral shifts due to unforeseen chemical differences among replicate measurements of colonies grown on rainbow agar. Studies on the effects of the mineral content of the water used in bacteria growth media showed that small differences in water pH and mineral content may lead to significant effects on the chemical makeup of the resultant bacterial cultures. Discussions are underway with Food Safety Inspection Service to design experiments to determine the impacts of water source, agar media chemical differences, and agar age on the ability to repeatably discriminate between non-O157 STECs with rainbow agar. Rapid identification of Salmonella serotypes with stereo and hyperspectral microscope imaging (HMI) methods. A HMI method, in conjunction with stereo microscope imaging, was developed to reduce Salmonella detection time to within 8 hours including incubation process. The early and rapid detection, with high throughput capabilities developed in this study, can be used to make the HMI method a prime candidate for implementation in the food industry. After bacterial colonies were picked from agar plates with a stereo microscope, HMI spectral data from five Salmonella serotypes were analyzed. The HMI and analytical methods can be used for quality assurance for in-house product safety assessments. Classification of gram-positive and gram-negative bacteria with HMI Analysis. Although traditional culture-based methods are still the most reliable and accurate, these methods are limited for practical use due to being time-consuming, cumbersome, and having sensitivity issues. Therefore, more sensitive, accurate and rapid pathogen detection methods are needed to prevent foodborne outbreaks. HMI was developed to enhance the presumptive-positive screening method with microscopic imaging of live bacterial cells from microcolonies grown on agar plate. The HMI, with machine learning algorithms, was able to classify serotypes of Salmonella and Staphylococcus with their spectral signatures from the cells. Detection of food toxin with atomic force microscopy (AFM) and surface plasmon resonance (SPR). Surface conformations and specific binding affinity of an anti-ricin aptamer was studied by AFM and SPR. The effect of a surface modification of a gold nanosubstrate on the aptamer affinity was estimated. AFM topography images showed high-enough resolution to distinguish different aptamer conformations. The specific binding site on the aptamer molecule was clearly located by the AFM recognition images. The results showed that single-molecule measurements can obtain different reaction parameters from bulk solution measurements. In AFM single- molecule measurements, the various conformations of aptamers immobilized on the gold surface, determined the availability of each specific binding site to the ricin molecules. The bulk solution measurements with SPR averaged the signals from specific and non-specific interactions which can be used for further development of a biosensor for pathogen and toxin detection in food matrices. Development of a HSI reconstruction method from red-green-blue (RGB) color. Numerous hyperspectral imaging models have been developed for detecting pathogens on agar plates. However, hyperspectral imaging is an expensive method and a simpler, less expensive color vision method might be suitable in some situations. A color vision technique was developed for detecting pathogens on agar plates with hyperspectral image classification models originally created from broadband hyperspectral image spectrometer data. A study demonstrated the feasibility of a RGB color-based prediction of pathogens growing on agar plates with hyperspectral classification models based on the visible spectral range from 400 and 700 nm. A multivariate linear regression method was used to estimate hyperspectral curves from the original three RGB bands. The performance of the RGB-to-hyperspectral estimation was evaluated by a simulation with the hyperspectral data in terms of its estimation accuracy and pathogen detection accuracy. Development of a Universal Hyperspectral Imaging System. A hyperspectral machine vision sensor was developed as a common imaging platform for high- speed, online multispectral imaging applications such as poultry carcass inspection and foreign material detection during food production. All necessary hardware and software components for real-time image spectroscopy were incorporated into an imaging enclosure. A benchmark test was performed to assess the speed and resolution of the imaging system. Accomplishments 01 Sixty egg hairline crack detection system. Human graders are having difficulty keeping up with high-speed packaging facilities and detecting fresh hairline cracks in table eggs. An imaging-based system to detect these cracks was developed to inspect sixty eggs per batch and includes numerous other design improvements from previous versions. The system is very accurate, has enhanced white LEDs to better candle the eggs, has been further automated, and is integrated with control and database software to manage the entire system. 02 Label-free surface enhanced Raman spectroscopy (SERS) detection of Salmonella Typhimurium on aptamer modified silver nanorod substrates. Current molecular-based methods face some constraints, particularly when detecting pathogens from real food samples, due to the need for sample pre-enrichment and presence of inhibitors in food matrices. Therefore, the development of rapid, alternative methods is still needed. ARS researchers at Athens, Georgia and their collaborators at the University of Georgia, developed SERS with nanostructured metal materials for amplifying the Raman scattering signals. Using the SERS spectra, various bacterial species or even strains can be differentiated. An aptamer-based, label-free SERS technique was able to detect Salmonella Typhimurium. The anti-S. Typhimurium aptamer, attached to the highly uniform silver nanorod array substrates, was able to identify S. Typhimurium from other control bacteria (Escherichia coli, Enterobacter, and S. Enteritidis). With a SERS method and classification models, contaminated products can be identified quickly and help reduce illness from foodborn pathogens.

Impacts
(N/A)

Publications

  • Wang, B., Park, B., Kwon, Y., Xu, B. 2014. Determining the elastic properties of aptamer-ricin single molecule multiple pathways. Applied Physics Letters. 104, 193702; doi:10.10631/1.4876603.
  • Park, B., Windham, W.R., Ladely, S.R., Gurram, P., Kwon, H., Yoon, S.C., Lawrence, K.C., Narang, N., Cray Jr, W.C. 2014. Classification of non-O157 shiga toxin-producing escherichia coli(STEC) serotypes with hyperspectral microscope imaging. Transactions of the ASABE. 57(3).
  • Appell, M.D., Jackson, M.A. 2014. Preface. In: Park, B; Appell, M., Eds. ACS Symposium #1143: Advances in Applied Nanotechnology for Agriculture. Washington, DC: American Chemical Society. p. ix.
  • Wu, X., Chen, J., Park, B., Huang, Y., Zhao, Y. 2013. The use of silver nanorod array based surface enhanced Raman scattering sensor for food safety applications. ACS Book Series 1143: Advances in Applied Nanotechnology for Agriculture. 5:85-108. DOI:10.1021/bk-2013-1143.ch005.
  • Sundaram, J., Park, B., Kwon, Y. 2013. Nanocolloid substrate for surface enhanced Raman scattering sensor for biological applications. ACS Book Series 1143: Advances in Applied Nanotechnology for Agriculture. 2:21-41. DOI: 10.102/bk-2013-1143.ch002.
  • Chen, G., Park, B., Xu, B. 2013. Food toxin detection with atomic force microscope. ACS Book Series 1143: Advances in Applied Nanotechnology for Agriculture. 7:125-143. DOI:10.1021/bk-2013-1143-ch007.
  • Seo, Y., Yoon, S.C., Park, B., Hinton Jr, A., Windham, W.R., Lawrence, K.C. 2015. Development of hyperspectral imaging technique for salmonella enteritidis and typhimurium on agar plates. Applied Engineering in Agriculture. 30(3):495-506. (doi:10.13031/aea.30.10435).


Progress 10/01/12 to 09/30/13

Outputs
Progress Report Objectives (from AD-416): 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. Approach (from AD-416): Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Differentiating Non-0157 STEC Colonies from Background Microflora in Ground Beef by Hyperspectral Imaging (HSI). Detection and recovery of non- O157 Shiga-toxin producing Escherichia coli (STEC) in high background ground beef is difficult due to the competing microflora and the lack of a suitable selective and differential agar media. A HSI technique was developed to detect and differentiate each of the �big-six� non-O157 serogroups from background microflora. The results provide the location of the suspect target STEC colony on the agar plate such that an analyst can pick the colony for further conformational tests. Relationship of Eggshell Microstructure to Chemical Composition. Low eggshell strength leads to development of cracks, allowing contamination of eggs by pathogenic bacteria and loss to the industry. A Laser Induced Breakdown Spectroscopy (LIBS) method was developed to correlate the chemical composition of the eggshell with microstructural features that enhance the propensity of eggshells to become damaged. Eggshell hardness was related to the ratio of calcium components detected by the LIBS system. Classification of Salmonella serotypes with Laser Induced Breakdown Spectroscopy (LIBS). A LIBS method was developed to identify five Salmonella serotypes based on the atomic spectral signatures emitted from the laser induced plasma. Colony films of the different serotypes were created on stainless steel and the LIBS spectra of the resulting films were subsequently treated via a hierarchical clustering method which successfully identified each serotype. Classification of Foodborne Pathogens with Hyperspectral Microscope Imaging (HMI). A HMI method was developed to classify foodborne pathogens from the optical properties of bacterial cells in micro colony samples using their spectral signatures. The HMI method was able to differentiate five serotypes of Salmonella and Staphylococcus with a support vector machine classification algorithm. The results can be used for a rapid presumptive screening method for foodborne pathogens. Salmonella Typhimurium (ST) Identification with DNA aptamers using Surface Enhanced Raman Scattering (SERS). A special substrate was fabricated and tested with SERS to produce a label-free, specific detection of ST from poultry carcass rinses. The substrate uses a silver nanorod array that have special binding reagents, known as aptamers, to capture the ST cells which are then detected by the SERS system. Further optimization for binding conditions and fabrication of device is needed for improving the lower limit of detection. Nanobiosensor for Ricin Detection with Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR). Nucleic acid aptamers was used as binding reagents for the label-free detection of biomolecules. The specific interactions between ricin and its corresponding aptamer were measured with AFM and SPR spectrometry. The specific binding site on the aptamer molecule was clearly recognized by the AFM recognition images. Aptamers can be integrated into various detection platforms and provide versatile applications for fast detection and analysis related to biosafety and biosecurity. Accomplishments 01 Hyperspectral imaging for detection of foreign materials. Hyperspectral machine vision sensor to detect foreign materials in specialty crops and to remove them by a robot was jointly developed under a cooperative agreement. Foreign material detection methods and computer algorithms, including their source codes, were developed and transferred to industry. Also, the application software and robot control software were developed and transferred. Preliminary tests of product moving on a conveyor were successful. The technology was further developed with a more universal platform which can be used in real-time poultry inspection applications and other material detection applications in the future. 02 Methods and device for early and rapid detection of foodborne pathogens on agar media. A hyperspectral microscope imaging (HMI) technique was developed for early and rapid detection of Salmonella serotypes ( Enteritidis, Heidelberg, Infantis, Kentucky, and Typhimurium) at incubation times of 8, 10, 12, and 24 hrs on brilliant green sulfa (BGS) agar plates. Pearson correlation values indicate virtually identical spectral patterns for varying incubation times and advanced multivariate statistical analysis techniques show good separation at all incubation times. Additionally, a soft independent modeling of class analogy classification method also produced very high classification values. Thus, Salmonella serotypes can be identified by applying these techniques to HMI data, regardless of incubation time, which can speed up detection time by reducing the time needed for incubation. 03 Automated colony counting with hyperspectral imaging. Computer algorithms were developed to automatically detect and count bacterial colonies growing on agar plates. The software uses image segmentation algorithms to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (commonly known as the �big six�), Salmonella Enteritidis and Typhimurium, and background microflora on agar plates. An algorithm to rapidly count cells from microscope images that fluoresce was also developed.

Impacts
(N/A)

Publications

  • Yoon, S.C., Windham, W.R., Ladely, S.R., Heitschmidt, G.W., Lawrence, K.C., Park, B., Narang, N., Cray Jr, W.C. 2013. Hyperspectral imaging for differentiating colonies of non-O157 shiga-toxin producing echerichia coli (STEC) serogroups on spread plates of pure cultures. Journal of Near Infrared Spectroscopy. 21(2):81-95.
  • Yoon, S.C., Windham, W.R., Ladely, S.R., Heitschmidt, G.W., Lawrence, K.C., Park, B., Narang, N., Cray Jr, W.C. 2013. Differentiation of big-six non- O157 shiga-toxin producing escherichia coli (STEC) on spread plates of mixed cultures using hyperspectral imaging. Journal of Food Measurement & Characterization. 7(2):47-59.
  • Park, B., Yoon, S.C., Lee, S., Sundaram, J., Windham, W.R., Hinton Jr, A., Lawrence, K.C. 2012. Acousto-optic tunable filter hyperspectral microscope imaging for identifying foodborne pathogens. Transactions of the ASABE. 55(5):1997-2006.
  • Sundaram, J., Park, B., Hinton Jr, A., Lawrence, K.C., Kwon, Y. 2013. Detection and differentiation of salmonella serotypes using surface enhanced Raman scattering (SERS) technique. Journal of Food Measurement & Characterization. 7(1):1-12.
  • Sundaram, J., Park, B., Yongkuk, K. 2013. Stable silver/biopolymer hybrid plasmonic nanostructures for high performance surface enhanced raman scattering (SERS). Journal of Nanoscience and Nanotechnology. 13(8):5382- 5390.
  • Sundaram, J., Park, B., Kwon, Y., Lawrence, K.C. 2013. Surface enhaced raman scattering (SERS) with biopolymer encapsulated silver nanosubstrates for rapid detection of foodborne pathogens. International Journal of Food Microbiology. DOI:10.1016/j.ijfoodmicro.2013.05.013.
  • Windham, W.R., Yoon, S.C., Ladely, S.R., Haley, J.A., Lawrence, K.C., Park, B., Narang, N., Cray Jr, W.C. 2013. Hyperspectral imaging of shiga toxin- producing escherichia coli serogroups O26, O45, O103, O111, O121, and O145 on Rainbow Agar. Journal of Food Protection. 76(7):1129-1136.


Progress 10/01/11 to 09/30/12

Outputs
Progress Report Objectives (from AD-416): 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. Approach (from AD-416): Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Improved automatic lighting for egg crack detection: Previously developed egg hairline crack detection system suffered from lower intensity candling lights that were only manually controlled in a few zones. The lighting system was totally upgraded to include higher-intensity, white light-emitting diodes (LEDs) and a custom-designed and developed programmable control board that used the existing camera system as part of feedback control. The result was automatic light control that allowed uniform illumination for all eggs in the crack detection system. Classification of Shiga Toxin producing Escherichia Coli (STEC) other than non-escherichia coli. O157 with hyperspectral microscope imaging: Non-O157 STEC serotypes (O26, O45, O103, O111, O121 and O145) have been recently recognized in an outbreak to cause human illness due to their toxicity. A hyperspectral microscope imaging method was developed to identify these pathogenic bacteria from the optical properties (spectral signatures) of the bacterial cells in micro-colony samples. The acousto- optic tunable filters-based hyperspectral microscope imaging method was able to identify STEC serotypes with several different classification algorithms. Salmonella detection with Surface Enhanced Raman Spectroscopy (SERS) with silver particle encapsulated biopolymer nanosubstrate: A nano- colloidal substrate developed from silver particle encapsulated biopolymer was tested for Salmonella detection with SERS. To confirm uniformity and reproducibility of the nanosubstrates, optical properties of the substrate plasma were measured with ultra-violet, visible spectroscopy, and hyperspectral microscopy. The biopolymer nanosubstrate has a longer shelf-life and generated stable SERS signals from Salmonella and enabled the differentiation of Salmonella serotypes Typhimurium and Enteritidis. Nanobiosensor for food toxin detection with DNA aptamers: Aptamers are single-stranded oligonucleotides generated from in vitro selection and have high affinities to their targets, which can be small molecules, proteins, virus, or cells. The protocol for detecting a single ricin protein based on these DNA aptamers was developed with a modified atomic- force microscope and the binding affinity of the aptamer to ricin was identified. The affinity was slightly higher than that with an antibody, which means aptamers could replace antibody as sensing agents. The methods will be used in further nanobiosensor development. Accomplishments 01 Differentiating non-O157 STEC serogroups in pure culture and from ground beef on agar media. A hyperspectral imaging technique was developed to detect and differentiate the Shiga Toxin-Producing Escherichia coli (STE serogroups other than E. coli O157 on Rainbow agar plates. These non-O15 STEC (O26, O111, O45, O121, O103, and O145) are known as the �big six�. number of different classification techniques were developed to distinguish these E. coli colonies on spread plates of pure and mixed cultures. The classification models were tested on inoculated ground bee to measure the performance of the imaging technique. The average sensitivity and specificity was 95% and 92%, respectively. Test results obtained from these studies showed the potential of the imaging techniqu for rapid screening of STEC positive colonies to ensure that the correct colonies were selected for further confirmatory techniques.

Impacts
(N/A)

Publications

  • Sundaram, J., Park, B., Yoon, S.C., Hinton Jr, A., Windham, W.R., Lawrence, K.C. 2012. Classification and structural analysis of live and dead salmonella cells using fourier transform infrared (FT-IR) spectroscopy and principle component analysis (PCA). Journal of Agricultural and Food Chemistry. DOI: 10.1021/jf204081g.
  • Wang, B., Guo, C., Zhang, M., Park, B., Xu, B. 2012. High-resolution single-molecule recognition imaging of the molecular details of ricin- aptamer interaction. Journal of Physical Chemistry. B116(17):5316-22.
  • Windham, W.R., Poole, G.H., Park, B., Heitschmidt, G.W., Albano, J.P., Gottwald, T.R., Lawrence, K.C., Hawkins, S.A. 2011. Rapid screening of huanglongbing-infected citrus leaves by near infrared reflectance spectroscopy. Transactions of the ASABE. 54(6):2253-2258.
  • Windham, W.R., Yoon, S.C., Ladley, S.R., Heitschmidt, G.W., Lawrence, K.C., Park, B., Narang, N., William, C.C. 2012. The effect of regions of interest and spectral pre-processing on the detection of non-O157 shiga- toxin producing escherichia coli serogroups on agar media by hyperspectral imaging. Near Infrared Spectroscopy Journal. 20(4):10. DOI:1255/jnirs.1004, 2012.
  • Yoon, S.C., Park, B., Lawrence, K.C., Windham, W.R., Heitschmidt, G.W. 2011. Line-scan hyperspectral imaging system for real-time inspection of poultry carcasses with fecal material and ingesta. Computers and Electronics in Agriculture. 79:159-168.
  • Yoon, S.C., Lawrence, K.C., Jones, D.R., Heitschmidt, G.W., Park, B. 2012. Motion compensated image processing and optimal parameters for egg crack detection using modified pressure. Sensing and Instrumentation for Food Quality and Safety. 5(5):172-184.
  • Wang, B., Guo, C., Chen, G., Park, B., Xu, B. 2012. Following aptamer- ricin specific binding by single molecule recognition and force spectroscopy measurements. ChemComm. 48:1644-1646.
  • Lawrence, K.C., Jones, D.R., Yoon, S.C., Heitschmidt, G.W., Anderson, K.E. 2011. Improved hairline crack detector and poor shell-quality eggs. Applied Engineering in Agriculture. 28(1):153-158.
  • Bowker, B.C., Fahrenholz, T.M., Sarnoski, P.J., Solomon, M.B. 2012. Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing . Journal of Food Science. 77(6):C594-C602.


Progress 10/01/10 to 09/30/11

Outputs
Progress Report Objectives (from AD-416) 1. Develop methods and instruments to identify food safety hazards throughout various stages of poultry and egg production and processing. 2. Detect and characterize foodborne pathogens, toxins, and bacterial threat agents with rapid optical methods. 3. Develop and evaluate detection methods for foodborne pathogens and toxins with nanotechnology. Approach (from AD-416) Various optical (imaging) methods will be used for detecting intentional and unintentional contaminants and bacterial pathogens of food products. A real-time in-line hyperspectral imaging system will be used to rapidly detect diseased and contaminated broiler carcasses in processing plants. A monochromatic imaging system will be used for detecting cracks in table and hatching eggs. Hyperspectral microscopic imaging system, Raman imaging instrument and Fourier transform infrared spectrometer will be used to detect biofilms and foreign materials on the surfaces of food processing equipment. Visible/near-infrared hyperspectral imaging systems will be used to rapidly detect and characterize foodborne pathogens associated with poultry products and bacterial threat agents. Nanotechnology will be used for detection of foodborne pathogens and toxins. Collaboration with ARS Environmental Microbial and Food Safety Laboratory, BARC, FSIS, AMS, and the University of Georgia Nano Science and Engineering Center will be used to enhance the research. Screening Non-O157 Shiga Toxin-producing E. coli Serotypes on Agar Media by Hyperspectral Imaging. Six serotypes of enterohemorrhagic Escherichia coli (O26, O111, O45, O121, O103, and O145) other than serotype O157:H7 have been identified as a serious threat to food safety. Screening these pathogens grown on agar plates is not effective because fermentable carbohydrates sources for discrimination are not available. A hyperspectral imaging technique was developed to discriminate each of the 6 serotypes on rainbow agar plates. The method will aid the analyst by reducing the number of colonies picked for further conformational testing. Acousto-Optic Tunable Filter Hyperspectral Microscope for Foodborne Pathogenic Bacteria Detection. A hyperspectral microscope imaging system could be an effective tool to understand the optical properties of foodborne pathogenic bacteria at the cell level. A hyperspectral microscope imaging method was developed for image acquisition with a dark field illumination light source. Salmonella, E.coli and biofilms were tested and their spectral characteristics were analyzed to identify spectral characteristics of different bacteria. Further development of this technique could be used for in-situ foodborne pathogen detection. Rapid Detection of Salmonella using Surface Enhanced Raman Spectroscopy (SERS) with Silver Nanorods. SERS with silver nanosubstrate deposited on thin titanium coated glass slides can improve sensitivity for pathogen detection. Salmonella typhimurium were tested in the forms of live and dead cells. Spectra from the cells on the silver nanosubstrate were acquired with a confocal Raman microscope. Spectral signatures of the dead and live cells were compared to differentiate them. The spectral signatures from each cell type indicated structural changes in bacteria cell components. Thus, the SERS method could differentiate between live and dead bacteria cells. Accomplishments 01 Shiga toxin-producing Escherichia coli (STEC) Detection on Agar Plates. Culture methods as a presumptive positive screening tool of non-O157 Shi toxin-producing Escherichia coli (STEC) are time-consuming and not effective in testing large amount of food samples. A hyperspectral imagi method has been under development to discriminate each of 6 non-O157 Shi toxin-producing Escherichia coli (STEC) serotypes (O26, O111, O45, O121, O103, and O145) on agar plates. Spectral libraries of pure pathogens cultures on agar plates were built and classification models were developed. Tests with Rainbow agar plates showed the potential of the imaging technique for rapid screening of food samples contaminated by Shiga toxin-producing Escherichia coli (STEC) organisms.

Impacts
(N/A)

Publications

  • Berrang, M.E., Windham, W.R., Meinersmann, R.J. 2011. Campylobacter, Salmonella and Escherichia coli on broiler carcasses subject to a high pH scald and low pH postpick chlorine dip. Poultry Science. 90(4):896-900.
  • Park, B., Yoon, S.C., Windham, W.R., Lawrence, K.C., Heitschmidt, G.W., Kim, M.S., Chao, K. 2011. Line-scan Hyperspectral Imaging for Real-time on- line Poultry Fecal Detection. Sensing and Instrumentation for Food Quality and Safety. 5:25-32.
  • Ko, S., Kim, J., Park, B., Cho, Y. 2011. Nanotechnology in Food Quality and Safety Evaluation Systems. In: Cho, Y.J., Kang, S., editors. Emerging Technologies for Food Quality and Food Safety Inspection. CRC Press. p. 376.
  • Park, B., Lawrence, K.C., Windham, W.R. 2010. Imaging: Hyperspectral Contaminant Detection. In: Heldman, D.R., Moraru, C.I., editors. Encyclopedia of Agricultural, Food, and Biological Engineering. 2nd edition. Boca Raton, FL:Taylor & Francis. p. 854-857.
  • Park, B., Yoon, S.C., Windham, W.R., Lawrence, K.C. 2011. In-plant test of in-line multispectral imaging system for fecal detection during poultry processing. Applied Engineering in Agriculture. 27(4):623-630.
  • Samuel, D., Park, B., Sohn, M., Wicker, L. 2011. Visible/Near-Infrared Spectroscopy to Predict Pale Broiler Breast Meat by Measuring Water Holding Capacity. Poultry Science. 90:914-921.