Progress 10/01/11 to 11/30/15
Outputs
Progress Report Objectives (from AD-416): 1. Detection and control of fungi in post-harvest, stored corn. 2. Evaluate commercially available plant compounds for their in vitro activity against agriculturally important bacteria and fungi. Approach (from AD-416): This project investigates new technologies to detect Aspergillus (A.) flavus and Fusarium (F.) verticillioides growth on corn and novel methods to inhibit their growth and mycotoxin production. Secondary metabolic volatiles unique for the growth of toxigenic strains of the fungi will be identified and shared with our stakeholder who will develop electronic, �real-time� sensors to detect the growth of these fungi. Safe, commercially available plant antifungal volatiles will be tested for their in vitro activity against these fungi. Promising compounds will be further tested to determine their ability to prevent fungal growth in small-scale, bench models of stored corn. These same compounds in the liquid form will be studied for their in vitro activity against the bacterium, Leuconostoc (L.) mesenteroides (a problem in sugar cane factories) and the fungi Penicillium (P.) digitatum and P. italicum which cause post harvest citrus rot. ARS collaborators will use this data to develop novel technologies to prevent the growth of these fungi in raw sugar cane juice and stored oranges. This serves as a final report for project 6435-41000-107-00D. Progress was made on all proposed research in the new project plan. Research was carried out on the identification of unique volatiles (compounds with ability to evaporate) produced by toxigenic strains of Fusarium (F.) verticllioides as compared to non-toxigenic strains and corn controls, when grown separately on sterile and non-sterile corn. Initial results from separate multiple runs using one strain each of toxigenic and one atoxigenic F. verticllioides indicate that unique volatiles are produced by the former. This initial study was followed with studies using non- sterile, wet corn as the growth substrate for the same fungal isolates. In order to identify safe, inexpensive, commercially available antimicrobials, the following were used in in vitro assays: (1) volatile compounds to protect stored corn from the growth of mycotoxin producing fungi, (2) non-volatile compounds to add to food grade protective films, and (3) water soluble compounds to prevent Leuconsotoc mesenteroides (a bacteria) growth. This initial step in our program continued during FY 2013. During early FY 2013, research was completed on the ability of intermittently pumped, volatilized trans-2-hexenal (T-2-H), a chemical compound, to prevent bacterial and fungal growth on non-sterile wet corn in a bench-top model of stored corn. Data showed that intermittent pumping (30 min every 2 hr) prevented bacterial and fungal growth from time zero. Pumping (30 min every 12 hr) prevented all fungal growth from time zero but only killed bacteria sometime between 24 and 72 hrs after pumping commenced. Also during FY 2013, research continued on the ability of blue light (peak: 470 nm) to prevent Aspergillus (A.) flavus fungal growth inoculated onto non-sterile corn. The initial study, using a single layer of corn in a Petri dish, clearly showed that growth of inoculated A. flavus as well as the naturally-occurring fungi was prevented by blue light treatment. Next we began studies of 8 oz of corn being turned in a glass cylinder between two banks of blue light to determine whether the light can prevent fungal growth on multiple layers of corn. At this time we found that the lights had lost 50% of their energy (mW) and that this level was insufficient to prevent growth. Accomplishments 01 Detection of unique volatiles produced by Fusarium (F.) verticillioides could be used to remove contaminated corn from storage and thereby reduce the threat of Fusarium species mycotoxins from food and feed. Under certain conditions of moisture and temperature, the naturally present mycotoxin fungi (e.g., Aspergillus (A.) and Fusarium spp.) can grow on stored corn and render it unsafe. After successfully completing a study of A. flavus produced volatiles unique to toxigenic strains, ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, in FY 2013, began a multi- year study to identify novel volatiles produced by toxigenic strains of F. verticllioides. Studies progressed with sterile corn as a growth substrate. After several toxigenic and atoxigenic isolates were studied and the unique volatiles for the toxigenic isolates determined, similar experiments were performed with non-sterile corn. Our stakeholder, Sensor Development Corporation, used data from a previously completed project to develop an electronic sensor for volatiles unique to aflatoxin-producing A. flavus. A similar sensor could be constructed using data obtained from this study with F. verticllioides upon completion. 02 Safe, effective, and inexpensive plant volatiles prevent microbial, especially toxigenic fungal, growth and toxin production in a stored corn model. In FY 2011-12, ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, completed a study that showed volatilized trans-2-hexenal (T-2-H) was highly effective against Aspergillus flavus both in vitro and when applied to wet, sterile corn in small models of stored corn. 03 In vitro research on the antifungal and antibacterial properties of safe, commercially available plant compounds to determine which are suitable candidates to protect post-harvest food and feed. Work was completed by ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, on the antifungal properties of three such compounds (limonene, carvacrol, bisabolol, dragosantol, pinene, linalool) against the non-germinated (NG) and germinating (G) conidia (equivalent of seed in plants) of Fusarium (F.) verticillioides, Aspergillus (A.) niger, Penicillium (P.) digitatum, and P. italicum). The NG and G conidia of F. verticillioides and P. italicum were resistant to dragosantol and bisabolol at = 20 �M. The NG conidia of A. niger were significantly susceptible to carvacrol (15 �M) and linalool (20 �M) while the G conidia were significantly susceptible at 5 �M for both compounds. Pinene and limonene cause significant mortality to NG and G conidia of F. verticillioides beginning at 5 �M. Bioassays defined the antimicrobial activity of eugenol and vanillin against a panel of bacteria and toxigenic fungi having agricultural importance. 04 Visible blue light (470 nm) is antimicrobial with potential use in food protection. Ultraviolet light (wavelength 10-400 nm) has antimicrobial properties but is a Group 1 carcinogen which causes cancer in humans. However, visible blue light (450-475 nm) is not carcinogenic and has antibacterial properties. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, performed initial studies that showed this wavelength prevented fungal growth, both naturally occurring and inoculated Aspergillus (A.) flavus, on single layers of non-sterile, wet corn. Next, initial studies were performed using 8 oz of similar corn being turned in a glass cylinder with two blue light arrays in parallel on the outside. Lack of antifungal activity lead to a check of the light (LED) energy (mW) level which showed the output was about 50% lower than specifications.
Impacts
(N/A)
Publications
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1. Detection and control of fungi in post-harvest, stored corn. 2. Evaluate commercially available plant compounds for their in vitro activity against agriculturally important bacteria and fungi. Approach (from AD-416): This project investigates new technologies to detect Aspergillus (A.) flavus and Fusarium (F.) verticillioides growth on corn and novel methods to inhibit their growth and mycotoxin production. Secondary metabolic volatiles unique for the growth of toxigenic strains of the fungi will be identified and shared with our stakeholder who will develop electronic, �real-time� sensors to detect the growth of these fungi. Safe, commercially available plant antifungal volatiles will be tested for their in vitro activity against these fungi. Promising compounds will be further tested to determine their ability to prevent fungal growth in small-scale, bench models of stored corn. These same compounds in the liquid form will be studied for their in vitro activity against the bacterium, Leuconostoc (L.) mesenteroides (a problem in sugar cane factories) and the fungi Penicillium (P.) digitatum and P. italicum which cause post harvest citrus rot. ARS collaborators will use this data to develop novel technologies to prevent the growth of these fungi in raw sugar cane juice and stored oranges. Progress was made on all planned research. Research was completed on the identification of unique secondary metabolic volatiles produced by toxigenic strains of Aspergillus (A.) flavus, as compared to non- toxigenic strains and corn controls, when grown separately on sterile and non-sterile corn. During FY 2011, results were reported to our stakeholder, Sensor Development Corporation, which used this data to develop a real-time sensor which is undergoing Beta testing by a large grain company in a silo. Later in 2012, work will commence on the study of unique secondary metabolic volatiles produced by the fungus Fusarium (F.) verticillioides on sterile corn. Investigations continue on the antifungal and antibacterial properties of safe, inexpensive plant compounds, especially those with volatile properties, and specifically those having significant activity at low concentrations (= 20 �M) against fungi that produce toxins on corn. These fungi include A. flavus, F. verticillioides. Other fungi targeted include F. graminearum (produces mycotoxins on wheat), and Penicillium (P.) digitatum and P. italicum, both of which cause post-harvest rot on citrus. Significant viability loss against these fungi was observed with concentrations as low as 1 �M with the compounds citral, carvacrol, and linalool (natural compounds that are safe and readily available from commercial sources). Significant bacterial (e.g., Leuconostoc mesenteroides, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli) viability loss was obtained at concentrations as low as 0.4 �M of these compounds. Data from these in vitro tests will determine which compounds will be used in future testing in (1) the small scale corn silo model; (2) prevention of Leuconostoc mesenteroides in raw sugar cane juice; and (3) safe, food grade edible films, respectively. In FY 2012, research using a small model of stored corn showed that volatilized compound trans-2-hexenal (produced by soybeans when infected with A. flavus) intermittently pumped into non-sterile, wet, whole corn inoculated with A. flavus prevented the growth of this fungus and subsequent aflatoxin production. Results obtained from the study of the antifungal properties of blue light (470 nm). Experiments showed that blue light inhibits the growth of fungi naturally present on whole corn as well as A. flavus inoculated onto the corn kernels. Accomplishments 01 Detection of unique volatiles produced by toxigenic Aspergillus (A.) flavus could be used to protect stored corn from fungal growth. Aspergillus flavus, commonly found on corn, produce aflatoxins (the most potent natural liver cancer-causing compounds known). When conditions a conducive, this fungus readily grows on stored corn and renders it unsaf ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, in FY 2012 completed a multi- year effort to identify unique volatiles produced by toxigenic A. flavus isolates on both sterile and non-sterile cracked corn. The first study identified the volatiles produced under optimal conditions when this fungus is grown alone on sterile corn. Later research determined the volatile profile of the A. flavus isolates when grown on non-sterile cor containing naturally-occurring bacteria and fungi. Unique volatile compounds consisted of alcohols, aldehydes, alkanes, terpenes, and a few other chemical families. The number and identity of the specific volatiles were dependent on the toxigenic isolate tested. Our stakehold Sensor Development Corporation, used this data to modify a sensor under development and undergoing Beta testing (testing outside lab in real conditions). In late 2012 or early 2013, work will begin on the identification of volatiles produced by Fusarium verticillioides, anothe toxin-producing fungus commonly found on corn. Detection of these volatiles could be used by companies to remove the contaminated corn bat before it contaminates wholesome corn, thus increasing the supply of wholesome corn for national and international use. 02 Safe, effective, and inexpensive plant volatiles prevent microbial, especially toxigenic fungal, growth and toxin production in a stored cor model. Improperly stored corn can become wet resulting in the growth of toxin-producing fungi (e.g., Aspergillus (A.) flavus, Fusarium verticillioides) that render the corn unsafe for consumption. Trans-2- hexenal (T2H) is a natural antifungal soybean volatile whose production induced during infection by A. flavus. In FY 2011, ARS scientists in th Food and Feed Safety Research Unit, Southern Regional Research Center, N Orleans, LA, found that T2H in the liquid and volatile forms was highly effective against A. flavus both in vitro and when applied to wet, steri corn in small models of stored corn. During FY 2012, the in vivo study was expanded where the volatile T2H was intermittently pumped (30 min pe two hour or 12 hour cycle over 1-7 seven days) in a large jar containing wet, non-sterile corn. Results showed that this volatile as compared to the control samples, no fungal growth occurred 24 hours after incubation began. The volatile T2H did not remain in the corn after it was removed from the container, indicating that residual T2H would not be a problem used as a fumigant. This research could aid grain (especially corn) storage companies to protect stored grains from the growth of naturally- occurring fungi and contamination with mycotoxins. 03 In vitro research on the antifungal and antibacterial properties of safe commercially available plant compounds to determine which are suitable candidates to protect post-harvest food and feed. It is important to prevent the growth of Aspergillus (A.) flavus and Fusarium (F.) verticillioides which are naturally present on corn and produce dangerou toxins that render the corn unsafe for use as food or feed. In vitro testing continued to discover the most active, safe commercially inexpensive plant compounds with activity against these fungi. Examples include citral, found in lemon oil and used in perfumes and flavoring, which kills nearly 100% of A. flavus and F. verticillioides at 0.75 and �M, respectively. Other natural compounds, linalool and carvacrol, that are known antimicrobial plant compounds were not as effective against these fungi. Investigations began on the elimination of Leuconostoc mesenteroides from raw sugarcane juice, and studies continued to find compounds that could be used either as a volatile or in food grade films to protect citrus from post-harvest rot caused by the fungi Penicillium ) digitatum and P. italicum. These compounds were also tested against bacteria such as Escherichia coli. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA found that citral (5 �M) within 24 hr killed approximately 100% L. mesenteroides inoculated into raw, sterile sugarcane juice. Carvacrol, another natural antimicrobial compound, significantly decreased Leuconostoc (L.) mesenteroides viability, but at a higher concentration than that seen with citral. It is expected that successful research wil provide the sugar industry with a safe, natural compound at low concentrations that effectively prevents L. mesenteroides growth and subsequent dextran formation and sucrose loss. 04 Visible blue light (470 nm) is antimicrobial with potential use in food protection. Ultraviolet light (wavelength 10-400 nm) has antimicrobial properties but is a Group 1 carcinogen which causes cancer in humans. However, visible blue light (450-475 nm) is not carcinogenic and has antibacterial properties. ARS scientists in the Food and Feed Safety Research Unit, Southern Regional Research Center, New Orleans, LA, conducted experiments over five days showing blue light light-emitting diodes (LEDs) (470 nm), in contrast to corn kernels not exposed to the LEDs, inhibit the growth of naturally occurring fungi as well as the growth of Aspergillus (A.) flavus inoculated onto the kernels. These results indicate that light of this wavelength could be used to prevent the growth of such fungi on corn while the grain is being moved on conveyor belts. Blue light could also be used to prevent fungal growth finished baked goods or other products where fungal growth can render th foods inedible. ARS scientists in the Food and Feed Safety Research Uni Southern Regional Research Center, New Orleans, LA, completed a collaborative study with the University of New Orleans on the antifungal properties of FD&C # 3 red food color (a photosensitive compound) analog in the presence of blue light. Data showed that the over 25 new analogs produced by the university were not as effective as photosensitizers as the parent compound in the presence of blue light. Results therefore sh that the currently used food color, but not the novel analogs, could be used in the presence of blue light (peak: 470 nm) to prevent spoilage fungi from growing on baked goods.
Impacts
(N/A)
Publications
- De Lucca II, A.J., Boue, S.M., Carter Wientjes, C.H., Bhatnagar, D. 2012. Volatile profiles and aflatoxin production by toxigenic and non-toxigenic isolates of Aspergillus flavus grown on sterile and non-sterile cracked corn. Annals of Agriculture and Environmental Medicine. 19(1):91-98.
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