Dox-Al Subsidiaries Doxal Swiss Doxal France Doxal Iberica

Dox-Al Subsidiaries Doxal Swiss Doxal France Doxal Iberica
Doxal Taiwan Doxal Australia

Dox-Al Distributors

FOODS WHERE TOXIGENIC MOULDS AND THEIR TOXINS MAY BE FOUND
Fungal species Toxin Foods which may be contaminated Aspergillus Flavus Aflatoxins Corn, peanuts, copra Aspergillus parasiticus Some nuts Fusarium moniliforme Fumonisins Corn Fusarium graminearum Trichothecenes zearalenone Wheat, corn Claviceps purpurea Ergot alkaloide Rye Penicillium verrucosum Ochratoxins barley

FOODS WHERE TOXIGENIC MOULDS AND THEIR TOXINS MAY BE FOUND
Fungal species Toxin Foods which may be contaminated Penicillium expansum Patulin Apples, pears Penicillium crustosum Penitrem A Bread, feeds Fusarium sporotrichioides T-2 Rye, barley Phomopsis letostomiformis Phomopsin Lupin seed Stachybotrys spp. Stachytrytoxins Feeds

TARGET ORGANS OF SOME MYCOTOXINS
Aflatoxin Liver Ochratoxin A Kidney Zearalenone Female genital tract

MAXIMUM LIMITS OF AFLATOXINS IN FOODS (ASIA) (µ/kg)
China 50 Hong kong 20 (Peanuts/peanut products) India 30 Japan 10 Malaysia Philippines 20 Singapore Absence Sri lanka 30 (ground nuts, oil-seed, flour and cereals Thailand

Minimum aw for growth of toxigenic mould species
Aspergillus ochraceus 0.78 Penicillium verrucosum 0.79 Aspergillus flavus 0.80 Fusarium moniliforme 0.87 Stachybotrys atra 0.94

Temperature range for growth of toxigenic mould species
Min. Opt. Max °C Penicillium verrucosum 20 31 Aspergillus ochraceus 8 28 37 Aspergillus flavus 10 32 42 (aflatoxin formation) 12 25 Fusarium moniliforme 3

MYCOTOXIN CONCENTRATIONS IN COMPLETE FEED IN CHINA
AF FUMONISIN OTA T-2 DON ZEA % positive samples 100 92 94 Av. Concentration (ppb) 8.27 1020 12.75 41.23 600 83.96 Wang et al. (2003) - Asian Pork Magazine

MYCOTOXIN CONCENTRATIONS IN COMPLETE FEED IN EUROPE (600 samples from 12 countries)
AF FUMONISIN OTA T-2 DON ZEA % positive samples 91 74 65 46 84 Av. Concentration (ppb) 6 115 26 35 415 165 (in house data, 2004)

INDIVIDUAL EFFECT OF AFLATOXIN IN RATS
3.5mg aflatoxins (AF)kg of diet Male rats of 3 week of age, for 28 days. Body weight gains were reduced 28% FCR –18% Increased weights of both kidney, heart Decreased serum concentrations of total protein, albumin and inorganic phosphorus. Increased concentrations of creatinine. Vopato I. Bizzini B Italian Project M.S.T./09/96

Vopato I. Bizzini B. -1998 Italian Project M.S.T./09/96
Effects of a Modified Hydrated Sodium Calcium Aluminosilicate on Mycotoxicosis in Rats 2 groups: control and captex 0.2% Male rats of 3 weeks age Diets containing AF 5mg/kg diet Vopato I. Bizzini B Italian Project M.S.T./09/96

Effects of a Modified Hydrated Sodium Calcium Aluminosilicate on Mycotoxicosis caused by D.O.N. in Rats 2 groups: control and Captex T2 0.2% Male rats of 3 weeks age DON at 2 mg/kg of diet Vopato I. Bizzini B Italian Project M.S.T./09/96

DUODENUM – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

ILEUM - NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

KYDNEY-NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

LIVER - NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

STOMAC- NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

Effects of a Modified Hydrated Sodium Calcium Aluminosilicate (Captex T-2) on Mycotoxicosis in Rats 2 groups: control and Captex T2 0.2% Male rats of 3 weeks age T2 at 5 mg/kg diet Vopato I. Bizzini B Italian Project M.S.T./09/96

Effects of Captex T2 (Modified Hydrated Sodium Calcium Aluminosilicate enriched with Glucomannans and Chitinase) on Mycotoxicosis caused by Fumonisin in Rats 2 groups: control and Captex T2 0.2% Male rats of 3 weeks age FB1 3 mg/kg of diet Vopato I. Bizzini B Italian Project M.S.T./09/96

ILEUM – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

KYDNEY – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

LIVER – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

STOMAC – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

DUODENUM – NO ALTERATIONS Vopato I. Bizzini B Italian Project M.S.T./09/96

Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel
Effects of CAPTEX T-2 on Aflatoxin toxicity in chickens (Gallus gallus) 3 groups (180 birds each) Standard feed and water ad libitum for 20 consecutive days Group A: standard blank feed Group B: standard feed contaminated by 3 ppm AFLATOXIN Group C: standard feed contaminated by 3 ppm Aflatoxin and fortified by 0.3% CAPTEX T-2 Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

RESULTS: Mean individual feed consumption (g/day) GROUP A 24.7 GROUP B 16.4 GROUP C 23.1 Mean Individual Body weight (g) 344.0 265.4 326.4 Mortality 3 birds: 1.66% 15 birds: 8.33% Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

RESULTS: Kidney degeneration GROUP A None GROUP B 18 (severe) 7 (mild) GROUP C 2 (mild) Bursa fabricii Correct Atrophy in 38 birds Atrophy in 1 bird Thymus conditions Atrophy in 36 birds Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

RESULTS: Spleen conditions GROUP A Correct GROUP B Depletion of follicles in 19 birds GROUP C Increase in 1 bird Liver conditions Hydropic degeneration (25 severe, 21 mild) Hydropic degeneration (6 mild) Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

RESULTS: Carcass grading GROUP A A1 78% - A2 22% GROUP B A1 56% - A2 33% - rejected 11% GROUP C A1 76% - A2 24% Plasmatic calcium GROUP A 2.18 ±0.16 mM/L GROUP B 1.64 ±0.66 mM/L GROUP C 2.09 ±0.22 mM/L Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

DISCUSSION: It has been found that the addition of 3 ppm Aflatoxin to a standard feed for broilers, caused dramatic lesions to most of the organs of the chicks exposed to the toxin, without any protection (Group B). The lesions found in liver, kidney, spleen, thymus and bursa of Fabricius were significantly more severe in Group B than in Group C. In fact, in Group C, not only the lesions that were found were of less severity, but, in most of the cases were concentrated to one bird. Carcasses of birds of Group B showed clear signs of micro-haemorrhages and evident bruises, to the extent that 20 birds were rejected (11%) at Carcass grading. A1 grading in Group B was 28% lower than in Group A and 26% lower than in Group C. Feed consumption and the final body weight of group C were similar to the ones of Group A. Group B mortality was 5 times higher than in the other two Groups. Plasmatic levels of calcium were similar in Groups A and C, while Group B showed a remarkable decrease, thus confirming a direct influence of Aflatoxicosis on calcium metabolism. Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

TOXINS BINDING CAPACITY OF THREE FEED ADDITIVES IN VITRO
Each mycotoxin was solved at the level of 50 microgram 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 mcg 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 Chromathgraphy, Column Kromasyl, fluorescence detector, excitation 274 nm, flow rate 1 mL/min, emission 440 nm. Dr. Gautieri and Dr. G. Colajanni

Dr. Gautieri and Dr. G. Colajanni
Results obtained: 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 Dr. Gautieri and Dr. G. Colajanni

Results obtained: 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 Dr. Gautieri and Dr. G. Colajanni

Results obtained: 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 Dr. Gautieri and Dr. G. Colajanni

Results obtained: D.O.N. CLAY AT 100 MCG 49.20 MCG CLAY AT 250 MCG 49.10 BENTONITE AT 100 MCG 49.40 BENTONITE AT 250 MCG 49.35 CAPTEX-T2 AT 100 MCG 16.00 CAPTEX-T2 AT 250 MCG 12.35 BLANK 50.05 Dr. Gautieri and Dr. G. Colajanni

TOXINS BINDING CAPACITY OF THREE FEED ADDITIVES IN VITRO
Each mycotoxin was solved at the level of 50 microgram 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 mcg and 250mcg aliquots of each MYCOSORB , TOXISORB 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 Chromathgraphy, Column Kromasyl, fluorescence detector, excitation 274 nm, flow rate 1 mL/min, emission 440 nm. Dr F Pedrazzini and Dr M Portali

Dr F Pedrazzini and Dr M Portali
Results obtained: AFLATOXIN MYCOSORB AT 100 MCG 6.90 MCG MYCOSORB AT 250 MCG 3.10 TOXISORB AT 100 MCG 7.00 TOXISORB AT 250 MCG 2.95 CAPTEX-T2 AT 100 MCG 3.60 CAPTEX-T2 AT 250 MCG 0.20 BLANK 50.10 Dr F Pedrazzini and Dr M Portali

Results obtained: ZEARALENONE MYCOSORB AT 100 MCG 37.80 MCG MYCOSORB AT 250 MCG 35.20 TOXISORB AT 100 MCG 33.20 TOXISORB AT 250 MCG 32.80 CAPTEX-T2 AT 100 MCG 16.10 CAPTEX-T2 AT 250 MCG 10.45 BLANK 48.80 Dr F Pedrazzini and Dr M Portali

Results obtained: FUMONISIN MYCOSORB AT 100 MCG 34.50 MCG MYCOSORB AT 250 MCG 35.20 TOXISORB AT 100 MCG 43.90 TOXISORB AT 250 MCG 43.60 CAPTEX-T2 AT 100 MCG 14.10 CAPTEX-T2 AT 250 MCG 12.70 BLANK 46.70 Dr F Pedrazzini and Dr M Portali

Results obtained: D.O.N. MYCOSORB AT 100 MCG 40.10 MCG MYCOSORB AT 250 MCG 39.10 TOXISORB AT 100 MCG 41.30 TOXISORB AT 250 MCG 40.15 CAPTEX-T2 AT 100 MCG 17.50 CAPTEX-T2 AT 250 MCG 10.00 BLANK 49.50 Dr F Pedrazzini and Dr M Portali

EFFECT OF THE ADMINISTRATION OF FEED CONTAMINATED BY MYCOTOXINS ON SERUM Ig CONCENTRATIONS IN YOUNG PIGS Feed Group IgA IgM Control 0.39 3.12 ZEN 0.88 5.30 DON 1.16 7.05 ZEN+CAPTEX 0.1 0.45 3.28 ZEN+CAPTEX 0.2 0.32 3.24 DON+CAPTEX 0.1 0.48 4.02 DON+CAPTEX 0.2 3.38 M.Veneroni -Doxal Italia s.p.a. Italy C.C.Chen - Hey Ma Company ltd - Hong Kong

M.Romasevic-Canovic- A.Takovic - Belgrade Yugoslavia
The effect of exchangeable cations in clinoptinolite and montmorillonite on the adsorption of Aflatoxin B1 Micro Element Ct (mg/g) Ca Ca-rich clinoptilolite Ca-rich montmorillonite Cu 2 0.23 0.80 Zn 20 0.18 Co 0.16 0.10 0.87 Mn 16 0.03 0.15 M.Romasevic-Canovic- A.Takovic - Belgrade Yugoslavia

CAPTEX T2- Composition Esterified glucomannans Enzymatic complex
CLINOPTILOLITE (Modified Aluminosilicate) Esterified glucomannans Enzymatic complex + ACETIC ACID PROPIONIC ACID

Archives of Animal Nutrition 2005 - IN PRESS
Reduction of the concentrations of ZON in the supernatant buffer solution by various detoxifying agents in the in vitro detoxification test ZON PRODUCT MEAN SD ACTIVATED CARBON 100 CHOLESTYRAMINE 94 1 CAPTEX 81 6 Toxisorb 55 Mycosorbextra 24 Klinosan 20 4 Mycofix PLUS 17 2 BENTONITE 13 12 Fix a Tox 5 Likratox  Archives of Animal Nutrition IN PRESS

NOT ALL GLUCOMANNANS ARE THE SAME !
Prior to their inclusion into CAPTEX-T2, Doxal’s Esterified Glucomannans are treated by 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. The Journal of Food Protection (Volume 67, Number 6, 1 June 2004, pp (6)) reports an assay carried out by ,Yiannikouris A. et al., which title is: “Adsorption of Zearalenone by -D-Glucans in the Saccharomyces cerevisiae Cell Wall”;

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 Vopato I. Bizzini B Italian Project M.S.T./09/96

DOX-AL ITALIA S.p.A Thanks

Safety for the Environment:
C o n c l u s i o n s Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed on a request from the Commision on the Safety of the product “BIOMIN BBSH 797” for piglets, pigs for fattening and chickens for fattening Safety for the Environment: Eubacteria are found as one of the major groups of bacteria in the digestive tract of livestock and humans and so are naturally occurring within the environment.Although little is known about the specific ecology of strain DSM it would be expected to behave as any other eubacteria and, as a strict anaerobe, would not be expected to survive in the wider environment.

European Project CERTIFOOD

NUTRITIONAL GOALS Modified yeasts with pre-biotic action and shuttle function Organic adesines, of vegetable origin, as competitive substances against pathogens Aminoacidic and polyaminoacidic growth promoters

BIOLOGICAL GOALS IMMUNOLOGIC GROWTH PROMOTERS
IMMUNO-DETOXICANTS FOR SPECIFIC SUBSTANCES

“CLEARANCE OF OCHRATOXIN A BY MEANS OF ANTI-OCHRATOXIN A ANTIBODIES IN THE INTOXICATED RAT”

PRODUCTION OF ANTI- OCHRATOXIN (OTA) ANTIBODIES
1st step PRODUCTION OF ANTI- OCHRATOXIN (OTA) ANTIBODIES

CONFIRMATION OF OTA PRESENCE IN VARIOUS LIQUIDS
2nd step CONFIRMATION OF OTA PRESENCE IN VARIOUS LIQUIDS

DECONTAMINATION OF LIQUIDS
3rd step DECONTAMINATION OF LIQUIDS

DETOXICATION OF ANIMALS
4th step DETOXICATION OF ANIMALS

MYCOTOXIN LABORATORY

OTA CHEMICAL STRUCTURE

B.Zimmerli, R. Dich, Ochratoxin A
in table wine and grape-juice: occurrence and risk assessment. Foods additives and contaminants, 1996: 13, P.Majeus, H.Otteneder. Nachweis und Vorkammen von Ochratoxin A in Wein und Tranbensaft. Deutsche Lebensmittel Rundschan, 1996: 92, M. Ospital, J-M. Cazabeil, A-M. Betbeder, C. Tricard, E. Creppy, B. Medina – L’Ochratoxine A dans les vins Rev. Franc.Oenologie, 1998: 168

Anti-Ochratoxin antibody production
Ochratoxin A-BSA conjugate in PBS was emulsified with Complete Freund’s Adjuvant (CFA) in the ration of 1:1 (vol/vol). Five rabbits were primed by administering 1mg of emulsified conjugate in 20 sites on both shaven flanks under the volume of 0.1 ml intradermallyy. Rabbits were boostered 21 days later by an intramuscular injection of 0.25 mg conjugate emulsified in Incomplete Freund’s Adjuvant (IFA). Ten days after the last booster injection blood was harvested by the intracardiac route and the antibody level determined by ELISA.

WATER SOLUTION 25°C O T A 2 h ANTIBODY 100% CLEARANCE

WINE 25°C 290mg/L O T A 745mg/L ANTIBODY 2h 100% CLEARANCE

BEER SOY SAUCE MILK TEMPERATURE?

DOXAL ITALIA PATENT “NOVEL INDUSTRIAL PROCESS FOR FOOD LIQUIDS DECONTAMINATION FROM CHEMICAL AND/OR BIOLOGICAL CONTAMINANTS”

Structure of Rat IgG2a

16 Wister Rats – 150 g - Male Control Negative (C) Saline Solution
0.3 ml, s.c. Treated (T) Saline Solution 0.3 ml + 25mg IgG, s.c.

TIME (h) C T 0.3 SALINE 0.3 SALINE + 25 mg IgG 2 O T A 22 mcg/head i.m.

TIME (h) C (mcg/ml) T (mcg/ml) 2 0.280 0.260 5 0.340 0.200

OTA kinetic, in mcg/ml Assay by HPLC
Group 3h 5h 8h 24h 36h 1 0.210 0.340 0.560 0.380 0.260 2 0.510 0.615 0.415 0.270 3 0.290 0.390 0.600 0.360 4 0.215 0.620 0.315 0.220 m4 0.244 0.400 0.599 0.367 0.240

OTA kinetic, in mcg/ml – Assay by HPLC

DOXAL ITALIA PATENT “IgG IMMUNOGLOBULINS AND F (ab’)2 FRANGMENTS THEREOF, SPECIFIC FOR DRUGS AND METABOITES THEREOF, AND THEIR USE FOR DETOXICATION PURPOSES”

Protocol of the test Oral Intoxication
25 mcg OTA/day, for 10 consecutive days. Toxin has been administered by masking it inside a chocolate drop. All Groups received the same amount of toxin.

Protocol of the test – Treatment
At the end of the 10th day of intoxication, subjects were treated as follows: GROUP A- IgG administered orally, 1mg/subject/day, for 5 consecutive days, in a chocolate drop. GROUP B - IgG administered by i.m. 0.5 mg/subject. GROUP C – no IgG administration (Control Negative), i.m. injection of physiological solution. GROUP D - IgG administered by i.m., 1.0mg/subject. GROUP E – IgG administered by i.c., 0.5mg/subject. GROUP F – IgG administered by i.p., 0.5mg/subject.

C GROUP – Control Negative
0.76 C2 1.18 C3 3.51 C4 2.44 OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

OTA kinetic, expressed in mcg/ml blood,
B GROUP – i.m. 0.5 mg T0 T2 T7 T24 B1 0.81 B2 2.12 B3 1.04 B4 0.11 OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

D GROUP – i.m. 1.0 mg T0 T2 T7 T24 D1 0.94 D2 1.05 D3 0.82 D4 0.16 OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

E GROUP – i.c. 0.5 mg T0 T2 T7 T24 E1 0.89 E2 1.14 E3 0.75 E4 0.14 OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

F GROUP – i.p. 0.5 mg T0 T2 T7 T24 F1 0.68 F2 1.32 F3 0.80 F4 0.16 OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

A GROUP - oral T0+ T72 T120 A1 0.60 A2 0.36 A3 0.30

A GROUP – LIVER – mcg OTA/g liver
0.40 A2 0.19 A3 0.11

C o n c l u s i o n s AFLATOXIN FUMONISIN ZEARALENONE D.O.N.
The same principle, here applied to OTA, could be, theoretically, applied also to other mycotoxins such as: AFLATOXIN FUMONISIN ZEARALENONE D.O.N.

Dox-Al Subsidiaries Doxal Swiss Doxal France Doxal Iberica

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