5/18/2015. Outline. Microbes in rice mold contamination and links to rice kernel discoloration. Background. Background Grain cooling units

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1 Outline Microbes in rice mold contamination and links to rice kernel discoloration Supriya Thote Dr. Griffiths G. Atungulu Date May 21, 2015 Background Research Objectives Materials and Methods Mold growth Mold identification Results Conclusion Acknowledgements Large percentage of the Arkansas rice is dried in bin Natural/slightly heated air is pushed through the rice to promote drying Dry to storage moisture content 12 to 14% w.b. Background Natural Air Drying Background Grain cooling units Provide a method independent of weather conditions vs NAD methods Store rice for short periods between processing Decreased microbial growth Decreased pests and insects Potential drying effects through cooling Energy savings Background The weather may not allow complete drying of the rice (1) mold growth in the grain mass (2) quality deterioration including staining, milling yield reduction, and sensory and functional problems Why worry about mold? Mold may produce mycotoxins. Mycotoxins, especially aflatoxin, are known carcinogens. 1

2 Depending on drying conditions the grain quality may get very bad. 1. Heat damage? 2. Mold related? 3. If mold which type? 4. Where does the blame really start, field or dryer? Traditional Mold Identification Next Generation DNA Sequencing The catch all term used to describe modern sequencing technologies A Metagenomics Approach Allows sequencing of the entire fungal metagenome Ion torrent: Proton / PGM sequencing Research Objectives (1) Determine mold on rice at different MCs and storage temperatures over a storage period of 16 weeks (2) Determine the kinetics of mold growth (3) Identify and establish a profile of the type of mold linked to discoloration "DNA Structure+Key+Labelled.pn NoBB" by Zephyris Own work. Licensed under CC BY SA 3.0 via Wikimedia Commons Overall design Discoloration Lab experiment data Mold growth Field observed data Experimental Design Cultivar MC Temperature ( C) Storage Duration CL XL Week 0 XL Week Week Week 6 40 Week 8 Week 10 Week 12 Week 16 Week 0 = Initial control conditions 12.5 MC = Control condition where rice is dried to the recommended limit 2

3 Temperature Control Field Samples Mold growth Kitchen cooler 10 C BAEG PG&C 15 C Pilot Plant PG&C 20 C EMC Chamber 27 C Incubator 40 C Newport Burdette Dermott Discoloration samples Separated kernels Mold identification sample Newport Ion Torrent Sequencer Materials and Methods Mold Counts Rough rice 2 hybrid cultivars Temperature was monitored by placing two HOBO TM sensors in each environment MC Oven drying method Mold counts 3M Petrifilms AOAC Method for counting mold Experimental Procedure Steps for Sequencing with Ion Torrent DNA Extraction DNA quantification PCR procedure PCR product quality check PCR product clean up Library preparation Sequencing 3

4 Ion Torrent Output.fastq file Results analyzed using Galaxy White kernels Yellow kernels Total Fungi Penicillium+Apergillus White Kernels Yellow Kernels Penicillium citrinum 9412 Penicillium citrinum 9866 Aspergillus amstelodami 6049 Aspergillus amstelodami 6379 Aspergillus ruber 4295 Aspergillus fumigatus 5270 Aspergillus pseudoglaucus 3815 Aspergillus ruber 4693 Aspergillus niger 3767 Aspergillus pseudoglaucus 4073 Fusarium culmorum 3619 Nigrospora oryzae 3120 Aspergillus fumigatus 3562 Aspergillus penicillioides 2809 Fusarium graminearum 2955 Aspergillus niger 2731 Nigrospora sphaerica 2663 Aspergillus restrictus 2536 Penicillium steckii 2284 Aspergillus chevalieri 2115 Aspergillus chevalieri 1988 Penicillium griseofulvum 1903 Penicillium griseofulvum 1790 Penicillium steckii 1879 Aspergillus niveoglaucus 1593 Aspergillus niveoglaucus 1704 Penicillium herquei 1592 Aspergillus clavatus 1619 Aspergillus penicillioides 1540 Aspergillus cristatus 1337 Alternaria alternata 1474 Aspergillus caesiellus 1320 Penicillium roseopurpureum 1440 Penicillium sumatrense 1310 Fusarium equiseti 1439 Penicillium chrysogenum 1304 Aspergillus restrictus 1436 Penicillium corylophilum 1293 Penicillium corylophilum 1395 Penicillium roseopurpureum 1259 Penicillium sumatrense 1349 Aspergillus proliferans 1249 Penicillium copticola 1318 Penicillium herquei 1249 Aspergillus cristatus 1277 Leptosphaeria sclerotioides 1208 Stagonosporopsis cucurbitacearum 1270 Penicillium copticola 1164 Cladosporium cladosporioides 1260 Aspergillus glaucus 1145 Penicillium janthinellum 1250 Parastagonospora nodorum 1009 Fusarium sporotrichioides 1231 Penicillium sclerotiorum 997 Aspergillus proliferans 1187 Eurotium halophilicum 994 Leptosphaeria sclerotioides 1167 Aspergillus flavus 983 Penicillium sclerotiorum 1132 Penicillium spinulosum 970 Fusarium oxysporum 1117 Penicillium janthinellum 963 uncultured Pleosporales 1072 Neosartorya fischeri 960 Epicoccum nigrum 1068 Stagonosporopsis cucurbitacearum 882 Aspergillus glaucus 1056 Penicillium citreonigrum 879 Phoma herbarum 1017 Aspergillus wentii 841 RESULTS Experimental Design Cultivar MC Temperature ( C) Storage Duration CL XL Week 0 XL Week Week Week 6 40 Week 8 Week 10 Week 12 Week 16 Results Moisture content data Actual MCs Set Value XL 745 XL Week 0 = Initial control conditions 12.5 MC = Control condition where rice is dried to the recommended limit 4

5 Statistical Analysis Mold growth curves for different MCs XL 745 XL 753 P value is significant MC (%) XL745 XL Mold growth curves for different MCs Relationship between MC and Mold Counts Actual MCs XL 745 XL Interaction Temperature and MC Mold growth curves for different Temperatures XL745 XL753 Actual MCs XL 745 XL

6 Comparison of Field and Lab data Newport (Sept 3) Burdette (Oct 12) Dermott (Oct 17) Log10 CFU/g Week Week Week Week Week Week Lab Experiment XL745 XL753 Log10 CFU/g Week Week Week Week Week Week Week Week Week HITS Comparison of Molds Observed of White and Yellow Rice Kernels TYPES OF MOLD White kernels Yellow kernels Top ten molds Penicillium steckii Nigrospora sphaerica Fusarium graminearum Aspergillus fumigatus Fusarium culmorum Aspergillus niger Aspergillus pseudoglaucus Aspergillus ruber Aspergillus amstelodami Penicillium citrinum Yellow kernels White kernels Aspergillus wentii Aspergillus clavatus Aspergillus flavus Aspergillus glaucus Aspergillus proliferans Aspergillus cristatus Aspergillus restrictus Aspergillus penicillioides Aspergillus niveoglaucus Aspergillus chevalieri Aspergillus fumigatus Aspergillus niger Aspergillus pseudoglaucus Aspergillus ruber Aspergillus amstelodami Major Aspergillus Molds Yellow Kernels White kernels Conclusions The mold counts over 16 weeks was dependent upon the MC of the samples For low MC samples, mold counts were not significantly different For high MC samples, mold counts were significantly different The moisture content of stored rice is directly correlated with mold growth The relationship temperature and mold growth is dependent on the MC level of the rice Temperature independently had no effect on mold counts Conclusions Although the number of hits for yellow and white kernels are similar The Penicillium and Aspergillus profiles look different for yellow and white kernels 6

7 Acknowledgements Dr. Griffiths Atungulu The research group Arnelia Couch, HouMin Zhong, Shantae Wilson, Anne Okeyo, Zach Young, Deandrae Smith Dr. Burt Bluhm and group Rice Processing Program Rice Research and Promotion Board Questions 7