MARKETING AND DELIVERY OF QUALITY GRAINS AND BIOPROCESS COPRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001948
• Project No.: IDA01489
• Multistate No.: NC-_old213
• Project Start Date: Nov 14, 2013
• Project End Date: Sep 30, 2018

Project Director
Ryu, DO, .

Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803

Performing Department
School of Food Science

Non Technical Summary
Mycotoxins are toxic compounds produced by certain filamentous microfungi or molds. Mycotoxins considered to be important, including aflatoxins, ochratoxins, fumonisins, deoxynivalenol and zearalenone, are produced mainly by three fungal genera, Aspergillus, Penicillium and Fusarium. Some of these fungi may invade plants and cereal grains in the field during the growing season, as well as during postharvest handling processes such as drying and storage. Cereal grains are the most frequently affected commodity while all major crops and commodities may be contaminated with one or more mycotoxins. Moreover, the stable nature of mycotoxins often leads to contamination of products in downstream processes including finished products destined for human and animal consumption. Infection of mold and subsequent toxin production may occur naturally virtually at any conditions. However, among the various environmental factors, temperature and precipitation are considered most critical for invasion, growth, and toxin production in grain in the field. For instance, cool and wet weather during the growing season seems to be the most important factor for wheat in the development of Fusarium head blight (FHB) as well as accumulation of trichothecenes (including deoxynivalenol). The climate condition in Pacific Northwest region, or Palouse, allow wheat and other commodities to grow naturally without irrigation by seasonal precipitation. Fertile soil and lack ofsevere temperature fluctuation also contribute to the high yield of the important staple food crop. However, recent variability in climate prompted better understanding of agroecosystem to ensure grain quality and food safety since the fungal infection and mycotoxin production may not remain the same under the varying environmental conditions. It is particularly true in Pacific Northwest where natural precipitation in the past allowed agricultural commodities, e.g. wheat, to be cultivated without irrigation system. The lack of extreme fluctuation in temperatures during the growing season also contributed to the higher yield of crops in comparison to those from other regions. However, increasing daily and seasonal fluctuation of temperature in addition to the variability in precipitation due to climate change would affect mycotoxin production. Variability in climate may cause changes in soil system, including organic and inorganic matters, where plants are rooted to grow. As many plant pathogenic fungi and mycotoxin producers, e.g. Fusarium species, are soilborne and their population is largely affected by the climate and soil system, it would be plausible to investigate the impact of climate changes on the soil system that will determine toxigenic fungal population and their potential in growth and survival of the organism as well as mycotoxin production. Therefore, Pacific Northwest region may provide a unique model system to study impact of climate variability on agroecosystem particularly in fungal population in the soil and quality of grains by assessing potential for mycotoxin production in wheat and other agricultural commodities.

Animal Health Component

0%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5010	1150	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5010 - Food;

Field Of Science
1150 - Toxicology;

Keywords

food safety

mycotoxins

climate variability

pacific northwest

Goals / Objectives
To characterize quality and safety attributes of cereals, oilseeds, and their processed products, and to develop related measurement systems.

Project Methods
Objective 1: Soil samples from various parts of Pacific Northwest will be collected annually. These samples will be transported to the PD's lab for the analysis of fungal population. The total mold count will be performed by appropriate serial dilution of soil samples in sterile peptone water followed by plating on dichloran rose bengal chloramphenicol agar. In addition, the number of Fusarium species will be counted by using Czapek iprodione agar and further identified to the species level. Objective 2: Available HPLC and LC-MS methods for detection and quantification of mycotoxins in cereal grains and other commodities will be evaluated employ advances in analytical instruments and techniques. Evaluation of rapid methods (immunochemical) for detecting and quantifying mycotoxins in a variety of foods will also be performed. Several foods will be analyzed using these rapid methods and the results compared to those obtained using the HPLC and LC-MS method. Objective 3: Sampling -Samples will be purchased from retail stores and bulk suppliers across the Pacific Northwest region though the geographic location will be concentrated in the two states of Idaho and Washington. In the case of wheat, winter, spring and Durum wheat samples will be collected from the annual wheat quality survey conducted through the Wheat Quality Laboratory at Washington State University or Idaho Wheat Commission and Washington Grain Commission. Analysis of mycotoxins - Quantification of mycotoxins will be carried out by the method verified in Objective 2. Two major toxins of interest in this study would be DON and OTA though other mycotoxins of concern for food safety will be included. The procedures for wheat prior to the optimization and verification are demonstrated as below. Objective 4: Quantitative risk assessment for the mycotoxins will be carried out with the steps described below. In general, methodology will be followed to estimate the impact to human health from mycotoxins in foods obtained in the Pacific Northwest. Risk assessment is the process of estimating the magnitude and the probability of a harmful effect to individuals or populations from certain agents or activities. Objective 5: Strategies to reduce exposure from mycotoxin in the food supply will be developed depending on the results of risk assessment in Objective 4. The strategies can be considered as a risk management plan based on the framework proposed by FAO and WHO (FAO/WHO, 1995) rather than investigation of measures to detoxify contaminated foods. Risk management is aimed to achieve practical solutions, e.g. guidelines regarding maximum residue levels or procedural guidelines to prevent the problem. In this project, however, regulatory measures will not be sought. Instead, the strategies will be developed with emphasis on postharvest practices including storage, processing, and surveillance.

Progress 11/14/13 to 09/30/18

Outputs
Target Audience:Farmers in the area where wheat and soil samples were collected for this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate and one undergraduate students were trained for microscopic and molecular method to identify Fusarium species. How have the results been disseminated to communities of interest?Publication in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Deoxynivalenol (DON), known as "vomitoxin", is a mycotoxin which can contaminate wheat and causes economic losses for growers who are unable to sell such low quality grain. Wheat grains become contaminated with DON after suffering an infection by toxigenic Fusarium species which causes a disease known as Fusarium Head Blight (FHB). In the Midwestern region of the United States where the majority of wheat is grown the period of maximum rainfall occurs in the spring. Humid conditions during flowering increase the risk of fungal infection and subsequent DON contamination. In the Inland Pacific Northwest (INW) region of the United States the period of maximum rainfall occurs during the winter. The typically dry conditions during flowering in this region makes the occurrence of FHB rare. However, the climate conditions of the INW are forecasted to change in the next century, and it remains unknown if the INW will continue to produce high quality grains free of mycotoxins such as DON. To address the risk of DON in wheat grown in the INW under climate change, a two-year soil survey quantifying the amount of Fusarium culmorum, a potentially toxigenic fungus capable of causing Fusarium head Blight (FHB) has been completed by sampling from agricultural fields across the Inland Pacific Northwest (INW). A total of 20,765 soil Fusaria have been collected from 9 fields across a precipitation gradient and 2,293 have been putatively identified as F. culmorum using morphological criteria. We conducted the survey with 9 fields across a precipitation gradient in the INW, and quantified the amount of F. culmorum present each season. Collecting these isolates allows for a better understanding of the amount and type of mycotoxins these fungi are capable of producing. We utilized a novel approach to model soil populations of F. culmorum and relate them to climate conditions. We treat the fungal soil population measurements in a similar manner to flood records. Hydrologists report 100-year floods so that builders can decide how tall to construct levies within a flood plain. In a similar manner, we are able to report the percentage of a grower's field which exceeds a given amount of inoculum present. Growers can be notified of the percentage of their field which exceeds a threshold amount, and use that information to make managerial decisions. The difference in isolation frequency of F. culmorum based on sampled field, quadrat, and sampling iteration has been analyzed using Bayesian inference with a varying intercepts approach. This allows for the quantification of uncertainty in isolation frequency of F. culmorum based on the different experimental clusters within the soil survey. Isolation frequency varied the most be the field being sampled from, followed by the sampling quadrat and sampling iteration. Isolation frequency of F. culmorum was lower in the higher regions of sampled fields. Differences in annual climate summaries for each field, such as precipitation and growing-degree days, were investigated by testing both linear and polynomial equations with either single or multiple annual climate variables. Annual climate summaries are too coarse of a resolution, and the remaining variability due to differences in sampling quadrat and iteration make the predictions too variable for use by growers. In addition, isolation frequency alone is insufficient in characterizing the magnitude of F. culmorum present in an agricultural field. The results are currently in review with the journal Phytopathology. The soil dilution factor used during plating allowed for a population density estimate as propagules per gram soil (PPG). This additional information enhances the isolation frequency data and allows for the quantification of the population density. Since differences in isolation frequency varied the most be sample field, the population densities from all sampled quadrats within a field during a sampling iteration were pooled together. An exponential distribution was used to model the population density which properly captures the variability in sampling quadrats which is not possible when only using isolation frequency. A predictive model for PPG values based on local climate has been developed. The model utilizes past precipitation and potential evapotranspiration data to compute an atmospheric water balance. The atmospheric water balance was calculated using either the prior 35 years or prior 90 days reference to the sampling date to create a historical term and a seasonal term. F. culmorum was isolated more frequently from cool and wet climate regimes which is captured by the historical atmospheric water balance term. F. culmorum was also isolated more frequently during the winter and spring compared to the summer and fall which is captured by the seasonal atmospheric water balance term. This approach also accounts for the issues with multicollinearity of weather variables which was demonstrated when evaluating model equations with isolation frequency data. The model demonstrated good predictive accuracy and was used along with 10 global climate models for two different climate change scenarios. RCP 4.5 is a low climate change scenario that assumes greenhouse gas emissions stabilize by mid-century and fall afterwards. RCP 8.5 is a high climate change scenario that assumes greenhouse gas emissions continue through the end of the 21st century. Population densities of F. culmorum are forecasted to remain constant across all 9 sampled fields for both climate change scenarios. The balancing effect of hotter and drier summers with warmer and wetter winters suggests that this potentially toxigenic plant pathogen will remain in the INW under the tested climate change scenarios. Treating a fungal soil population like flood data also allows for more informative comparisons to be made with climate conditions. We were able to relate seasonal changes in F. culmorum soil populations to seasonal weather patterns of moisture coming into the soil system through rainfall and moisture leaving the soil system through potential evapotranspiration. Creating models which quantify the relationship between seasonal climate and F. culmorum soil populations allows for the ability to generate forecasts for the next century using data from climate change models for the INW as inputs. Soil populations of F. culmorum in the INW are likely to persist through the next century under climate change and remain a source of inoculum capable of causing FHB. There is a balancing effect as forecasted fall conditions are likely to decrease F. culmorum soil populations while the forecasted winters are likely to be more favorable for increases in F. culmorum soil populations. Future work is needed to determine if the INW climate will become more favorable for the development of FHB under climate change and related to the amount of inoculum present.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Utility of a multilevel modelling approach to investigate differences in isolation frequency of Fusarium culmorum in agricultural soil across the Inland Pacific Northwest. Phytopathology.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Farmers in the area where wheat and soil samples were collected for this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Attended Washington Association for Food Protection Annual Meeting, Chelan, WA, September 21-22, 2017. How have the results been disseminated to communities of interest?The results have been disseminated through professional meetings. What do you plan to do during the next reporting period to accomplish the goals?Finish collecting seasonal soil samples and isolate possible toxigenic strains including Fusarium culmorum. Theseisolates will be identified by their morphological characteristics and confirmed by molecular methods. In addition,isolates will be tested for theproduction of deoxynivalenol.

Impacts
What was accomplished under these goals? Objective 1. Optimization and verification of methods for detecting and quantifying fungal population in soil There have been 6 completed iterations of seasonal soil sampling, starting from June 2016 through September 2017. There remain 2 further sampling dates, December 2017 and March 2018, to complete the proposed 2 years of continual sampling. The goal is to both enumerate the amount of potentially toxigenic F. culmorum present in fields across the Inland Pacific Northwest, and to collect a culture collection for future experiments concerning toxigenic potential. The amount of F. culmorum isolates collected, out of total Fusaria analyzed for each season are as follows: June 2016 (167/2598), September 2016 (230/2494), December 2016 (303/2799), March 2017 (320/2670), June 2017 (305/2683), and September 2017 (158/2307). The sampling workflow has proven robust throughout each seasonal iteration of soil sampling. Plating onto peptone-pentachloronitrobenzene agar and transferring colonies displaying unrestricted growth, indicative of Fusarium species, onto homemade potato dextrose agar (PDA) is staggered over 3 weeks with 3 fields analyzed each week. Red-pigmented colonies on PDA are transferred onto carnation leaf agar (CLA) and identified to species level using microscopic features including macroconidia chape and phialide structure. F. culmorum isolates typically sporulate heavily on CLA which allows them to be easily differentiated prior to microscopic identification. The workload can be completed in its entirety by a single graduate student, with time remaining to teach labs for the department. In addition to the collection of approximately 1300 F. culmorum isolates, the corresponding location and soil dilution for each isolate has allowed for a soil population estimate expressed as propagules / g soil (ppg). The sampling region for each field consists of 9 1 acre quadrats, and the distribution of ppg counts across all quadrats for each field is used as the response variable during statistical modelling when using site-specific climate variables as predictors. Objective 2. Development and validation of analytical method to detect mycotoxins using HPLC and LC-MS Development and validation of analytical methods to detect trichothecene mycotoxins using HPLC and LC-MS is in progress. Determination of mycotoxin concentration in both grains and extracts from fungal cultures is currently being assessed using an HPLC method with UV detection. The current limit of detection (LOD) and limit of quantification (LOQ) for deoxynivalenol (DON) using this method are currently 50 and 100 ng/mL in standard solutions injected directly into an Agilent 1260 infinity HPLC system. LOD and LOQ in sampled grains are currently being determined, as modifications to the extraction protocol can change those values for sampled grains. Robust and high-throughput workflows for analyzing toxin production for the large number of collected F. culmorum isolates are being developed. Approximately 400 F. culmorum isolates have already been transferred to yeast extract sucrose agar to determine toxigenic potential. A method using agar plug extracts to quantify the amount of DON produced has been developed. Some F. culmorum isolates are also likely to be nivalenol (NIV) producers, so the incorporation of NIV standards and the development of a multi-mycotoxin method using the same HPLC-UV parameters are in progress. Objective 3. Determination of the mycotoxin concentrations and the presence of toxigenic fungi in soil and foods The presence of F. culmorum in agricultural soils within 9 separate fields across the Inland Pacific Northwest has been determined over 6 seasons. The experimental design incorporated 3 fields in each of 3 agroecological classes: annual crop, transitional, and grain-fallow. Classes differ primarily by annual precipitation, with fields in the annual cropping class receiving enough rainfall to plant annually, while crops grown within the grain-fallow class are grown every other year so that enough moisture has been accumulated. F. culmorum populations were cosmopolitan in the annual crop and transitional class, but very residual to nonexistent in the grain-fallow class. While soil populations of F. culmorum are highly variable within and across fields, it appears that soil populations are maximal in the spring and minimal in the fall. Since F. culmorum is most typically found in cool and wet climates, it follows that the soil populations would follow similar annual trends in temperature and moisture. Fields which were fallow over the winter witnessed greater F. culmorum soil population increases compared to fields with winter wheat present.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Robinson, A. L. and Dojin Ryu. 2017. Predicting seasonal soil populations of Fusarium culmorum using site-specific climate data. Washington Association for Food Protection Annual Meeting, Chelan, WA, September 20, 2017.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Farmers in the area where wheat and soil samples were collected for this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One full-time graduate student and one half-time undergraduate student were trained to analyze isolate and identify fungal species from the soil samples collected in the area. How have the results been disseminated to communities of interest?We published original research papers as well as a review paper in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?The large amount of F. culmorum isolates will be analyzed for toxin production using an agar-plug extraction method coupled with HPLC analysis with UV detection. Reproductive fitness of F. culmorum isolates will be used as a response variable in controlled laboratory environments to better understand data collected from the field. While population levels were found to be correlated with climatic variables, it remains unknown how F. culmorum populations change over the seasons and if that change is related to the local climate from which they are isolated. Seasonal sampling has already begun, and the time-series analysis for F. culmorum populations at 9 different locations will yield a better understanding of how climate is able to influence the inoculum level of this toxigenic plant pathogen. Data gathered on seasonal population changes will also provide reference for determining if climate change in the Pacific Northwest could cause and increase in the incidence of Fusarium head blight in the region.

Impacts
What was accomplished under these goals? Soil-borne populations of Fusarium culmorum, a plant pathogen capable of producing deoxynivalenol during infection of grains, were elucidated for 9 fields across three different agro-ecological classes within the Pacific Northwest. By sampling from multiple quadrats within each field, it was discovered that F. culmorum populations show a very high degree of spatial aggregation. The inoculum level of F. culmorum, represented as propagules per gram of soil, was found to significantly decrease in agro-ecological classes that commonly experience warmer winters and less annual precipitation. The reproductive fitness of all F. culmorum isolates was tested in vitro, and will provide a baseline comparison for future experiments under controlled laboratory conditions.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee, H.J. and D. Ryu. 2015. Advances in mycotoxin research: Public health perspectives. J. Food Sci. doi: 10.1111/1750-3841.13156.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Al-Taher, F., J. Zweigenbaum, J. Cappozzo, H.J. Lee, L.S. Jackson*, and D. Ryu*. 2016. Detection and quantitation of mycotoxins in infant cereals in the U.S. market by LC-MS/MS using a stable isotope dilution assay. Food Control doi:10.1016/j.foodcont.2016.07.027.

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

Outputs
Target Audience:Farmers and scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One full-time and one half-time graduate student was trained to conduct this research project. How have the results been disseminated to communities of interest?Original research papers as well as a review paper were published in peer-reviewed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?It is planned to complete Objective 1 and 2 during the next reporting period. Objective 3 will also be pursued by coordinating effort to collect samples from the field.

Impacts
What was accomplished under these goals? To address above goal, following specific objectives are proposed in this study. 1) Optimization and verification of methods for detecting and quantifying fungal population in soil to obtain reliable data in varying climate and environmental conditions. 2) Deployment of analytical methods to detect mycotoxins for efficient determination of mycotoxins in foods. 3) Determination of the mycotoxin concentrations and the presence of toxigenic fungi in soil and foods with a survey using analytical methods established in Objective 2 and a mycological analysis. The objective 1, optimization and verification of methods for detecting and quantifying fungal population in soil, is in progress. Tested different semi-selective media for their ability to efficiently isolate Fusarium species present in agricultural soils. Media tested were different peptone-pentochloronitrobenzene agar (PPA) and Czapek-Dox agar with iprodione and dichloran (CZID) formulations. Soil dilutions on CZID plates were overrun with non-Fusarium genera and no Fusarium species could be isolated. Soil dilutions on PPA also grew non-Fusarium genera, but Fusarium colonies were easily differentiated by their unrestricted growth. Colonies suspected to be Fusarium species were transferred to synthetic nutrient agar (SNA) to sporulate. Single spores were separated using micromanipulation under a compound microscope and used to inoculate carnation leaf agar (CLA) and homemade potato dextrose agar (PDA) for species level identification. Colony color on PDA was used to confirm species identity. Multiple isolates of deoxynivalenol (DON) producing fungi were identified including F. graminearum and F. culmorum. Soil dilution plating using PPA will be used to isolate, identify, and quantify toxigenic Fusarium species present in agricultural soils. Quantification method of the population of fungal strains in other genera using polymerase-chain reaction (PCR) based technique is in progress. The objective 2, development and validation of analytical method to detect mycotoxins using HPLC and LC-MS is in progress. In order to determine levels of mycotoxins contamination of cereal grains (especially, winter wheat) grown in sampled fields, and to assess mycotoxins production in grain cultures, a HPLC method is being developed and validated. Inmmunoaffinity column (IAC) technique was used for sample preparation and Agilent 1260 infinity HPLC system with UVD for DON or FLD for ochratoxin A (OTA) was used for mycotoxin determination. The limit of detection (LOD) was the concentration of mycotoxins that gave a peak height 3 times the average height of the equipment noise (i.e. the signal-to-noise (S/N) ratio was 3:1). The limit of quantification (LOQ) was the concentration of mycotoxins that gave an S/N ratio of 10:1. The LOD and LOQ values for OTA are 0.032 and 0.10 ng/g, respectively. The LOD and LOQ values for DON are 50 and 100 ng/g, respectively. For the objective 3, determination of the mycotoxin concentrations and the presence of toxigenic fungi in soil and foods, a collaboration between multiple winter wheat growers was formed to seasonally sample soil and collect harvested grains. The test plots were selected based on the amount of rainfall and agricultural conditions. Three agroecological zones of the Pacific Northwest (annual crop, grain-fallow transition, and grain-fallow) displaying different annual precipitation regimes are represented by three growers each. Locations of sampling sites summarized in Table 1. Sampling sites were chosen based on grower cooperation and representative location within different agroecological zones. Each site has a unique local climate differing in mean annual precipitation and snowfall, as well as temperature regime. Grain samples will be analyzed for fungal population and mycotoxin concentration upon harvest. Soil samples from each testing plots will be collected and analyzed for the fungal population. Table 1: Summary of sampling locations Zone* Site Mean Max Temp Range (°C) Mean Max Temp (°C) Mean Min Temp Range (°C) Mean Min Temp (°C) Precipitation Range (mm) Mean Annual Precipitation (mm) Snowfall Range (mm) Mean Annual Snowfall (mm) Field Location GPS coordinates 1 1 13.6 - 19.1 19.1 -0.3 - 5.1 5.1 599 - 605 599 1257 - 2515 1257 Genessee, ID 46.49356 -116.97205 2 14.5 2.6 605 1265 Moscow, ID 46.68661 -116.89479 3 13.6 -0.3 949 2515 Troy, ID 46.67649 -116.78328 2 4 14.2 - 15.7 14.2 2.3 - 2.6 2.6 500 - 540 540 719 - 726 719 Pullman, WA 46.76238 -117.05108 5 14.2 2.6 540 719 Albion, WA 46.76055 -117.17069 6 15.7 2.3 500 726 Colfax, ID 46.87574 -117.45165 3 7 16.9-18.3 16.9 2.8 - 6.4 2.8 289 - 359 359 61 - 422 422 Lacroose, ID 46.77597 -117.72181 8 18.3 6.4 289 61 Hay, ID 46.71484 -117.80174 9 18.3 6.4 289 61 Hay, ID 46.70976 -117.79111 * 1 = annual crop; 2 = annual crop-fallow transition; 3 = grain-fallow

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee, H.J. and D. Ryu. 2015. Significance of ochratoxin A in breakfast cereals from the United States. J. Agric. Food Chem. doi: 10.1021/jf505674v.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Kuruc, J.A., J. Hegstad, H.J. Lee, K. Simons, D. Ryu, and C. Wolf-Hall. 2015. Infestation and quantification of ochratoxigenic fungi in barley and wheat naturally contaminated with ochratoxin A. J. Food Prot. 78(7):1350-1356.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Robinson, A.L., H.J. Lee, and D. Ryu. 2015. Polyvinylpolypyrrolidone reduces cross-reactions between antibodies and phenolic compounds in an enzyme-linked immunosorbent assay for the detection of ochratoxin A. Food Chem.
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Ryu, D. and C. Wolf-Hall. 2015. Yeast and Molds. In: Compendium of Microbiological Examination of Foods, 5th Edition. American Public Health Association, Washington, DC.

Progress 11/14/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Threearticles were published in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? Research work will be focused on address specific objective 1. optimization and merification of methods for detecting and quantifying fungal pupulation in soil.

Impacts
What was accomplished under these goals? Due to unforeseen problems with a graduate student who returned to her home country, reportable data for Objective 1 (Optimization and verification of methods for detecting and quantifying fungal population in soil) has not been generated during this reporting period. Instead, another graduate student has been hired and being trained. Normal research activities are expected to resume in January 2015. Objective 2, Development of analytical method to detect mycotoxins, has been conducted with focus on detection of ochratoxin (OTA). A LC-MS/MS method was developed to shorten the time of analysis by extracting samples with ultrasonication and eliminating purification steps. As a continued effort to improve analytical methods, an HPLC method using stir bar sorptive extraction was developed for the detection of OTA in beer. This method also eliminates purification steps and need of expensive immunoaffinity columns used for the most types of sample preparation in chromatography, i.e. HPLC and LC-MS/MS. Using a stir bar sorptive extraction method in detection of OTA was reported for the first time.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nguyen, K.T.N. and D. Ryu. 2014. Ultrasonic extraction with liquid chromatography-tandem mass spectrometry for the analysis of ochratoxin A in processed cereal products. J. AOAC Int. 97(5):1384-1386.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Lee, H.J., A.D. Meldrum, N. Rivera, and D. Ryu. 2014. Cross-reactivity of antibodies with phenolic compounds in pistachios during quantification of ochratoxin A by commercial ELISA kits. J. Food Prot. 77(10):1754-1759.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nguyen, K.T.N. and D. Ryu. 2014. Development of a stir bar sorptive extraction method for analysis of ochratoxin A in beer. J. AOAC Int. 97(4):1092-1096.