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

About Mycotoxins Apspergillus flavus → produces aflatoxins 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 Apspergillus flavus → produces aflatoxins Aspergillus flavus growing on corn Aspergillus flavus magnified Mycotoxins are elaborated (produced) by molds that grow on grains and other feedstuffs. There are over 370 different mycotoxins which have been identified thus far. Mycotoxin are specific chemical compounds. Once they are produced, they remain in the sample even if the mold is killed. * Handbook of Toxic Fungal Metabolites – Cole / Cox (1981) DRTaylor Consulting

About Mycotoxins Fusarium toxins - Deoxynivalenol (vomitoxin), T-2, zearalenone Fusarium graminearum → produces vomitoxin, T-2, zearalenone Mycotoxins are elaborated (produced) by molds that grow on grains and other feedstuffs. There are over 370 different mycotoxins which have been identified thus far. Mycotoxin are specific chemical compounds. Once they are produced, they remain in the sample even if the mold is killed. 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. Mycotoxins are hard to avoid and almost impossible to get rid of. And they are potent at very low concentrations in an animal’s feed. DRTaylor Consulting

About Mycotoxins Different mycotoxins affect different organs. Here we see the typically fatty liver caused by aflatoxicosis compared to a normal liver. Fatty liver due to aflatoxin (left) compared to normal liver (right) (Ledoux UMC) DRTaylor Consulting

About Mycotoxins Only six mycotoxins are of real commercial concern… Aflatoxin B1 Deoxynivalenol (“vomitoxin”) Fumonisin B Ochratoxin A Zearalenone T-2 toxin The six mycotoxins of commercial interest are: aflatoxins (particularly B1), a known carcinogen, deoxynivalenol (abbreviated DON and sometimes called “vomitoxin” because it causes certain animals to vomit when ingested), fumonisin which many also be a natural carcinogen, ochratoxin, zearalenone and T-2 toxin. DRTaylor Consulting

Effects of Mycotoxins on Animals Affected Commercial Species EFFECTS on ANIMALS Aflatoxins B1, B2, G1, and G2 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. Deoxynivalenol (Vomitoxin) Swine, cattle, chicken, turkey, horse, human Food refusal by swine; vomiting and diarrhea; reduction in weight gain. T-2 Toxin Oral lesions. Severe inflammation of gastrointestinal tract and possible hemorrhage; edema; infertility; degeneration of bone marrow; reduced weight gain; slow growth; sterility. This table shows the manifestations of the various mycotoxins on various animals. 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 B1, B2 Horses Leucoencephalomalacia, "blind staggers," in horses. DRTaylor Consulting

Recommended Acceptable Levels FDA Guidelines on maximum levels of Aflatoxin, Vomitoxin and Fumonisin in feedstuffs for animals Aflatoxin M1 Aflatoxin B1 < 0.5 ppb in milk < 20 ppb in feeds Vomitoxin 5 ppm swine 10 ppm cattle, poultry The FDA (USA) has made recommendations for acceptable levels of various mycotoxin in feedstuffs for animals. 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 8

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 EU has made recommendations for permissible levels of aflatoxin and ochratoxin in various commodities. 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 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. There are limited options for dealing with the problem of mycotoxins in animal feeds. The use of mycotoxin binders is the most cost effective. DRTaylor Consulting

Early History of HSCAS as Aflatoxin Binder 1988 – Phillips, Taylor, Kubena, Harvey show 0.5 wt% hydrated sodium calcium aluminosilicate (HSCAS) protects CHICKENS against 7.5 ppm AFB1 (Poultry Sci., 67, 243-247) 1989 – Harvey, Phillips, Kubena, et al. show HSCAS protects SWINE against AFB1 (Amer. J. Vet. Res., 50, 416-420) 1991 – Kubena, Huff, Harvey, et al. show HSCAS protects TURKEYS against AFB1 (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 AFB1 (J. Anim. Sci., 72, 677-682) At last count (2006) there were over 200 peer-reviewed articles devoted to the subject of mycotoxin binding by various binding agents. 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. Approaches to evaluating the efficacy of mycotoxin binders. DRTaylor Consulting

Comparison: in-vitro vs. in-vivo testing 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. In-vivo vs. in-vivo testing (pros and cons). 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. DRTaylor Consulting

Correlation: In-vivo Wt. Gain vs. In-vitro Binding Activity1 Broilers: Aflatoxin Challenge - 3000 PPB Conclusion: In-vitro binding correlates with in-vivo response Pos. Control (0 ppb AFB1/ 0 binder) Binders: Increasing Binding Activity (3000 ppb AFB1/ 0.5% binder) In this plot, the in-vivo weight gain (by broilers challenged with 3 ppm aflatoxin) is compared to the in-vitro binding by a series of binders of increasing aflatoxin binding activity. As shown, in-vivo weight gain increases as the binding activity of the binders increases. Neg. Control (3000 ppb AFB1/ 0 binder) 1 2002, Dr. Carlos Mallman, Univ. Federal de Santa Maria, Brazil DRTaylor Consulting

An in-vitro approach to finding the answer 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/m3) • Porosity (cc/g) • Pore diameter (µm) • Surface area (m2/g) Here are shown pictures of the type of binding clay (montmorillonite) used to make Flo-Bond. Also listed are some of the physical properties that can be measured such as density, porosity, pore diameter and surface area. This type of clay is characterized by extensive porosity and high surface area compared to other minerals. Scanning Electron Microscopy at high magnification DRTaylor Consulting

Chemical properties of montmorillonite • Surface acidity (pKa) • Cation Exchange Capacity (meq/100 g) • Exchange cations (Na+, K+, H+, Mg++, Ca++) • pH of clay slurry in water Exchangeable cations → Ca++ Here is shown a diagram of the (montmorillonite) clay structure on the atomic level. Such clays exhibit a number of chemical properties including surface acidity, cation exchange capacity, and differences in the types of exchangeable cation in the interlayer region. DRTaylor Consulting

Protocol of the study Obtain a representative group of commercially available binders. Measure aflatoxin B1 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. This slide presents the protocol for an in-vitro methodology for studying groups of commercially available mycotoxin binders to determine which physical or chemical properties (or combinations of physical and chemical properties) are important for binding of mycotoxins. DRTaylor Consulting

Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Mineralogy Product Aflatoxin-% Binding Mineralogical Composition Code ( 20 µg / mg / mL ) ( basis XRD analysis ) ----------------------------------------------------------------------------------------------------------------------------------------------- A 99.2 Ca montmorillonite…………..……. + Qz, FS B 97.1 Ca/Na montmorillonite………..….. + Qz 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 J 86.4 Na montmorillonite K 85.2 Attapulgite (low level) L 83.6 Attapulgite M 80 Ca montmorillonite……….…….….. + Qz N 73.9 Ca montmorillonite…………….…… + Opal CT O 70.5 Ca montmorillonite (low level)….… + Qz, FS P 66.2 Ca montmorillonite Q 55.9 Clinoptilolite, mordenite…….…….. + Qz R 47.7 Kaolinite + mica/illite S 44 Ca montmorillonite (low level) T 25.2 Amorphous silica U 16.9 Amorphous silica Excellent Binding Good Binding Two major points are illustrated by this slide. 1) The majority (62%) of products used to bind aflatoxin are montmorillonite clay. 2) Just because one has a montmorillonite clay does not insure it will be an excellent, or even a good binder. This points to the need for any supplier of a mycotoxin binder to provide in-vitro and most importantly, in-vivo data supporting his claims. Poor Binding Inadequate Binding DRTaylor Consulting

Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Physical Properties Product Slurry Loose Density Hg PV Hg Pore BET Surface Code pH (Kg/m3) (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 C 4.0 602 0.4156 0.09; 0.015a 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 T 10 215 0.9556 0.19; 1.0a 66 U 6.8 350 1.6362 0.11 72 a Bimodal distribution of porosity; two maxima in pore volume versus pore diameter plots. This slide lists the physical properties measured (slurry pH, loose density, mercury pore volume, mercury pore diameter and BET surface area). DRTaylor Consulting

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 pKa >1.5 Total 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 0.080 0.160 28.6 0.4 48 D 0.080 0.130 0.210 20.0 3.0 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 0.020 41.2 21.1 75 T 0 0.060 0.060 1.3 183.2 185 U N/A N/A N/A 1.2 245.9 248 This slide lists the chemical properties measured (surface acidity; for strong acid sites - below pKa 1.5, for weak acid sites – above pKa 1.5; total cation exchange capacity; and specific exchange capacity for Ca++ and Na+ cations).

ln [ (<1.5 pKa) x (Mg++conc.) ] 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 surface acidic sites (of pKa <1.5) and Mg++ concentration 20 40 60 80 100 In-Vitro Binding (%) This slide shows that the best correlation between in-vitro binding and physicochemical properties is obtained from a mathematical function derived as the natural log of the product between concentration of strong surface acidity sites (pKa < 1.5) and Mg+2 20 40 60 80 100 120 ln [ (<1.5 pKa) x (Mg++conc.) ]

Optimum binding – like tumblers in a lock… 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… DRTaylor Consulting

compare to the competition? 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

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 ncy Mycotoxins / Concentration Binder Level: Binding Conditions: % Efficie Binders Studied 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 % De sorption @ pH 8.0 Flo - Bond, Flo Bond AZ (experimental), Flo Bond X (experimental), NovaSil Plus, MycoAd This slide shows the protocol for a recent in-vitro binding study of Flo-Bond (including some new experimental products) versus two well-known mycoto New experimental products (Brookside-Agra currently conducting in-vivo trials) DRTaylor Consulting 28

2010 Competitive in-vitro Mycotoxin Binder Study RESULTS AFB1 FUM DON OCHRA T-2 ZONE 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 3 100 95 20.6 77.8 23.7 31 87 15.9 30.8 8 4.7 0.2 100 90 22.6 94.4 67.2 96.5 49.4 15.6 93.3 7.3 4.4 40.6 7 1.1 59.9 92.1 99.9 95.3 36.7 98.1 98.3 99.8 0.3 7.9 12.8 9.2 99.6 87.4 23.9 88.9 99.5 This slide shows that FLO-BOND is an effective in-vitro binder for a number of key mycotoxins including: aflatoxin B1 and T-2 toxin as are the competitive binders. Note that the new experimental FLO-BOND products are also effective for zearalenone (FB AZ) and a combination of other mycotoxins (FB X) 100 99.2 27.3 95.2 40.8 74.9 0.1 86.2 20.8 95.1 35.6 99.9 13 6.5 39.3 100 98.4 17.6 91.8 35.4 50.7 94.7 14.6 89.3 42.6 3.7 3 2.5 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) 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 DRTaylor Consulting 30

Why does aflatoxin bind so strongly compared to other mycotoxins? Some similarity here Some similarity between these and here …but none here Deoxynivalenol This grouping causes strong binding Although ochratoxin and zearalenone contain a related 1,3-ketoalcohol moiety which is structurally similar to the 1,3-diketone found in aflatoxin B1, other mycotoxins like deoxynivalenol (vomitoxin), T-2 toxin and fumonision lack this similarity. Ochratoxin A Zearalenone DRTaylor Consulting

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 Ca++ Clay Structure Diagram showing the binding of 1,3-diketone moiety of aflatoxin to cationic site in montmorillonite structure. This forms a type of complex called a CHELATE DRTaylor Consulting 6

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 Ca++ Clay Structure DRTaylor Consulting 6

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

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

In-Vitro Binding Data - Binding Conditions Very Important !! Make Sure You Read the Fine Print ! Let’s look a little more carefully at the binding conditions Percentage Binding 10 20 30 40 50 60 70 80 90 100 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) When you compare the specific binding conditions employed for Binder #1, you see that 10 – 50 times as much binder was used for fumonisin, zearalenone and ochratoxin binding as was used for Binder #2. In fact, Binder #1 and Binder #2 were the same binder – just different binder levels were used. In the case of Binder #2, all four toxins were utilized at the same binding level (4 μg toxin/1 mL solution/1 mg binder) and this experiment more clearly shows the relative capacity for binding of mycotoxins is: aflatoxin >>> fumonisin > zearalenone > ochratoxin. Binder #1 Binder #2 Binder #1 = #2 = Flo-Bond: Just Different Levels of Binder DRTaylor Consulting

IN-VIVO DATA USING FLO-BOND DRTaylor Consulting

Chicken Feeding Trial: FLO-BOND vs. NovaSil : 2500 ppb Aflatoxin B1 0 ppb AFB1 / no binder 2500 ppb AFB1 / .75% FB 50 100 150 200 250 300 350 Average Body Weight (gms) 2500 ppb AFB1 / .5% NS 2500 ppb AFB1 / .5% FB 2500 ppb AFB1 / .25% FB 2500 ppb AFB1 / no binder Therefore 0.25% FLO-BOND = 0.5% NovaSil 0.5% FLO-BOND better than 0.5% NovaSil This graph shows the results of an in-vivo binding study with chickens using Flo-Bond versus NovaSil against a 2.5 ppm aflatoxin B1 challenge. Note that by week 4, 0.5% FB has returned the aflatoxin-challenged birds to a body weight equal to the unchallenged birds. Note also that by week 4, 0.25% FB is equal to 0.5% NovalSil and 0.5% FB is superior to 0.5% NovaSil. 1 2 3 4 Week DRTaylor Consulting

Protective Effect 0.275% Flo-Bond vs. 2000 PPB Ochratoxin: Broilers* Liver: Macroscopic Results 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Percentage (%) GRADE 1 GRADES 2-4 Control Control + Flo-Bond Control + 2000 ppb Ochratoxin + Flo-Bond Control + 2000 ppb Ochratoxin This slide shows that 0.275% Flo-Bond provides some protection against the effects of a 2 ppm ochratoxin challenge (broilers) on the basis of liver grading scores. Test period: 7 – 28 days of age Groups: 4 rations (see graph) * 64 male broilers/ 16 chicks per ration DRTaylor Consulting

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 This slide shows the protocol for a recent in-vivo study with swine using 0.25% and 0.5% Flo-Bond against deoxynivalenol (0.9 ppm and 1.8 ppm). DRTaylor Consulting

2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge RESULTS FLO-BOND vs. DON @ 0.9 ppm FLO-BOND lbs/ton 5 10 DON (ppm) 0.9 Day 0-7ADG (lbs/d) 1.982a 1.893a 0.693c 1.430b 1.307b Day 0-7 Feed/Gain (lb/1b) 1.324a 1.284a 2.415c 1.632b 1.663b Feed Consumption (lb) 18.36a 16.99ab 12.30c 16.66b 15.09b FLO-BOND vs. DON @ 1.8 ppm This slide shows that 0.25% as well as 0.5% Flo-Bond provides protection against the DON challenges of 0.9 ppm and 1.8 ppm. The best results were obtained when Flo-Bond was used at the 0.25% level. FLO-BOND lbs/ton: 5 10 DON (ppm): 1.8 Day 0-10 ADG (lbs/d) 1.982a 1.893a 0.566c 1.079b 0.779bc Day 0-10 Feed/Gain (lb/lb) 1.324a 1.284a 8.544c 1.834b 2.050b Feed Consumption (lb) 18.36a 16.99a 11.41d 13.4b 10.61c DRTaylor Consulting

FLO-BOND Does Not Hinder Uptake of Nutrients Effect on Nutrient Level of HSCAS Nutrient Utilization Riboflavin 0.5 % None 1.0 % Vitamin A Manganese Zinc Slight tibia Zn decrease Phosphorus, inorganic Phosphorus, phytate The question comes up as to whether HSCAS clays will interfere with the utilization of other essential nutrients (i.e. water soluble vitamins, oil-soluble vitamins, and minerals). Experiments conducted by Baker et al. showed no effect on the utilization of riboflavin, vitamin A, manganese, zinc or phosphorus, when HSCAS was used at levels of 0.5 wt% in the diets. There was a very slight negative effect on zinc utilization at the 1 wt% level. In practice, very few farmers use binders at this high a level. 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 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

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

Effect of Thermal Processing on Surface Area vs Effect of Thermal Processing on Surface Area vs. Binding of Aflatoxin B1 Increasing Processing Temperature 40 50 60 70 80 90 100 Aflatoxin Binding (%) Therefore, it is very important not to overheat clay that is to be used as mycotoxin binder While there is no correlation between in-vitro binding of aflatoxin and surface area over a range of different binders, it is quite clear that high surface area for a given binder is desirable. In this experiment, the effect of increasing processing temperature causes the surface area to drop (from about 95 m2/g down to about 65 m2/g). Notice that aflatoxin binding drops as well. This indicates the importance of having good quality control during the manufacturing of a mycotoxin binder. 50 60 70 80 90 100 BET Surface Area (m²/g) DRTaylor Consulting

Effect of Particle Size (Grind) vs. Binding of Aflatoxin B1 70 75 80 85 90 95 % AFB1 Bound 65 50 55 60 % -325 Mesh Increasing level of binding Increasing fineness of grind Therefore, it is very important to get good grinding in order to get maximum binding This slide shows that in-vitro binding of aflatoxin (and other toxins) will increase with increasing fineness of the grind. 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. 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) DRTaylor Consulting

Thank you for your kind attention The End DRTaylor Consulting