1. DRTaylor Consulting Mycotoxins and Use of Mycotoxin Binders to Alleviate Mycotoxicoses Sponsored by BROOKSIDE-AGRA By Dennis R. Taylor, Ph.D.

2. DRTaylor Consulting About Mycotoxins  Over 370 known mycotoxins *  Mycotoxins are produced by molds and fungi that grow on grains  Mycotoxins are specific chemicals (called “metabolites”) produced by the molds and fungi Aspergillus flavus growing on corn * Handbook of Toxic Fungal Metabolites – Cole / Cox (1981) Aspergillus flavus magnified Apspergillus flavus → produces aflatoxins

3. DRTaylor Consulting About Mycotoxins  Fusarium toxins - Deoxynivalenol (vomitoxin), T-2, zearalenone Fusarium graminearum → produces vomitoxin, T-2, zearalenone

4. DRTaylor Consulting About Mycotoxins  Mycotoxins can adversely affect animal health and performance.  Mycotoxins are potent chemicals and can produce toxic effects at very low levels – parts per million (ppm) and parts per billion (ppb) range.  Mycotoxins are very common – it is hard to avoid eventually getting some contaminated grains.  Once mycotoxins are produced, they are hard to get rid of… They cannot be destroyed by heating – even to 340 °C. They cannot be washed off – low solubility in water. There are no effective chemical treatments.

5. DRTaylor Consulting About Mycotoxins Fatty liver due to aflatoxin (left) compared to normal liver (right) (Ledoux UMC)

6. DRTaylor Consulting About Mycotoxins  Only six mycotoxins are of real commercial concern… T-2 toxin Aflatoxin B1 Deoxynivalenol (“vomitoxin”) Ochratoxin A Zearalenone Fumonisin B

7. DRTaylor Consulting Effects of Mycotoxins on Animals Leucoencephalomalacia, "blind staggers," in horses. Horses Fumonisin B1, B2 Estrogenic effects (edema of vulva, prolapsed vagina, enlargement of uterus), abortion, infertility, stunting. Atrophy of testicles, ovaries, enlargement of mammary glands. Swine, dairy cattle, turkey, lamb Zearalenone Oral lesions. Severe inflammation of gastrointestinal tract and possible hemorrhage; edema; infertility; degeneration of bone marrow; reduced weight gain; slow growth; sterility. T-2 Toxin Food refusal by swine; vomiting and diarrhea; reduction in weight gain. Swine, cattle, chicken, turkey, horse, human Deoxynivalenol (Vomitoxin) Toxic to kidneys and liver; abortion; poor feed conversion, reduced growth rate, reduced immunity to infection. Swine,duckling, chicken, human Ochratoxins Carcinogenic; attacks liver; reduced growth rate; hemmorrhagic enteritis; suppression of natural immunity to infection; decreased production of meat, milk, and eggs. Duckling, turkey poult, chicks, mature chickens, piglets, calves, pregnant sows, sheep, human, fish Aflatoxins B1, B2, G1, and G2 EFFECTS on ANIMALS Affected Commercial Species TOXIN

8. DRTaylor Consulting Recommended Acceptable Levels Aflatoxin M1 Aflatoxin B1 < 0.5 ppb in milk Vomitoxin 5 ppm swine 10 ppm cattle, poultry Fumonisins 5 ppm horses 10 ppm swine 50 ppm beef cattle and poultry FDA Guidelines on maximum levels of Aflatoxin, Vomitoxin and Fumonisin in feedstuffs for animals THE ROLE OF MYCOTOXINS IN FOOD AND FEED SAFETY Jon Ratcliff — Food and Agriculture Consultancy Services, UK www.facs.org.uk < 20 ppb in feeds

9. DRTaylor Consulting Recommended Acceptable Levels EU Maximum permitted levels of mycotoxins in animal feed and foods for human consumption Aflatoxin B1 5 ppb animal feedstuffs – cattle, sheep 2 ppb animal feeding stuffs – adult poultry and swine 1 ppb animal feeding stuffs – piglets and chicks Ochratoxin A 5 ppb dried fruit and nuts THE ROLE OF MYCOTOXINS IN FOOD AND FEED SAFETY Jon Ratcliff — Food and Agriculture Consultancy Services, UK www.facs.org.uk

10. DRTaylor Consulting What can be done? Limited options  Only buy uncontaminated grains But difficult to accomplish because even if you analyze for toxins you may miss them. Usually contamination is not uniformly distributed throughout the sample. Sometimes nothing but contaminated grains are available. Remember – not possible to remove toxins by heating or washing.  Use mycotoxin binding sorbents to sequester toxins This approach – first reported in 1988 by Phillips & Taylor, et al. – has over 30 years of peer reviewed research and commercial use proving its viability and utility. At last count, there were over 100+ companies world-wide offering mycotoxin binders – and new offerings are made practically every day.

11. DRTaylor Consulting  1988 – Phillips, Taylor, Kubena, Harvey show 0.5 wt% hydrated sodium calcium aluminosilicate (HSCAS) protects CHICKENS against 7.5 ppm AFB 1 (Poultry Sci., 67, 243-247)  1989 – Harvey, Phillips, Kubena, et al. show HSCAS protects SWINE against AFB 1 (Amer. J. Vet. Res., 50, 416-420)  1991 – Kubena, Huff, Harvey, et al. show HSCAS protects TURKEYS against AFB 1 (Poultry Sci., 70, 1823-1830)  1991 – Harvey, Kubena, Phillips, et al. show HSCAS protects LAMBS against AFB1 (Amer. J. Vet. Res., 57, 152-156)  1994 – Phillips, Harvey, Kubena, et al., show HSCAS protects GOATS against AFB 1 (J. Anim. Sci., 72, 677-682) Early History of HSCAS as Aflatoxin Binder

12. DRTaylor Consulting Evaluating Mycotoxin Binders  Two possible approaches… In-vivo testing Uses live animals Uses mycotoxin contaminated feeds Uses mycotoxin binder mixed with contaminated & uncontaminated feeds. In-vitro testing Does not use live animals Generally uses low level of mycotoxin dissolved in water. Uses mycotoxin binder to remove the mycotoxin from the water. Usually does not use mycotoxin contaminated feeds.

13. DRTaylor Consulting Comparison: in-vitro vs. in-vivo testing  IN-VIVO In-vivo tests are very expensive. It usually cost $15K-$20K to conduct an in-vivo evaluation with perhaps 3 or 4 treatment groups (poultry least expensive). In-vivo tests take time (~40 days / evaluation for poultry) There are too many competitive mycotoxin binders to evaluate in a single in-vivo test.  IN-VITRO In-vitro tests are much less expensive. It usually costs about $350 to conduct an in-vitro evaluation of a sorbent (+4 toxins). In-vitro tests are much quicker – usually about 1 week. In-vitro tests are much more reproducible because all conditions can be carefully controlled. Any number of competitive sorbents can be evaluated, and at different periods in time.

14. DRTaylor Consulting Does in-vitro binding correlate with in-vivo binding?  The answer is YES… …at least for aflatoxin B1 in-vitro versus in-vivo testing.

15. Correlation: In-vivo Wt. Gain vs. In-vitro Binding Activity 1 DRTaylor Consulting Broilers: Aflatoxin Challenge - 3000 PPB Neg. Control (3000 ppb AFB1/ 0 binder) Binders: Increasing Binding Activity (3000 ppb AFB1/ 0.5% binder) Pos. Control (0 ppb AFB1/ 0 binder) 1 2002, Dr. Carlos Mallman, Univ. Federal de Santa Maria, Brazil Conclusion: In-vitro binding correlates with in-vivo response

16. DRTaylor Consulting So what makes one mycotoxin binder Good – and another not so good – or even Poor? An in-vitro approach to finding the answer

17. Physical properties of montmorillonite DRTaylor Consulting Scanning Electron Microscopy at high magnification Raw clay Density (Kg/m 3 ) Porosity (cc/g) Pore diameter (µm) Surface area (m 2 /g)

18. Chemical properties of montmorillonite DRTaylor Consulting Ca ++ Exchangeable cations → Surface acidity (pK a ) Cation Exchange Capacity (meq/100 g) Exchange cations (Na +, K +, H +, Mg ++, Ca ++ ) pH of clay slurry in water

19. DRTaylor Consulting Protocol of the study  Obtain a representative group of commercially available binders.  Measure aflatoxin B 1 binding under a constant set of conditions … (20 µg toxin / 1 mg binder / 1 mL)  Obtain XRD & measure complete set of physical & chemical properties.  Determine if aflatoxin binding correlates to any particular property … or combination of properties.

20. DRTaylor Consulting Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Mineralogy Product Aflatoxin-% Binding Mineralogical Composition Code ( 20 µg / mg / mL ) ( basis XRD analysis ) ----------------------------------------------------------------------------------------------------------------------------------------------- A99.2Ca montmorillonite…………..……. + Qz, FS B97.1Ca/Na montmorillonite………..….. + Qz C95.2Ca montmorillonite (low level)….. + Opal CT, Qz D95.9Attapulgite + Ca montmorillonite. + Qz E94.6Sepiolite F92.6Ca montmorillonite G90.7Ca/Na montmorillonite……………. + Qz ----------------------------------------------------------------------------------------------------------------------------------------------- H87.5Na montmorillonite………………… + FS I87.3Na/Ca montmorillonite……………. + Qz J86.4Na montmorillonite K85.2Attapulgite (low level) L83.6Attapulgite M80Ca montmorillonite……….…….….. + Qz ----------------------------------------------------------------------------------------------------------------------------------------------- N73.9Ca montmorillonite…………….…… + Opal CT O70.5Ca montmorillonite (low level)….… + Qz, FS P66.2Ca montmorillonite Q55.9Clinoptilolite, mordenite…….…….. + Qz R47.7Kaolinite + mica/illite S44Ca montmorillonite (low level) T25.2Amorphous silica U16.9Amorphous silica Excellent Binding Good Binding Poor Binding Inadequate Binding

21. DRTaylor Consulting Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Physical Properties A6.9 806 0.5119 0.02 65 B9.2 1050 0.1533 0.028 25 C4.0 602 0.4156 0.09; 0.015 a 97 D7.5 607 0.5081 0.05140 E8.5 559 0.4432 0.028195 F9.5 751 0.1213 0.03 57 G9.82 701 0.2713 0.03 77 H9.1 1048 0.2473 0.038 21 I9.12 632 0.5546 0.065 31 J9.2 1067 0.0848 0.07 21 K9.5 663 0.3849 0.03 32 L6.8 812 0.1785 0.035 64 M8.35 777 0.1292 0.03 81 N6.84 761 0.212 0.03 82 O8.4 657 0.3088 0.4 14 P9.1 830 0.2414 0.04 75 Q9.7 990 0.1844 0.05 18 R5.1 342 0.6061 0.68 25 S6.4 693 0.1776 0.025 8 T10 215 0.9556 0.19; 1.0 a 66 U6.8 350 1.6362 0.11 72 a Bimodal distribution of porosity; two maxima in pore volume versus pore diameter plots. Product Slurry Loose Density Hg PV Hg Pore BET Surface Code pH (Kg/m 3 ) (cc/g) Diameter (µm) Area (m²/g) --------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------

22. Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Chemical Properties Product Surface Acidity ( meq/g ) Cation Exchange Capacity ( meq/100 g ) Code pKa 1.5 Total Ca ++ Na + Total ------------------------------------------------------------------------------------------------------------------------------------------- A0.0790.0890.16852.09.076 B0.0210.0530.07462.053.0119 C0.0800.0800.16028.60.448 D0.0800.1300.21020.03.039 E0.1800.0400.22054.11.577 F0.0600.2000.26059.83.0102 G0.0210.0290.049110.086.0207 H0.0390.1420.18138.075.0121 I0.0100.0320.04249.053.0127 J0.0420.1060.14842.067.0125 K0.0200.1100.13063.626.3121 L0.0800.0200.10039.331.879 M0.0300.1400.17068.012.0102 N0.1170.0760.193111.02.0122 O0.0390.1000.13976.02.082 P0.0500.1300.18038.719.963 Q0.0210.0200.040144.057.0215 R0.0200.0010.0202.80.67 S00.0200.02041.221.175 T00.0600.0601.3183.2185 UN/AN/AN/A1.2245.9248

23. In-Vitro Binding vs. Physical / Chemical Properties of Mycotoxin Binders RESULTS  No single physical property correlates with in-vitro binding of aflatoxin  No single chemical property correlates with in-vitro binding of aflatoxin  … however, there is a weak correlation with combination of strong 020406080100120 ln [ (<1.5 pKa) x (Mg ++ conc.) ] 0 20 40 60 80 100 In-Vitro Binding (%) surface acidic sites (of pKa <1.5) and Mg ++ concentration

24. Optimum binding – like tumblers in a lock… DRTaylor Consulting Optimum set of physical / chemical properties in binder … …like tumblers in a lock… Aflatoxin Aflatoxin fits perfectly……so lock and key mechanism work together to bind aflatoxin Another toxin If toxin does not match physical / chemical properties of binder - lock and key mechanism won’t work…

25. DRTaylor Consulting So - how does FLO-BOND compare to the competition? Some more in-vitro and in-vivo studies

26. DRTaylor Consulting Flo-Bond is the only mycotoxin binder with Organic approval in the USA

27. Recent Dioxin Analysis on Flo-Bond DRTaylor Consulting All samples below 1.5 ppt EEC limit for sum (dioxins + PCB’s) Except sample B&N 8511 which was mining sample not used for Flo-Bond

28. DRTaylor Consulting 2010 Competitive in-vitro Mycotoxin Binder Study METHODOLOGY New experimental products (Brookside-Agra currently conducting in-vivo trials) Aflatoxin B1, Deoxynivalenol, Fumonisin B1, Ochratoxin A, T-2 Toxin, Zearalenone– all@ 2000 ppb 0.5 wt% Adsorption phase: 3 reps. @ pH 3.0 Desorption phase: 3 reps. @ pH 8.0 - = %Adsorption@ pH 3.0% Desorption @ pH 8.0 ncy Mycotoxins / Concentration Binder Level: Binding Conditions: %Efficie Binders Studied Flo-Bond, Flo-Bond AZ (experimental), Flo-Bond X (experimental), NovaSil Plus, MycoAd

29. 2010 Competitive in-vitro Mycotoxin Binder Study RESULTS Results by Trilogy Analytical Lab, Missouri, USA DRTaylor Consulting AFB1FUMDONOCHRAT-2ZONE Flo-Bond New Flo-Bond AZ (AFB1/ZONE) NEW Flo-Bond X (Full Spectrum) NS Plus MycoAd % Adsorption pH3 % Desorption pH 8 % Efficiency % Adsorption pH 3 % Desorption pH 8 % Efficiency % Adsorption pH 3 % Desorption pH 8 % Efficiency % Adsorption pH 3 % Desorption pH 8 % Efficiency % Adsorption pH 3 % Desorption pH 8 % Efficiency 1009520.677.823.731 08715.977.8030.8 10084.7023.70.2 10099.227.395.240.874.9 0.186.220.895.1035.6 99.9136.50.140.839.3 10098.417.691.835.450.7 094.714.689.3042.6 1003.732.535.48.1 1009022.694.467.296.5 049.415.693.37.34.4 10040.671.159.992.1 99.995.336.798.198.399.8 0.37.912.89.200.3 99.687.423.988.998.399.5

30. DRTaylor Consulting 2010 Competitive in-vitro Mycotoxin Binder Study CONCLUSIONS General While all mycotoxins studied (except DON) were significantly bound in-vitro under acidic (pH 3) conditions, some were significantly, or completely desorbed under basic (pH 8) conditions. Aflatoxin B1 Aflatoxin strongly bound by all binders in-vitro under acidic (pH 3) & basic (pH 8) conditions. Numerous studies support in-vivo efficacy. Experimental Blends… New experimental blends being studied by Brookside-Agra are showing promise for extending binder efficacy for other mycotoxins

31. DRTaylor Consulting Deoxynivalenol Zearalenone Aflatoxin B 1 Why does aflatoxin bind so strongly compared to other mycotoxins? Ochratoxin A Some similarity here …but none here and here Some similarity between these This grouping causes strong binding

32. DRTaylor Consulting Ca ++ Binding of the diketone moiety to cationic sites: the reason for the strong binding of aflatoxin The 1,3-diketone structure of aflatoxin possesses high electron density and is therefore strongly attracted to positively charged sites This forms a type of complex called a CHELATE Clay Structure

33. DRTaylor Consulting Ca ++ Binding of the diketone moiety to cationic sites: the reason for the strong binding of aflatoxin + Broken bonds at crystal edges can also generate cationic sites These sites can also bind aflatoxin as a CHELATED complex Clay Structure

34. DRTaylor Consulting “THE DEVIL IS IN THE DETAILS” A CAUTION ABOUT IN-VITRO BINDING STUDIES

35. DRTaylor Consulting In-Vitro Binding Data – Be careful about what you think you see! A Study of two binders Percentage Binding 0 10 20 30 40 50 60 70 80 90 100 Binder #1Binder #2 Aflatoxin B1 Fumonisin Zearalenone Ochratoxin WHICH WOULD YOU PICK?

36. DRTaylor Consulting In-Vitro Binding Data - Percentage Binding 0 10 20 30 40 50 60 70 80 90 100 Binder #1Binder #2 AFB1 (20 µg/ mL/ mg) Fumonisin (4 µg/ mL/ 10 mg) ZONE (4 µg/ mL/ 20 mg) Ochratoxin (4 µg/ mL/ 50 mg) AFB1 (4 µg/ mL/ mg) Fumonisin (4 µg/ mL/ 1mg) ZONE (4 µg/ mL/ 1mg) Ochratoxin (4 µg/ mL/ 1mg) Binder #1 = #2 = Flo-Bond: Just Different Levels of Binder Make Sure You Read the Fine Print ! Let’s look a little more carefully at the binding conditions Binding Conditions Very Important !!

37. DRTaylor Consulting IN-VIVO DATA USING FLO-BOND

38. DRTaylor Consulting Chicken Feeding Trial: FLO-BOND vs. NovaSil : 2500 ppb Aflatoxin B1 1234 Week 0 50 100 150 200 250 300 350 Average Body Weight (gms) Therefore 0.25% FLO-BOND = 0.5% NovaSil 0.5% FLO-BOND better than 0.5% NovaSil 2500 ppb AFB1 / no binder 2500 ppb AFB1 /.25% FB 2500 ppb AFB1 /.5% FB 2500 ppb AFB1 /.5% NS 2500 ppb AFB1 /.75% FB 0 ppb AFB1 / no binder

39. DRTaylor Consulting Protective Effect 0.275% Flo-Bond vs. 2000 PPB Ochratoxin: Broilers* 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Percentage (%) GRADE 1 GRADES 2-4 Liver: Macroscopic Results Control Control + Flo-Bond Control + 2000 ppb Ochratoxin + Flo-Bond Control + 2000 ppb Ochratoxin Test period: 7 – 28 days of age Groups: 4 rations (see graph) * 64 male broilers/ 16 chicks per ration

40. 2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge  Evaluation of FLO-BOND in growing pigs fed 0.9 ppm and 1.8 ppm deoxynivalenol (DON) contaminated diets  Test: A 7-d (64-pen) pig study was conducted comparing live performance (average daily gain, feed conversion, and feed consumption) of pigs fed FLO-BOND at levels of 0%, 0.25% and 0.50% in commercial type diets contaminated with DON mycotoxin at levels of 1.8 and 0.9 ppm  Location: Virginia Diversified Research, Corp., Harrisonburg, VA; investigator: Michael D. Sims DRTaylor Consulting

41. FLO-BOND lbs/ton050510 DON (ppm)000.9 Day 0-7ADG (lbs/d)1.982a1.893a0.693c1.430b1.307b Day 0-7 Feed/Gain (lb/1b)1.324a1.284a2.415c1.632b1.663b Feed Consumption (lb)18.36a16.99ab12.30c16.66b15.09b DRTaylor Consulting 2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge FLO-BOND vs. DON @ 0.9 ppm FLO-BOND lbs/ton:050510 DON (ppm):001.8 Day 0-10 ADG (lbs/d)1.982a1.893a0.566c1.079b0.779bc Day 0-10 Feed/Gain (lb/lb)1.324a1.284a8.544c1.834b2.050b Feed Consumption (lb)18.36a16.99a11.41d13.4b10.61c FLO-BOND vs. DON @ 1.8 ppm RESULTS

42. DRTaylor Consulting FLO-BOND Does Not Hinder Uptake of Nutrients Chung, T.K.et al., 1990 Poultry Science 69: 1364-1370 Chung, T.K. and Baker, D.H., 1990 J Animal Science 68: 1992-1998 Effect on NutrientLevel of HSCASNutrient Utilization Riboflavin0.5 %None Riboflavin1.0 %None Vitamin A0.5 %None Vitamin A1.0 %None Manganese0.5 %None Manganese1.0 %None Zinc0.5 %None Zinc1.0 % Slight tibia Zn decrease Phosphorus, inorganic0.5 %None Phosphorus, inorganic1.0 %None Phosphorus, phytate0.5 %None Phosphorus, phytate1.0 %None

43. Effect of Flo-Bond Plus on Mold Reduction DRTaylor Consulting Conclusions This evaluation demonstrates that Flo- Bond Plus can significantly reduce the mold count in a high moisture corn sample.

44. DRTaylor Consulting What about processing? What about quality control? Can they affect product quality?

45. DRTaylor Consulting Selective Mining of HSCAS Strata Manufacturing Plant Product Dried & Ground into Powder Quality Control Check Bagging & Sample Retention FLO-BOND Containerization & Shipping FLO-BOND Process – No Added Ingredients – Only Drying & Grinding

46. DRTaylor Consulting Effect of Thermal Processing on Surface Area vs. Binding of Aflatoxin B 1 5060708090100 BET Surface Area (m²/g) 40 50 60 70 80 90 100 Aflatoxin Binding (%) Increasing Processing Temperature Therefore, it is very important not to overheat clay that is to be used as mycotoxin binder

47. DRTaylor Consulting Effect of Particle Size (Grind) vs. Binding of Aflatoxin B 1 707580859095 % AFB1 Bound 65 50 55 60 65 70 75 80 85 90 95 % -325 Mesh Therefore, it is very important to get good grinding in order to get maximum binding Increasing level of binding Increasing fineness of grind

48. DRTaylor Consulting Important Considerations Before Buying a Mycotoxin Binder  Does the manufacturer have both in-vitro and in-vivo data demonstrating efficacy for his product?  Does the manufacturer have a proven track record for delivering a quality product?  Does the manufacturer have control over his source materials and manufacturing process?  Does the manufacturer maintain good quality control during the manufacturing process?  To all these questions, Brookside-Agra can say “ YES ” with regard to its FLO-BOND mycotoxin binding product.

49. DRTaylor Consulting SUMMARY AND CONCLUSIONS  Montmorillonite is the most common commercially available mycotoxin binder.  Aflatoxin B1 is the most strongly adsorbed mycotoxin.  FLO-BOND is (HSCAS) montmorillonite binder that possess superior mycotoxin binding characteristics for many different mycotoxins.  Manufacturing conditions (temperature, grind) affect binding performance, so good quality control is absolutely essential.  Brookside-Agra is committed to good quality control during the manufacturing of FLO-BOND  USDA approved as organic product  FLO BOND is dioxin free – (i.e. - below EEC limits for dioxin content)

50. DRTaylor Consulting Thank you for your kind attention The End