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

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

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

About Mycotoxins Ø Mycotoxins can adversely affect animal health and performance. Ø Mycotoxins are About Mycotoxins Ø Mycotoxins can adversely affect animal health and performance. Ø Mycotoxins are potent chemicals and can produce toxic effects at very low levels – parts per million (ppm) and parts per billion (ppb) range. Ø Mycotoxins are very common – it is hard to avoid eventually getting some contaminated grains. Ø 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. DRTaylor Consulting

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

About Mycotoxins Ø Only six mycotoxins are of real commercial concern… Aflatoxin B 1 About Mycotoxins Ø Only six mycotoxins are of real commercial concern… Aflatoxin B 1 Deoxynivalenol (“vomitoxin”) Fumonisin B Ochratoxin A Zearalenone T-2 toxin DRTaylor Consulting

Effects of Mycotoxins on Animals TOXIN Affected Commercial Species EFFECTS on ANIMALS Aflatoxins B Effects of Mycotoxins on Animals TOXIN Affected Commercial Species EFFECTS on ANIMALS Aflatoxins B 1, B 2, G 1, and G 2 Duckling, turkey poult, chicks, mature chickens, piglets, calves, pregnant sows, sheep, human, fish Carcinogenic; attacks liver; reduced growth rate; hemmorrhagic enteritis; suppression of natural immunity to infection; decreased production of meat, milk, and eggs. Ochratoxins Swine, duckling, chicken, human Toxic to kidneys and liver; abortion; poor feed conversion, reduced growth rate, reduced immunity to infection. Food refusal by swine; vomiting and diarrhea; reduction in weight gain. Deoxynivalenol (Vomitoxin) T-2 Toxin Swine, cattle, chicken, turkey, horse, human Oral lesions. Severe inflammation of gastrointestinal tract and possible hemorrhage; edema; infertility; degeneration of bone marrow; reduced weight gain; slow growth; sterility. Zearalenone Swine, dairy cattle, turkey, lamb Estrogenic effects (edema of vulva, prolapsed vagina, enlargement of uterus), abortion, infertility, stunting. Atrophy of testicles, ovaries, enlargement of mammary glands. Fumonisin B 1, B 2 Horses Leucoencephalomalacia, "blind staggers, " in horses. DRTaylor Consulting

Recommended Acceptable Levels FDA Guidelines on maximum levels of Aflatoxin, Vomitoxin and Fumonisin in Recommended Acceptable Levels FDA Guidelines on maximum levels of Aflatoxin, Vomitoxin and Fumonisin in feedstuffs for animals Aflatoxin M 1 Aflatoxin B 1 < 0. 5 ppb in milk < 20 ppb in feeds Vomitoxin 5 ppm swine 10 ppm cattle, poultry Fumonisins 5 ppm horses 10 ppm swine 50 ppm beef cattle and poultry THE ROLE OF MYCOTOXINS IN FOOD AND FEED SAFETY Jon Ratcliff — Food and Agriculture Consultancy Services, UK www. facs. org. uk DRTaylor Consulting

Recommended Acceptable Levels EU Maximum permitted levels of mycotoxins in animal feed and foods Recommended Acceptable Levels EU Maximum permitted levels of mycotoxins in animal feed and foods for human consumption Aflatoxin B 1 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 DRTaylor Consulting

What can be done? Limited options Ø Only buy uncontaminated grains • But difficult What can be done? Limited options Ø 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. Ø 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. DRTaylor Consulting

Early History of HSCAS as Aflatoxin Binder Ø 1988 – Phillips, Taylor, Kubena, Harvey Early History of HSCAS as Aflatoxin Binder Ø 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) Ø 1989 – Harvey, Phillips, Kubena, et al. show HSCAS protects SWINE against AFB 1 (Amer. J. Vet. Res. , 50, 416 -420) Ø 1991 – Kubena, Huff, Harvey, et al. show HSCAS protects TURKEYS against AFB 1 (Poultry Sci. , 70, 1823 -1830) Ø 1991 – Harvey, Kubena, Phillips, et al. show HSCAS protects LAMBS against AFB 1 (Amer. J. Vet. Res. , 57, 152 -156) Ø 1994 – Phillips, Harvey, Kubena, et al. , show HSCAS protects GOATS against AFB 1 (J. Anim. Sci. , 72, 677 -682) DRTaylor Consulting

Evaluating Mycotoxin Binders Ø Two possible approaches… • In-vivo testing • Uses live animals Evaluating Mycotoxin Binders Ø 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. DRTaylor Consulting

Comparison: in-vitro vs. in-vivo testing Ø IN-VIVO • In-vivo tests are very expensive. It Comparison: in-vitro vs. in-vivo testing Ø IN-VIVO • In-vivo tests are very expensive. It usually cost $15 K-$20 K 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. Ø 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. DRTaylor Consulting

Does in-vitro binding correlate with -vivo binding? in Ø The answer is YES… …at Does in-vitro binding correlate with -vivo binding? in Ø The answer is YES… …at least for aflatoxin B 1 in-vitro versus in-vivo testing. DRTaylor Consulting

Correlation: In-vivo Wt. Gain vs. In-vitro Binding Activity 1 Broilers: Aflatoxin Challenge - 3000 Correlation: In-vivo Wt. Gain vs. In-vitro Binding Activity 1 Broilers: Aflatoxin Challenge - 3000 PPB Conclusion: In-vitro binding correlates with in-vivo response Pos. Control (0 ppb AFB 1/ 0 binder) Binders: Increasing Binding Activity (3000 ppb AFB 1/ 0. 5% binder) Neg. Control (3000 ppb AFB 1/ 0 binder) 2002, Dr. Carlos Mallman, Univ. Federal de Santa Maria, Brazil 1 DRTaylor Consulting

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

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

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

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

Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Mineralogy Product Code Aflatoxin-% Binding ( Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Mineralogy Product Code Aflatoxin-% Binding ( 20 µg / m. L ) Mineralogical Composition ( basis XRD analysis ) -----------------------------------------------------------------------A 99. 2 Excellent Ca montmorillonite…………. + Qz, FS B 97. 1 Ca/Na montmorillonite………. . + Qz Binding C 95. 2 Ca montmorillonite (low level)…. . + Opal CT, Qz D 95. 9 Attapulgite + Ca montmorillonite. + Qz E 94. 6 Sepiolite F 92. 6 Ca montmorillonite G 90. 7 Ca/Na montmorillonite……………. + Qz -----------------------------------------------------------------------H 87. 5 Na montmorillonite………………… + FS I 87. 3 Na/Ca montmorillonite……………. + Qz Good J 86. 4 Na montmorillonite Binding K 85. 2 Attapulgite (low level) L 83. 6 Attapulgite M 80 Ca montmorillonite………. …. . + Qz -----------------------------------------------------------------------N 73. 9 Ca montmorillonite……………. …… + Opal CT Poor O 70. 5 Ca montmorillonite (low level)…. … + Qz, FS Binding P 66. 2 Ca montmorillonite Q 55. 9 Clinoptilolite, mordenite……. . + Qz Inadequate R 47. 7 Kaolinite + mica/illite S Binding 44 Ca montmorillonite (low level) T 25. 2 Amorphous silica U 16. 9 Amorphous silica DRTaylor Consulting

Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Physical Properties Product Slurry Loose Density Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Physical Properties Product Slurry Loose Density Hg PV Hg Pore BET Surface Code p. H (Kg/m 3) (cc/g) Diameter (µm) Area (m²/g) -------------------------------------------------------------A 6. 9 806 0. 5119 0. 02 65 B 9. 2 1050 0. 1533 0. 028 25 a C 4. 0 602 0. 4156 0. 09; 0. 015 97 D 7. 5 607 0. 5081 0. 05 140 E 8. 5 559 0. 4432 0. 028 195 F 9. 5 751 0. 1213 0. 03 57 G 9. 82 701 0. 2713 0. 03 77 H 9. 1 1048 0. 2473 0. 038 21 I 9. 12 632 0. 5546 0. 065 31 J 9. 2 1067 0. 0848 0. 07 21 K 9. 5 663 0. 3849 0. 03 32 L 6. 8 812 0. 1785 0. 035 64 M 8. 35 777 0. 1292 0. 03 81 N 6. 84 761 0. 212 0. 03 82 O 8. 4 657 0. 3088 0. 4 14 P 9. 1 830 0. 2414 0. 04 75 Q 9. 7 990 0. 1844 0. 05 18 R 5. 1 342 0. 6061 0. 68 25 S 6. 4 693 0. 1776 0. 025 8 a T 10 215 0. 9556 0. 19; 1. 0 66 U 6. 8 350 1. 6362 0. 11 72 a Bimodal distribution of porosity; two maxima in pore volume versus pore diameter plots. DRTaylor Consulting

Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Chemical Properties Product Code Surface Acidity Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Chemical Properties Product Code Surface Acidity ( meq/g ) p. Ka <1. 5 p. Ka >1. 5 Total Cation Exchange Capacity ( meq/100 g ) Ca ++ Na+ Total ---------------------------------------------------------------------A 0. 079 0. 089 0. 168 52. 0 9. 0 76 B 0. 021 0. 053 0. 074 62. 0 53. 0 119 C 0. 080 0. 160 28. 6 0. 4 48 D 0. 080 0. 130 0. 210 20. 0 39 E 0. 180 0. 040 0. 220 54. 1 1. 5 77 F 0. 060 0. 200 0. 260 59. 8 3. 0 102 G 0. 021 0. 029 0. 049 110. 0 86. 0 207 H 0. 039 0. 142 0. 181 38. 0 75. 0 121 I 0. 010 0. 032 0. 042 49. 0 53. 0 127 J 0. 042 0. 106 0. 148 42. 0 67. 0 125 K 0. 020 0. 110 0. 130 63. 6 26. 3 121 L 0. 080 0. 020 0. 100 39. 3 31. 8 79 M 0. 030 0. 140 0. 170 68. 0 12. 0 102 N 0. 117 0. 076 0. 193 111. 0 2. 0 122 O 0. 039 0. 100 0. 139 76. 0 2. 0 82 P 0. 050 0. 130 0. 180 38. 7 19. 9 63 Q 0. 021 0. 020 0. 040 144. 0 57. 0 215 R 0. 020 0. 001 0. 020 2. 8 0. 6 7 S 0 0. 020 41. 2 21. 1 75 T 0 0. 060 1. 3 183. 2 185 U N/A N/A 1. 2 245. 9 248

In-Vitro Binding vs. Physical / Chemical Properties of Mycotoxin Binders RESULTS Ø No single In-Vitro Binding vs. Physical / Chemical Properties of Mycotoxin Binders RESULTS Ø No single physical property correlates with in-vitro binding of aflatoxin Ø No single chemical property correlates with in-vitro binding of aflatoxin Ø … however, there is a weak correlation with combination of strong surface acidic sites (of p. Ka <1. 5) and Mg++ concentration In-Vitro Binding (%) 100 80 60 40 20 0 0 20 40 60 80 ln [ (<1. 5 p. Ka) x (Mg++conc. ) ] 100 120

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

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

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

Recent Dioxin Analysis on Flo-Bond All samples below 1. 5 ppt EEC limit for Recent Dioxin Analysis on Flo-Bond 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 DRTaylor Consulting

2010 Competitive in-vitro Mycotoxin Binder Study METHODOLOGY Mycotoxins / Aflatoxin B 1, Deoxynivalenol, Fumonisin 2010 Competitive in-vitro Mycotoxin Binder Study METHODOLOGY Mycotoxins / Aflatoxin B 1, Deoxynivalenol, Fumonisin B 1, Ochratoxin A, Concentration T-2 Toxin, Zearalenone – all @ 2000 ppb Binder Level: 0. 5 wt% Binding Conditions: Adsorption phase: 3 reps. @ p. H 3. 0 Desorption phase: 3 reps. @ p. H 8. 0 % Efficiency = % Adsorption @ p. H 3. 0 - % Desorption @ p. H 8. 0 Binders Studied Flo-Bond, Flo-Bond AZ (experimental), Flo-Bond X (experimental), Nova. Sil Plus, Myco. Ad New experimental products (Brookside-Agra currently conducting in-vivo trials) DRTaylor Consulting

2010 Competitive in-vitro Mycotoxin Binder Study RESULTS Flo-Bond % Adsorption p. H 3 % 2010 Competitive in-vitro Mycotoxin Binder Study RESULTS Flo-Bond % Adsorption p. H 3 % Desorption p. H 8 % Efficiency AFB 1 100 0 100 FUM 95 87 8 DON 20. 6 15. 9 4. 7 OCHRA 77. 8 0 T-2 23. 7 0 23. 7 ZONE 31 30. 8 0. 2 New Flo-Bond AZ (AFB 1/ZONE) % Adsorption p. H 3 % Desorption p. H 8 % Efficiency 100 0 100 90 49. 4 40. 6 22. 6 15. 6 7 94. 4 93. 3 1. 1 67. 2 7. 3 59. 9 96. 5 4. 4 92. 1 NEW Flo-Bond X (Full Spectrum) % Adsorption p. H 3 % Desorption p. H 8 % Efficiency 99. 9 0. 3 99. 6 95. 3 7. 9 87. 4 36. 7 12. 8 23. 9 98. 1 9. 2 88. 9 98. 3 0 98. 3 99. 8 0. 3 99. 5 NS Plus % Adsorption p. H 3 % Desorption p. H 8 % Efficiency 100 0. 1 99. 9 99. 2 86. 2 13 27. 3 20. 8 6. 5 95. 2 95. 1 0. 1 40. 8 0 40. 8 74. 9 35. 6 39. 3 Myco. Ad % Adsorption p. H 3 % Desorption p. H 8 % Efficiency 100 0 100 98. 4 94. 7 3. 7 17. 6 14. 6 3 91. 8 89. 3 2. 5 35. 4 0 35. 4 50. 7 42. 6 8. 1 Results by Trilogy Analytical Lab, Missouri, USA DRTaylor Consulting

2010 Competitive in-vitro Mycotoxin Binder Study CONCLUSIONS General While all mycotoxins studied (except DON) 2010 Competitive in-vitro Mycotoxin Binder Study CONCLUSIONS General While all mycotoxins studied (except DON) were significantly bound in -vitro under acidic (p. H 3) conditions, some were significantly, or completely desorbed under basic (p. H 8) conditions. Aflatoxin B 1 Aflatoxin strongly bound by all binders in-vitro under acidic (p. H 3) & basic (p. H 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 DRTaylor Consulting

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

Binding of the diketone moiety to cationic sites: the reason for the strong binding 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 Clay Structure Ca++ This forms a type of complex called a CHELATE DRTaylor Consulting

Binding of the diketone moiety to cationic sites: the reason for the strong binding 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 + Clay Structure Ca++ These sites can also bind aflatoxin as a CHELATED complex DRTaylor Consulting

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

Chicken Feeding Trial: FLO-BOND vs. Nova. Sil : 2500 ppb Aflatoxin B 1 0 Chicken Feeding Trial: FLO-BOND vs. Nova. Sil : 2500 ppb Aflatoxin B 1 0 ppb AFB 1 / no binder 2500 ppb AFB 1 /. 75% FB 2500 ppb AFB 1 /. 5% NS 2500 ppb AFB 1 /. 5% FB Average Body Weight (gms) 350 300 2500 ppb AFB 1 /. 25% FB 2500 ppb AFB 1 / no binder 250 200 Therefore 0. 25% FLO-BOND = 0. 5% Nova. Sil 0. 5% FLO-BOND better than 0. 5% Nova. Sil 150 100 50 0 1 2 3 4 Week DRTaylor Consulting

2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge Ø Evaluation of FLO-BOND in 2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge Ø Evaluation of FLO-BOND in growing pigs fed 0. 9 ppm and 1. 8 ppm deoxynivalenol (DON) contaminated diets Ø 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 Ø Location: Virginia Diversified Research, Corp. , Harrisonburg, VA; investigator: Michael D. Sims DRTaylor Consulting

2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge RESULTS FLO-BOND vs. DON @ 2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge RESULTS FLO-BOND vs. DON @ 0. 9 ppm FLO-BOND lbs/ton 0 5 10 DON (ppm) 0 0 0. 9 Day 0 -7 ADG (lbs/d) 1. 982 a 1. 893 a 0. 693 c 1. 430 b 1. 307 b Day 0 -7 Feed/Gain (lb/1 b) 1. 324 a 1. 284 a 2. 415 c 1. 632 b 1. 663 b Feed Consumption (lb) 18. 36 a 16. 99 ab 12. 30 c 16. 66 b 15. 09 b FLO-BOND vs. DON @ 1. 8 ppm FLO-BOND lbs/ton: 0 5 10 DON (ppm): 0 0 1. 8 Day 0 -10 ADG (lbs/d) 1. 982 a 1. 893 a 0. 566 c 1. 079 b 0. 779 bc Day 0 -10 Feed/Gain (lb/lb) 1. 324 a 1. 284 a 8. 544 c 1. 834 b 2. 050 b Feed Consumption (lb) 18. 36 a 16. 99 a 11. 41 d 13. 4 b 10. 61 c DRTaylor Consulting

FLO-BOND Does Not Hinder Uptake of Nutrients Nutrient Effect on Level of HSCAS Nutrient FLO-BOND Does Not Hinder Uptake of Nutrients Nutrient Effect on Level of HSCAS Nutrient Utilization Riboflavin Vitamin A Manganese Zinc Phosphorus, inorganic 0. 5 % 1. 0 % 0. 5 % Phosphorus, inorganic Phosphorus, phytate 1. 0 % 0. 5 % 1. 0 % None None Slight tibia Zn decrease None 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 DRTaylor Consulting

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

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

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

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

Effect of Particle Size (Grind) vs. Binding of Aflatoxin B 1 95 90 Increasing Effect of Particle Size (Grind) vs. Binding of Aflatoxin B 1 95 90 Increasing fineness of grind % -325 Mesh 85 80 75 70 65 60 55 f lev g sin o el g din bin ea r Inc Therefore, it is very important to get good grinding in order to get maximum binding 50 65 70 75 80 % AFB 1 Bound 85 90 95 DRTaylor Consulting

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

SUMMARY AND CONCLUSIONS Ø Montmorillonite is the most common commercially available mycotoxin binder. Ø SUMMARY AND CONCLUSIONS Ø Montmorillonite is the most common commercially available mycotoxin binder. Ø Aflatoxin B 1 is the most strongly adsorbed mycotoxin. Ø FLO-BOND is (HSCAS) montmorillonite binder that possess superior mycotoxin binding characteristics for many different mycotoxins. Ø Manufacturing conditions (temperature, grind) affect binding performance, so good quality control is absolutely essential. Ø Brookside-Agra is committed to good quality control during the manufacturing of FLO-BOND Ø USDA approved as organic product Ø FLO BOND is dioxin free – (i. e. - below EEC limits for dioxin content) DRTaylor Consulting

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