CAPTEX T2

Effect of the different mycotoxins in on the different farmed animals SPECIES Poultry Broilers Layers Breeder Sheep Goats Fish Shrimp Mycotoxins Aflatoxin (AFB) Fumonisin (FB) Ochratoxin (OTA) T-2 Toxin (T-2) Zearalenone (ZEN) Vomitoxin or Deoxynivalenol (DON)

MYCOTOXIN CONCENTRATIONS IN COMPLETE FEED IN EUROPE (318 samples from 10 countries, 2012) FEED AF FUMONISIN OTA T2 DON ZEA % positive samples 84 66 65 39 57 37 Av. Concentration (ppb) 18 106 21 111 76

How does CAPTEX T2 deactivates mycotoxins (mode of action) 3 different strategies to counteract a broad spectrum of mycotoxins Preventing further mold growth Adsorption by minerals Biotransformation by enzymatic activity CAPTEX T2 combines in one product 3 different strategies to counteract a broad spectrum of mycotoxins Preventing further mold growth Adsorption by minerals Biotransformation by enzymatic activity

1. Preventing further mold growth Sodium Propionate acts as a mold inhibitor to prevent mold growth and proliferation minimizes risk of having mycotoxin-producing molds further proliferating in feed. has no activity against mycotoxins, but, always, when you ascertain mycotoxins presence, there is still a residual presence of molds. Sodium Propionate (the sodium salt of propionic acid) acts as a mold inhibitor to prevent mold growth and proliferation Thereby minimizing the risk of having mycotoxin-producing molds that further proliferate in the feed. Sodium Propionate has no activity against mycotoxins, but, always, when you ascertain mycotoxins presence, there is still a residual presence of molds.

1. Preventing further mold growth Minimum inhibitory concentration of propionic acid (g/kg diet) on bacteria and molds Bacteria Propionic Salmonella typhimurium 1.5 Pseudomonas aeroginosa 2.0 Escherichia coli Staphylococcus aureus 2.5 Listeria monocytogenes Campylobcter jejuni Clostridum botulinum Clostridum perfringes Molds Propionic Aspergillus Niger 2.5 Penicillum expansum 1.3 Fusarium nivale Cladosporium sp.

2. ABsorption by minerals Through a number of screening studies the best aluminosilicate adsorbents with regard to mycotoxin deactivation and safe application where selected. A premium blend of minerals was designed that as part of CAPTEX T2 product guarantees maximum, pH-independent activity at an inclusion rate as low as 1 kg/ton without removing essential nutrients from the diet. Through a number of screening studies the best aluminosilicate adsorbents with regard to mycotoxin deactivation and safe application where selected. A premium blend of minerals was designed that as part of CAPTEX T2 product guarantees maximum, pH-independent activity at an inclusion rate as low as 1 kg/ton without removing essential nutrients from the diet.

2. Adsorption by minerals CAPTEX T2 contains 2 main aluminosilicates Calcium Bentonite (montmorillonite) Zeolite (clinoptilolite) Through a number of screening studies the best aluminosilicate adsorbents with regard to mycotoxin deactivation and safe application where selected. A premium blend of minerals was designed that as part of CAPTEX T2 product guarantees maximum, pH-independent activity at an inclusion rate as low as 1 kg/ton without removing essential nutrients from the diet.

Bentonite / Montmorillonite 2. ABsorption by minerals Bentonite / Montmorillonite originally created from the breakdown (weathering) of volcanic ash. is a silicate with a layered polar crystalline microstructure which adsorbs organic substances either on its external surfaces or within its interlaminar spaces. Used to bind mainly Aflatoxin.

Molecular structures of Bentonite (Montmorillonite) 2. ABsorption by minerals Molecular structures of Bentonite (Montmorillonite) 2:1 clay structure: one octahedral sheet sandwiched between two tetrahedral sheets The mycotoxins that are bound by the montmorillonite are those that can physically enter into the interlayer space. The width of the interlayer space is 0.25 to 0.7 nanometers in the dry state and 1 nanometer in the hydrated state. Aflatoxins and ochratoxins can enter into this space Vimonses, V., Lei, S., Jin, B., Chow, C., Saint, C. (2009) Kinetic study and equilibrium isotherm analysis of Congo Red adsorption by clay materials. Chemical production Journal, Volume 148, Issues 2–3, 15, Pages 354–364

Zeolites / Clinoptilolite 2. ABsorption by minerals Zeolites / Clinoptilolite is of (sea or lake) sedimentation origin with a unique, complex crystalline structure. honeycomb (tetrahedral) framework of cavities and channels act like cages, trapping mycotoxins. Used to deactivate Zearalenone, Ochratoxin and Fumonisin.

molecular structures of Clinoptilolite 2. ABsorption by minerals molecular structures of Clinoptilolite 3-dimentional crystalline Structure with an 8-ring and 10-ring channels has a cage-like structure, with pores and channels running through the crystal. The cage and surrounding mineral carries a net negative charge, making it one of the few negatively charged minerals found in nature Manley and Holmes, 1989, Minerals with natural advantage, New Scientist, March 25, 1989, pp. 39-43

molecular structures of Bentonite (Clinoptilolite) 2. ABsorption by minerals molecular structures of Bentonite (Clinoptilolite) Because of its cage-like structure and negative charge, clinoptilolite has the ability to draw and trap within and on itself positively charged toxic particles that fit into the pores and channels of the cage It acts as molecular sieves and absorb substances of a low-molecular compounds (mycotoxins) Used to adsorb Zearalenone, Ochratoxin and Fumonisin, Deoxynivalenol Manley and Holmes, 1989, Minerals with natural advantage, New Scientist, March 25, 1989, pp. 39-43

2. ABsorption by minerals CAPTEX Belongs to a new generation of mycotoxin deactivators, which are classified as highly purified and activated clays. The minerals undergo a special process that involves; Physical treatment (Micronisation) Chemical treatment which greatly increases its adsorbent efficiency

Physical Treatment and active surface of CAPTEX T2 2. ABsorption by minerals Physical Treatment and active surface of CAPTEX T2 The high affinity of binding is due to the fact that the product is modified by the micronisation process to be extremely fine, 150,000 particles/gram. This provides for a larger surface area that increases the possibility of interacting with mycotoxins. Though extremely fine, caution is given not to have any particles size of less than 5 microns in order to avoid dustiness as well as inhalation issues with the people that are handling the product.

Chemical Treatment and CEC of CAPTEX T2 2. ABsorption by minerals Chemical Treatment and CEC of CAPTEX T2 CEC (Cationic Exchange Capacity) is important as it explains the water absorption capacity of the product. The lower the CEC the better it is, as its water absorption capacity decreases. In the other hand, if CEC is below of 35 then it starts losing its affinity to mycotoxins. CEC over 100, means that the product has high in water absorption capacity and consequently some nutrients can be trapped. CAPTEX T2 has a CEC of approximately 55. IDEAL!!

2. ABsorption by minerals CAPTEX T2 does not bind either nutrient or drugs! Maximum size of Captex T2 holes is 50 microns Name of compound Minimum particle size (microns) VITAMIN A 350 VITAMIN D3 200 VITAMIN B12 120 FOLIC ACID IRON SULFATE 250 ZINC SULFATE 160 TYLOSINE 150 AMOXYCILLIN Name of mycotoxins Maximum particle size (microns) AFLATOXIN 1 ZEARALENONE OCHRATOXIN FUMONISIN T2 VOMITOXIN

Physical & Chemical Treatment 2. ABsorption by minerals Physical & Chemical Treatment These treatments enable the minerals to form a stable irreversible complex that immobilizes the target mycotoxins in the gastrointestinal tract of animals and by reducing their bio-availability, they are prevented from being absorbed through the gut and into the blood circulation, so thus eliminated through faeces. 

3. Biotransformation is the enzymatic degradation of mycotoxins that leads to non- toxic metabolites. In this case, Chitinase is this hydrolytic enzyme capable of deactivating mycotoxins by degrading their molecules. Chitinase is incorporated into yeast cells by our patented process and becomes active only at intestinal level when yeast cells are lyzed by the intestinal enzymes and cell content is released. Chitinase is able to form non toxic de-epoxy metabolite by removing oxygen from the epoxide group of the trichothecene mycotoxin. This action mimics the detoxifying process carried out by carboxylesterase (a microsomal enzyme from liver) that selectively hydrolyses the C-4 acetyl group of T-2 toxin. Biotransformation, is the enzymatic degradation of mycotoxins that leads to non-toxic metabolites. In this case, Chitinase is this hydrolytic enzyme capable of deactivating mycotoxins by degrading their molecules. Chitinase is incorporated into the yeast cell by the patented process and becomes active only at intestinal level when the yeast cell is lyzed by the intestinal enzymes and the cell content is released. Chitinase is able to form non toxic de-epoxy metabolite by removing oxygen from the epoxide group of the trichothecene mycotoxin. This action mimics the detoxifying process carried out by carboxylesterase (a microsomal enzyme from liver) that selectively hydrolyses the C-4 acetyl group of T-2 toxin.

3. Biotransformation Molecule of T-2 and what happens to it after Biotransformation by Chitinase

Biotransformation of T-2 toxin This action mimics the detoxifying process carried out by carboxylesterase (a microsomal enzyme from liver) that selectively hydrolyses the C-4 acetyl group of T-2 toxin to yield HT-2 toxin CAPTEX reduces the toxicity of T-2 toxin by been able to form de-epoxy metabolites (which are essentially non toxic compounds), by removing oxygen from the epoxide group of the trichothecene mycotoxin. This action seems to mimic the detoxifying process carried out by carboxylesterase, a microsomal enzyme from liver, that selectively hydrolyses the C-4 acetyl group of T-2 toxin, to yield HT-2 toxin.

Not all glucomannans are the same! CAPTEX T2 Not all glucomannans are the same! Prior to their inclusion into CAPTEX-T2, Doxal’s Esterified Glucomannans are treated by another Chitinase, an enzyme which is able to reduce, dramatically, their chitin content. Chitin content is supposed to increase the alkali insolubility of β-glucans and to decrease the cell wall flexibility, so that the toxin molecule has a restricted access to the complexing chemical sites. Our product contains Glucomannans, in an esterified form. Glucomannans are known to be able to protect the intestinal mucosa from toxins adhesion. But it is very important that the natural content of chitin, in glucomannans, be the minimum possible, in order to increase the capacity of the substance to be “flexible”.

Vopato I. Bizzini B. -1998 Italian Project M.S.T./09/96 Glucans and Chitin contents of various sources of Saccharomyces cerevisiae Yeast Sources Total Glucans (%) (1.3)-glucans (%) (1.3)-glucans (%) Chitin (%) Saccharomyces cerevisiae 1 13.2 7.2 6.0 9.0 Saccharomyces cerevisiae 2 14.4 7.4 7.0 9.2 Saccharomyces cerevisiae 3 13.5 7.3 6.2 9.1 Saccharomyces cerevisiae 4 13.9 7.6 6.3 10.0 Doxal’s S.C. 2234 18.2 10.6 2.7 Doxal’s Yeast source for Glucomannans has a very high content of glucans, specially beta-Glucans, and an extremely low level of Chitin. Vopato I. Bizzini B. -1998 Italian Project M.S.T./09/96

Toxins binding capacity of three feed additives in vitro CAPTEX T2 TRIALS Toxins binding capacity of three feed additives in vitro Each mycotoxin was solved at the level of 50 µg into 200 ml of methanol, and kept under gentle stirring throughout the test period, in a 250 ml volumetric flask. 100 mcg and 250 mcg aliquots of each Clay, Bentonite and CAPTEX-T2 were solved into the flasks, and kept under gentle stirring for 20 minutes; one flask of each mycotoxins was left as blank. After 20 minutes, small aliquots were collected from each volumetric flask and then assayed by High Performance Liquid Chromathography. A test, carried out in vitro, comparing the capacity of three compounds to bind various mycotoxins, under same conditions. Each mycotoxin was solved at the level of 50 µg into 200 ml of methanol, and kept under gentle stirring throughout the test period, in a 250 ml volumetric flask. Seven replicates for each mycotoxin were prepared. 100 µg and 250mcg aliquots of each Clay, Bentonite and CAPTEX-T2 were solved into the flasks, and kept under gentle stirring for 20 minutes; one flask of each mycotoxins was left as blank. After 20 minutes, small aliquots were collected from each volumetric flask and then assayed by High Performance Liquid Chromathography, Column Kromasyl, fluorescence detector, excitation 274 nm, flow rate 1 mL/min, emission 440 nm.

Toxins binding capacity of three feed additives in vitro CAPTEX T2 TRIALS Toxins binding capacity of three feed additives in vitro AFLATOXIN CLAY AT 100 MCG 7.40 MCG CLAY AT 250 MCG 2.40 BENTONITE AT 100 MCG BENTONITE AT 250 MCG 1.00 CAPTEX-T2 AT 100 MCG 4.05 CAPTEX-T2 AT 250 MCG 0.00 BLANK 49.90 These results indicate that, out of 50 mcg of toxin, Clay at first dosage has been able to bind most of it, as only 7.40 mcg, at last, were found not bound to it. These results tend to demonstrate also that there is a dose-depending response. When Aflatoxin is the only contaminant, also very cheap and common minerals, like clay and/or bentonite are effective in binding the toxin. Anyhow Captex T-2, as 250mcg, was the only one able to bind 100% of the toxin.

Toxins binding capacity of three feed additives in vitro CAPTEX T2 TRIALS Toxins binding capacity of three feed additives in vitro ZEARALENONE CLAY AT 100 MCG 42.60 MCG CLAY AT 250 MCG 36.60 BENTONITE AT 100 MCG 27.70 BENTONITE AT 250 MCG 20.00 CAPTEX-T2 AT 100 MCG 15.10 CAPTEX-T2 AT 250 MCG 9.90 BLANK 49.70 When the contaminating toxin is Zearalenone, administration of Clay is performing very poorly. Bentonite has a limited effect. Captex-T2 is very effective. Again, there is a dose-depending response (higher dose, better results).

Toxins binding capacity of three feed additives in vitro CAPTEX T2 TRIALS Toxins binding capacity of three feed additives in vitro OCHRATOXIN CLAY AT 100 MCG 6.90 MCG CLAY AT 250 MCG 3.10 BENTONITE AT 100 MCG 7.00 BENTONITE AT 250 MCG 2.95 CAPTEX-T2 AT 100 MCG 3.60 CAPTEX-T2 AT 250 MCG 0.20 BLANK 50.10

Toxins binding capacity of three feed additives in vitro CAPTEX T2 TRIALS Toxins binding capacity of three feed additives in vitro FUMONISIN CLAY AT 100 MCG 47.48 MCG CLAY AT 250 MCG 46.10 BENTONITE AT 100 MCG 44.90 BENTONITE AT 250 MCG 42.50 CAPTEX-T2 AT 100 MCG 27.40 CAPTEX-T2 AT 250 MCG 15.05 BLANK 49.30 In case of Fumonisin, both Clay and Bentonite are ineffective.

TOXINS BINDING CAPACITY OF CAPTEX on T-2 toxin IN VITRO CAPTEX T2 TRIALS TOXINS BINDING CAPACITY OF CAPTEX on T-2 toxin IN VITRO T-2 TOXIN Activated carbon AT 100 MCG 45.63 MCG Activated carbon AT 250 MCG 42.14 CAPTEX-T2 AT 100 MCG 22.42 CAPTEX-T2 AT 250 MCG 15.21 BLANK 49.20 In case of Fumonisin, both Clay and Bentonite are ineffective.

Trial in vivo Poultry field - Results CAPTEX T2 TRIALS Trial in vivo Poultry field - Results Feed consumption Body weight mortality Plasmatic calcium g/day g % mM/L group A 24,7 344 1,66 2,180 ± 0,16 group B 16,4 265,4 8,33 1,64 ± 0,66 group C 23,1 326,4 2,09 ± 0,22 A total of 540 day-old chicks (Ross)(Gallus gallus) was randomly divided into three groups, of 180 birds each. Standard feed and water were provided ad libitum, for 20 consecutive days. Group A was fed standard blank feed (Control Positive). Group B was fed standard feed, contaminated by 3 ppm Aflatoxin (Control Negative) Group C was fed standard feed, contaminated by 3 ppm Aflatoxin and fortified by 0.3% CAPTEX (Test). The Aflatoxin used in this test was supplied by Bidifarm - Italy, and was produced by Aspergillus parasiticus. Body weights and feed consumption were recorded every seven days. Mortality was recorded daily. We may conclude that: the addition of 3 ppm Aflatoxin in feed for day-old chicks, fed continuously for 20 consecutive days, causes lower Feed Intake, lower Growth, increases Mortality and moreover it decreases Calcium Plasmatic levels. The addition of Captex to feed for day-old chicks is very effective in significantly reducing the toxicity effects caused by 3 ppm Aflatoxin fed continuously for 20 days.

Thanks for the kind attention