![Mycotoxins Have A Crippling Effect On
Livestock Feed
25% of crops are affected
annually with mycotoxins
worldwide
~ 30 Million tons of animal feed
Ref [1-3]2](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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Soft Nanomaterial Technology to Measure Mycotoxin Contamination in Animal Feed
- 1. Using Molecularly Imprinted Polymers for the Detection of Mycotoxins in Animal Feed Adam Weinstein Dhilan Bekah Kai Slaughter Sahaj Dhamija
- 2. Mycotoxins Have A Crippling Effect On Livestock Feed 25% of crops are affected annually with mycotoxins worldwide ~ 30 Million tons of animal feed Ref [1-3]2
- 3. The most potent naturally occurring carcinogen in the world Up to 30% of liver cancer in Ref [34]3
- 4. Current Testing Methods Are Not Sufficient ELISA Kits, Thin-layer chromatography, Black light Quick tests must be followed up by confirmatory tests Aflatoxin FDA limit is ~ 20 ppb Ref [4-5]4
- 5. Molecularly Imprinted Polymers (MIPs): ‘Plastic Antibodies’ • Low Cost • High Stability • High Sensitivity Ref [6-9]5
- 6. Our Solution: MIP-Cellulose Composite Film Implemented in 5 steps: 1. Formation of porous cellulose film 2. Polymerization of MIP around template molecule 3. Removal of template molecule 4. Addition of animal feed to film 5. Observation of bound aflatoxin under black light Ref [10-11]6
- 7. Formation of Cellulose-MIP Composite Film Soxhlet Extraction Ref [12-13]7
- 8. Detection of aflatoxin in animal feed 8
- 9. Key Advantages • Superior to current antibody detection designs - Cost - Chemical Stability • On-site vs. In-lab • Possibly adaptable to other relevant testing Ref [8]9
- 10. Costs For Comparison Material Cost CNC $25/Kg Methacrylic Acid $2/Kg 3-methacryloxypropyltrimethoxysilane $1/Kg Ethyleneglycol dimethacrylate $1/Kg 5,7-Dimethoxycoumarin (DMC - Aflatoxin template) $130/g MIP Cost (Outsourced): $0.10-0.50/mg Antibody Cost: $100-1000/mg Ref [14-19]10
- 11. Market Analysis Modern agriculture requires vast amounts of animal feed: • 119 Million tons produced • ~ $25 Billion spending annually in just the US Worldwide business • Millions of tons of animal feed distributed worldwide Ref [3, 20]11
- 12. Future Directions Other mycotoxins (i.e. Ochratoxins) Other food industries affected by mycotoxins Bacteria (i.e. Salmonella, E.Coli, Lysteria) Ref [21]12
- 14. Questions?
- 15. References [1] “Mold and Mycotoxin Issues in Dairy Cattle: Effects, Prevention and Treatment - eXtension,” Mold and Mycotoxin Issues in Dairy Cattle: Effects, Prevention and Treatment - eXtension, 23-Sep-2016. [Online]. Available: http://articles.extension.org/pages/11768/mold-and-mycotoxin-issues-in-dairy-cattle:-effects-prevention-and-treatment. [Accessed: 02-Nov-2016]. [2] “Mycotoxins: Invisible Toxins with Visible Impacts”, 15-May-2015. [Online]. Available: https://agrilinks.org/blog/mycotoxins- invisible-toxins-visible-impacts. [Accessed: 02-Nov-2016]. [3] J. A. Crump, P. M. Griffin, and F. J. Angulo, “Bacterial Contamination of Animal Feed and Its Relationship to Human Foodborne Illness,” Clinical Infectious Diseases, vol. 35, no. 7, pp. 859–865, 2002. [4] R. S. Adams et al, “Mold and Mycotoxin Problems in Livestock Feeding (Dairy Cattle Nutrition),” Dairy Cattle Nutrition (Penn State Extension), 2016. [Online]. Available: http://extension.psu.edu/animals/dairy/nutrition/forages/mycotoxins-nitrates-and-other- toxicity-problems/mold-and-mycotoxin-problems-in-livestock-feeding-1. [Accessed: 02-Nov-2016]. [5] “Mycotoxin FAQs - CPN-2002 - Crop Protection Network,” Crop Protection Network. [Online]. Available: http://cropprotectionnetwork.org/corn/mycotoxin-faqs/. [Accessed: 02-Nov-2016]. [6] “Molecularly imprinted polymer,” Wikipedia. 26-Jun-2016.
- 16. References (Cont’d) [7] Regal, P., Díaz-Bao, M., Barreiro, R., Miranda, J. M., & Cepeda, A. (2015). Development of a novel molecularly imprinted stir- bar for isolation of aflatoxins. Proceedings of The 19th International Electronic Conference on Synthetic Organic Chemistry. doi:10.3390/ecsoc-19-d001hhhhhhhh [8] M. J. Whitcombe et al., “The rational development of molecularly imprinted polymer-based sensors for protein detection,” Chem. Soc. Rev., vol. 40, no. 3, pp. 1547–1571, Mar. 2011. [9] L. Uzun and A. P. F. Turner, “Molecularly-imprinted polymer sensors: realising their potential,” Biosens. Bioelectron., vol. 76, pp. 131–144, Feb. 2016. [10] R. Devonshire, “A Step by Step Guide to Using a Franking Machine,” LinkedIn Pulse, 31-Jul-2015. [Online]. Available: https://www.linkedin.com/pulse/step-guide-using-franking-machine-ryan-devonshire. [Accessed: 03-Nov-2016]. [11] Huang, Jennifer, and Diab Elmashni. "Analysis of Aflatoxins Using Fluorescence Detection." (2011): n. pag. Thermo Fisher Scientific. Web. [12] C. Bodhibukkana et al., “Composite membrane of bacterially-derived cellulose and molecularly imprinted polymer for use as a transdermal enantioselective controlled-release system of racemic propranolol,” J. Controlled Release, vol. 113, no. 1, pp. 43–56, Jun. 2006. [13] “Soxhlet extractor,” Wikipedia. 30-Oct-2016.
- 17. References (Cont’d) [14] https://www.alibaba.com/product-detail/Manufacturing-methacrylic-acid_549935670.html [15] https://www.alibaba.com/product-detail/ShinEtsu-KBM-503-CAS-2530-85_60421020692.html?s=p [16] https://www.alibaba.com/product-detail/Ethylene-glycol-dimethacrylate-EGDMA-factory-in_60450864212.html [17] Sigma-Aldrich – DMC [Online]. http://www.sigmaaldrich.com/catalog/product/aldrich/116238?lang=en®ion=CA [18] M. J. Whitcombe, “The Rational Development of Molecularly Imprinted Polymer-Based Sensors for Protein Detection,” Chemical Society Reviews, vol. 40, pp. 1547–1571, 2011. [19] Lohr, Rhiannon et al. "Improved Aneurysm Treatment With Nanocrystalline Cellulose Reinforced Polymers". 2015. Presentation. [20] Mycotoxins: Risks in Plant, Animal. and Human Systems. CAST - Council for Agricultural Science and Technology, 2003. [21] http://www.foodpoisonjournal.com/uploads/image/recall(3).jpg
- 18. References (Cont’d) [22] T. Piacham, C. Isarankura-Na-Ayudhya, and V. Prachayasittikul, “A simple method for creating molecularly imprinted polymer-coated bacterial cellulose nanofibers,” Chem. Pap., vol. 68, no. 6, pp. 838–841, Nov. 2013. [23] M. Jiang et al., “Aflatoxin B1 Detection Using a Highly-Sensitive Molecularly-Imprinted Electrochemical Sensor Based on an Electropolymerized Metal Organic Framework,” Toxins, vol. 7, no. 9, pp. 3540–3553, Sep. 2015. [24] A. Rachkov, S. McNiven, A. El’skaya, K. Yano, and I. Karube, “Fluorescence detection of β-estradiol using a molecularly imprinted polymer,” Anal. Chim. Acta, vol. 405, no. 1–2, pp. 23–29, Jan. 2000. [25] M. A. Klich, “Aspergillus flavus: the major producer of aflatoxin,” Mol. Plant Pathol., vol. 8, no. 6, pp. 713–722, Nov. 2007. [26] J. C. C. Yu and E. P. C. Lai, “Molecularly imprinted polymers for ochratoxin a extraction and analysis,” Toxins, vol. 2, no. 6, pp. 1536–1553, Jun. 2010. [27] F. Navarro-Villoslada, J. L. Urraca, M. C. Moreno-Bondi, and G. Orellana, “Zearalenone sensing with molecularly imprinted polymers and tailored fluorescent probes,” Sens. Actuators B Chem., vol. 121, no. 1, pp. 67–73, Jan. 2007. [28] B. Schyrr, S. Pasche, G. Voirin, C. Weder, Y. C. Simon, and E. J. Foster, “Biosensors Based on Porous Cellulose Nanocrystal–Poly(vinyl Alcohol) Scaffolds,” ACS Appl. Mater. Interfaces ACS Applied Materials & Interfaces, vol. 6, no. 15, pp. 12674–12683, 2014
- 19. References (Cont’d) [29] D. Flynn, “USDA: U.S. Foodborne Illnesses Cost More Than $15.6 Billion Annually | Food Safety News,” USDA: U.S. Foodborne Illnesses Cost More Than $15.6 Billion Annually, Aug-2014. [Online]. Available: http://www.foodsafetynews.com/2014/10/foodborne-illnesses-cost-usa-15-6-billion-annually/. [Accessed: 02-Nov-2016]. [30] “Burden of Foodborne Illness: Findings,” Estimates of Foodborne Illness in the United States, 2016. [Online]. Available: http://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html. [Accessed: 02-Nov-2016]. [31] Counting chickens. (2011, July/August). Retrieved October 20, 2016, from http://www.economist.com/blogs/dailychart/2011/07/global-livestock-counts [32] “What is the aflatoxin problem?”. [Online]. Available: http://www.aflatoxinpartnership.org/?q=aflatoxins. [Accessed: 02-Nov- 2016]. [33] “Aflatoxins in Kenya’s food chain: Overview of what researchers are doing to combat the threat to public health”. [Online]. Available: https://news.ilri.org/2014/05/06/aflatoxins-in-kens-food-chain/ [34] T. Lore, "New ILRI study finds high levels of aflatoxin in milk and dairy feeds in Greater Addis Ababa milk shed", AgHealth, 2016. . [35] “FDA Mycotoxin Regulatory Guidance: A Guide for Grain Elevators, Feed Manufacturers, Grain Processors and Exporters,” Aug-2011. [Online]. Available: https://www.ngfa.org/wp-content/uploads/NGFAComplianceGuide- FDARegulatoryGuidanceforMycotoxins8-2011.pdf. [Accessed: 26-Nov-2016].
- 20. Cellulose Porous Film Preparation
- 21. Foodborne Illness Foodborne illness is extremely prevalent CDC estimates that every year, 1 in 6 Americans (48 million people) contracts a foodborne illness, 128,000 are hospitalized, and 3000 die It is also extremely expensive The USDA estimates that foodborne illnesses annually cost the US economy more than $15.6 billion In reality these costs are much higher
- 23. Aflatoxin DMC
- 24. CNC-PVA film formation • Glass and quartz slides cleaned by sonication in 2-propanol and rinsed with deionized water • Substrates immersed in bath of concentrated sulfuric acid for 2 min to hydrophilize surface by revealing silanol groups on surface • Substrates rinsed for 5 min with DI water and dried in N2 • PVA dissolved in deionized water heating at 95°C for 10 h with stirring • PVA solution diluted to 1 mg/mL in DI water • Freeze-dried CNCs added to conc. of 4 mg/mL • Mixture sonicated for 1 h at room temperature • Thin films deposited on substrates by dip coating • Films dried by freeze-drying process CNC–composite film formation • Carried out in the presence of template molecule • React with 3-MPS (10% w/w in toluene) at 80°C for 5 h • Wash film in methanol and dry • Solution with 12 mmol MAA, 0.05 mol EDMA, 2mmol of template, 0.7 mmol AIBN in DMF (2 mL) • Purge with nitrogen, close and polymerize at 60°C for 18 h • Soxhlet extraction to remove the template molecule (10% w/w acetic acid in methanol for 72 h)
Editor's Notes
- Hello everyone, Today my group and I will be presenting our idea for addressing a huge problem in the livestock industry – mycotoxin feed contamination. More specifically, we will be talking about using Molecularly Imprinted Polymers for the Detection of Mycotoxins in Animal Feed.
- So What are Mycotoxins? • Mycotoxins are fungal-borne toxins, often produced by mould on food (as you can see from the image on the right), affecting 25% of crops annually worldwide • These mycotoxins have several detrimental consequences • They are even easily transferrable to cow’s milk – this presents a huge concern to humans. Mycotoxins: • Cause irritating tissues • Reduce feed consumption • Reduce nutrient utilization • Suppress immunity • Alter reproduction • Death
- One of the most prominent and naturally occurring mycotoxins is aflatoxin, as it has been reported to be the most potent naturally occurring carcinogen in the world Explain chart and importance for detecting aflatoxin: So we start with seeds, grain, and corn used in animal feed which are susceptible to aflatoxin contamination During storage, poor conditions (like high humidity) cause the development of aflatoxin producing mold on the food, this is especially a problem in developing countries where proper storage conditions are not always available If the contaminated crops are not tested they are then added to animal feed Animals that consume this feed become sick resulting in lower food production and income loss. If humans consume the contaminated food, they also experience toxic effects and possible cancer outcomes In fact, it is estimated that aflatoxins cause between 5% to 30% of all liver cancer in the world [32] In the USA alone, the cost associated with dealing with aflatoxins in animal feed has been estimated to be around $225 million/year
- There are several issues with regards to current testing. First, current testing is reactive. This means that testing is only done after incidence occurrence i.e. animals getting sick. There are 3 main types of quick tests: ELISA Kits, TLC, and Black light detection ELISA kits are much more expensive (sigma quotes prices ~$500-600 CAD), regular ELISA takes place in-lab TLC, while the response time is quick, still requires a skilled technician, pre-treatment of sample, and expensive equipment. What is often used is blacklight detection, which is poor. It is shone on food sample directly to detect aflatoxins since they are fluorescent, as demonstrated by the image on the right. However, this is unreliable as other chemicals are also fluorescent, and therefore it is not conclusive that all of the fluorescence is due to aflatoxins, and not other compounds stemming from fungi. Moreover, this test is evident only once the food has already reached a stage where it would be visible to the naked eye. On this note, it is important to recognize that the FDA Limit for aflatoxin is 20 ppb, which is much smaller than what you would see through the blacklight test, and this is dangerous. These quick tests are more often than not followed by confirmatory tests (HPLC or GC). There is therefore additional time and transportation costs to consider, and, in addition, Aflatoxin can develop during sample transport which can result in false positives.
- 3-MPS: 3-methacryloxypropyltrimethoxysilane MAA: Methacrylic acid EDMA: ethylene glycol dimethacrylate DMC: 5,7-Dimethoxycoumarin Methanol/acetic acid for soxhlet extraction
- - 1 ppb (reminder that FDA Limit is 20 ppb)
- The cost of raw materials in our design is fairly low The only exception being DMC (our Aflatoxin template for making the molecularly imprinted polymer) It is far less expensive and less dangerous than actual Aflatoxin Also only very small amounts are required for MIP preparation Even if we decide to outsource the fabrication of the MIP: The common figures for the fabrication of MIP are ~10-50 cents per mg Very small compared to the $100-1000 per mg of antibodies which are the major component in ELISA, the most relevant detection method currently on the market.
- Feeding livestock requires around 119 million tons of feed, or $25 billion in spending in the US, a huge industry Worldwide, countries import millions of tons of feed Mycotoxins become an even larger issue and cost even more in developing countries, where food is often not stored properly and expensive testing is not feasible While Aflatoxins cost $225 million yearly, Mycotoxins cost ~$1.4 billion yearly in just the US Our testing device would be a huge cost savings if proactive testing is done to avoid using feed contaminated with mycotoxins In addition to the loss of animal productivity and possible human cancer outcomes, regulatory agencies like the FDA and the European food safety authority can confiscate all contaminated grain, and in some cases can proceed with injunction or prosecution of the responsible parties.
- Ochratoxins are also fluorescent, so our sensor could easily be adapted for ochratoxins by simply changing the template used in MIP fabrication An obvious future direction would be expanding Mycotoxin detection to other industries (food or milk intended for human consumption) Another huge problem in livestock is contamination of meat with bacteria that cause foodborne illnesses like Salmonella, E.Coli, and Lysteria Studies have shown a significant link between these foodborne illness outbreaks and the presence of these bacteria in animal feed Every year, 1 in 6 Americans (48 million people) contracts a foodborne illness (CDC) Foodborne illnesses annually cost the US economy more than $15.6 billion (USDA) There would be a technical challenge since bacteria are not fluorescent the way mycotoxins are, but if the right molecule could be targeted and detected, the same MIP method could be applied to bacteria detection
- - These are some companies that produce animal feed and may be interested in using our technology to test their own products or sell the technology to farmers for testing during storage