dox-al subsidiaries

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Doxal Swiss

Doxal Iberica

Doxal France

Doxal Taiwan

Doxal Australia

C A P T E X

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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

Aflatoxins Some nuts

Fusarium moniliforme

Fumonisins Corn

Fusarium graminearum

Trichothecenes zearalenone

Wheat, corn

Claviceps purpurea

Ergot alkaloide Rye

Penicillium verrucosum

Ochratoxins barley

C A P T E X

C A P T E X

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

C A P T E X

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TARGET ORGANS OF SOME MYCOTOXINS

Mycotoxin Target

Aflatoxin Liver

Ochratoxin A Kidney

Zearalenone Female genital tract

C A P T E X

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MAXIMUM LIMITS OF AFLATOXINS

IN FOODS (ASIA) (µ/kg)

China 50

Hong kong 20 (Peanuts/peanut products)

India 30

Japan 10

Malaysia 10

Philippines 20

Singapore Absence

Sri lanka 30 (ground nuts, oil-seed, flour and cereals

Thailand 20

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Minimum aw for growth of toxigenic mould species

Minimum aw for growth

Aspergillus ochraceus 0.78

Penicillium verrucosum 0.79

Aspergillus flavus 0.80

Fusarium moniliforme 0.87

Stachybotrys atra 0.94

C A P T E X

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Temperature range for growth of toxigenic mould species

Min. Opt. Max

°C °C °C

Penicillium verrucosum 0 20 31

Aspergillus ochraceus 8 28 37

Aspergillus flavus 10 32 42

(aflatoxin formation) 12 25 37

Fusarium moniliforme 3 25 37

Wang et al. (2003) - Asian Pork Magazine

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MYCOTOXIN CONCENTRATIONS IN

COMPLETE FEED IN CHINA

FEED AF FUMONISIN OTA T-2 DON ZEA

% positive samples

100 92 94 100 100 100

Av. Concentration (ppb)

8.27 1020 12.75 41.23 600 83.96

(in house data, 2004)

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MYCOTOXIN CONCENTRATIONS IN

COMPLETE FEED IN EUROPE (600 samples from 12 countries)

FEED AF FUMONISIN OTA T-2 DON ZEA

% positive samples

91 74 65 46 84 65

Av. Concentration (ppb)

6 115 26 35 415 165

Vopato I. Bizzini B. -1998 Italian Project M.S.T./09/96

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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.


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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


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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%


• DON at 2 mg/kg of diet


C A P T E X

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DUODENUM – NO ALTERATIONS


C A P T E X

C A P T E X

ILEUM - NO ALTERATIONS


C A P T E X

C A P T E X

KYDNEY-NO ALTERATIONS


C A P T E X

C A P T E X

LIVER - NO ALTERATIONS


C A P T E X

C A P T E X

STOMAC- NO ALTERATIONS


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Effects of a Modified Hydrated Sodium Calcium Aluminosilicate

(Captex T-2) on Mycotoxicosis in Rats



• T2 at 5 mg/kg diet


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Effects of Captex T2 (Modified Hydrated Sodium Calcium

Aluminosilicate enriched with Glucomannans and Chitinase)

on Mycotoxicosis caused by Fumonisin in Rats



• FB1 3 mg/kg of diet


C A P T E X

C A P T E X

ILEUM – NO ALTERATIONS


C A P T E X

C A P T E X

KYDNEY – NO ALTERATIONS


C A P T E X

C A P T E X

LIVER – NO ALTERATIONS


C A P T E X

C A P T E X

STOMAC – NO ALTERATIONS


C A P T E X

C A P T E X

DUODENUM – NO ALTERATIONS

Riemschneider J. Chen C.C.- Doxal Italia 's Scientific Panel

C A P T E X

C A P T E X

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


C A P T E X

C A P T E X

RESULTS:

Mean individual feed consumption (g/day)

GROUP A 24.7

GROUP B 16.4

GROUP C 23.1

Mean Individual Body weight (g)

GROUP A 344.0

GROUP B 265.4

GROUP C 326.4

Mortality

GROUP A 3 birds: 1.66%

GROUP B 15 birds: 8.33%

GROUP C 3 birds: 1.66%


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RESULTS:Kidney degeneration

GROUP A None

GROUP B 18 (severe) 7 (mild)

GROUP C 2 (mild)

Bursa fabriciiGROUP A Correct

GROUP B Atrophy in 38 birds

GROUP C Atrophy in 1 bird

Thymus conditionsGROUP A Correct

GROUP B Atrophy in 36 birds

GROUP C Correct


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RESULTS:

Spleen conditionsGROUP A Correct

GROUP B Depletion of follicles in 19 birds

GROUP C Increase in 1 bird

Liver conditionsGROUP A Correct

GROUP B Hydropic degeneration (25 severe, 21 mild)

GROUP C Hydropic degeneration (6 mild)


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RESULTS:

Plasmatic calciumGROUP A 2.18 ±0.16 mM/L

GROUP B 1.64 ±0.66 mM/L

GROUP C 2.09 ±0.22 mM/L

Carcass grading GROUP A A1 78% - A2 22%

GROUP B A1 56% - A2 33% - rejected 11%

GROUP C A1 76% - A2 24%


C A P T E X

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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.

Dr. Gautieri and Dr. G. Colajanni

C A P T E X

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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.


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C A P T E X

Results obtained:

AFLATOXINCLAY AT 100 MCG 7.40 MCG

CLAY AT 250 MCG 2.40 MCG

BENTONITE AT 100 MCG 7.40 MCG


CAPTEX-T2 AT 100 MCG 4.05 MCG


BLANK 49.90 MCG


C A P T E X

C A P T E X

Results obtained:

ZEARALENONECLAY AT 100 MCG 42.60 MCG






BLANK 49.70 MCG


C A P T E X

C A P T E X

Results obtained:

FUMONISINCLAY AT 100 MCG 47.48 MCG






BLANK 49.30 MCG


C A P T E X

C A P T E X

Results obtained:

D.O.N.CLAY AT 100 MCG 49.20 MCG






BLANK 50.05 MCG

Dr F Pedrazzini and Dr M Portali

C A P T E X

C A P T E X

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.


C A P T E X

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Results obtained:

AFLATOXINMYCOSORB AT 100 MCG 6.90 MCG

MYCOSORB AT 250 MCG 3.10 MCG

TOXISORB AT 100 MCG 7.00 MCG




BLANK 50.10 MCG


C A P T E X

C A P T E X

Results obtained:

ZEARALENONEMYCOSORB AT 100 MCG 37.80 MCG






BLANK 48.80 MCG


C A P T E X

C A P T E X

Results obtained:

FUMONISINMYCOSORB AT 100 MCG 34.50 MCG






BLANK 46.70 MCG


C A P T E X

C A P T E X

Results obtained:

D.O.N.MYCOSORB AT 100 MCG 40.10 MCG






BLANK 49.50 MCG

M.Veneroni -Doxal Italia s.p.a. Italy C.C.Chen - Hey Ma Company ltd - Hong Kong

C A P T E X

C A P T E X

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 0.45 3.38

M.Romasevic-Canovic- A.Takovic - Belgrade Yugoslavia

C A P T E X

C A P T E X

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 0.18

Co 0.16 0.10 0.87

Mn 16 0.03 0.15

C A P T E X

C A P T E X

CAPTEX T2- Composition

• CLINOPTILOLITE

(Modified Aluminosilicate)

• Esterified glucomannans

• Enzymatic complex+ ACETIC ACID

PROPIONIC ACID

Archives of Animal Nutrition 2005 - IN PRESS

C A P T E X

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Reduction of the concentrations of ZON in the supernatant buffer solution by various detoxifying agents in the in vitro

detoxification testZON

PRODUCT MEAN SD

ACTIVATED CARBON 100 0

CHOLESTYRAMINE 94 1

CAPTEX 81 6

Toxisorb 55 1

Mycosorbextra 24 1

Klinosan 20 4

Mycofix PLUS 17 2

BENTONITE 13 12

Fix a Tox 5 1

Likratox 5 2

C A P T E X

C A P T E X

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. 1195-1200(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”;


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Glucans and Chitin contents of various sources of

Saccharomyces cerevisiaeYeast Sources Total

Glucans (%)(1.3)-

glucans (%)(1.3)-

glucans (%)Chitin (%)

Saccharomyces cerevisiae 1

13.2 7.2 6.0 9.0


14.4 7.4 7.0 9.2


13.5 7.3 6.2 9.1


13.9 7.6 6.3 10.0

Doxal’s

S.C. 223418.2 7.6 10.6 2.7

C A P T E X

C A P T E X

Thanks

www.doxal.com

DOX-AL ITALIA S.p.A

C A P T E X

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C o n c l u s i o n sOpinion 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 11798 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.

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 11798 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.

C A P T E X

C A P T E X European Project

CERTIFOOD

C A P T E X

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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

C A P T E X

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BIOLOGICAL GOALS

• IMMUNOLOGIC GROWTH PROMOTERS

• IMMUNO-DETOXICANTS FOR SPECIFIC SUBSTANCES

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“CLEARANCE OF OCHRATOXIN A BY MEANS OF

ANTI-OCHRATOXIN A ANTIBODIES

IN THE INTOXICATED RAT”

C A P T E X

C A P T E X

PRODUCTION OF

ANTI- OCHRATOXIN

(OTA) ANTIBODIES

C A P T E X

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CONFIRMATION OF

OTA PRESENCE

IN VARIOUS LIQUIDS

C A P T E X

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DECONTAMINATION OF LIQUIDS

C A P T E X

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DETOXICATION

OF ANIMALS

C A P T E X

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MYCOTOXIN LABORATORY

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OTA CHEMICAL STRUCTURE

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2) P.Majeus, H.Otteneder. Nachweis und Vorkammen von Ochratoxin A in Wein und Tranbensaft. Deutsche Lebensmittel Rundschan, 1996: 92, 388-390

3) M. Ospital, J-M. Cazabeil, A-M. Betbeder, C. Tricard, E. Creppy, B. Medina – L’Ochratoxine A dans les vinsRev. Franc.Oenologie, 1998: 168

1) B.Zimmerli, R. Dich, Ochratoxin A in table wine and grape-juice: occurrence and risk assessment. Foods additives and contaminants, 1996: 13, 655-668

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Anti-Ochratoxin antibody productionOchratoxin 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.

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WATER SOLUTION

O T A

ANTIBODY

100% CLEARANCE

25°C

2 h

C A P T E X

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WINE

O T A

ANTIBODY

100% CLEARANCE

290mg/L

745mg/L

25°C

2h

C A P T E X

C A P T E X

•BEER

•SOY SAUCE

•MILK TEMPERATURE?

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DOXAL ITALIA PATENT

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

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Structure of Rat IgG2a

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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.

8 RATS 8 RATS

C A P T E X

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TIME (h) C T

00.3

SALINE

0.3 SALINE

+ 25 mg IgG

2 O T A 22 mcg/head i.m.

C A P T E X

C A P T E X

TIME (h) C (mcg/ml) T (mcg/ml)

2 0.280 0.260

5 0.340 0.200

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C A P T E XGroup 3h 5h 8h 24h 36h

1 0.210 0.340 0.560 0.380 0.260

2 0.260 0.510 0.615 0.415 0.270

3 0.290 0.390 0.600 0.360 0.210

4 0.215 0.360 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

C A P T E X

C A P T E X

0,244

0,4

0,599

0,367

0,24

0,2

0,25

0,3

0,35

0,4

0,45

0,5

0,55

0,6

3 5 8 24 36T I M E

OTA kinetic, in mcg/ml –

Assay by HPLC

C A P T E X

C A P T E X

0,244

0,4

0,599

0,367

0,240,2

0,3

0,4

0,5

0,6

0,7

3 5 8 24 36

OTA kinetic, in mcg/ml –

Assay by HPLC

C A P T E X

C A P T E X

DOXAL ITALIA PATENT

“IgG IMMUNOGLOBULINS AND F (ab’)2 FRANGMENTS THEREOF, SPECIFIC FOR DRUGS AND METABOITES THEREOF, AND THEIR USE

FOR DETOXICATION PURPOSES”

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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.

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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.

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C GROUP – Control Negative

T0 T2 T7 T24

C1 0.76

C2 1.18

C3 3.51

C4 2.44

OTA kinetic, expressed in mcg/ml blood, Assayed by HPLC

C A P T E X

C A P T E X

B GROUP – i.m. 0.5 mg

T0 T2 T7 T24

B1 0.81

B2 2.12

B3 1.04

B4 0.11


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C A P T E X

D GROUP – i.m. 1.0 mg

T0 T2 T7 T24

D1 0.94

D2 1.05

D3 0.82

D4 0.16


C A P T E X

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E GROUP – i.c. 0.5 mg

T0 T2 T7 T24

E1 0.89

E2 1.14

E3 0.75

E4 0.14


C A P T E X

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F GROUP – i.p. 0.5 mgT0 T2 T7 T24

F1 0.68

F2 1.32

F3 0.80

F4 0.16


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0,2

0,6

1

1,4

1,8

2,2

2,6

3

3,4

3,8

T0 T2 T7 T24

C B D E F

C A P T E X

C A P T E X

C A P T E X

C A P T E X

A GROUP - oral

T0+ T72 T120

A1 0.60

A2 0.36

A3 0.30

C A P T E X

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0,6

0,36

0,3

0,2

0,25

0,3

0,35

0,4

0,45

0,5

0,55

0,6

0,65

T0+ T72 T120

C A P T E X

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A GROUP – LIVER – mcg OTA/g liver

T0+ T72 T120

A1 0.40

A2 0.19

A3 0.11

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C o n c l u s i o n sThe same principle, here applied to OTA,

could be, theoretically, applied also to other mycotoxins such as:

AFLATOXINAFLATOXIN

FUMONISINFUMONISIN

ZEARALENONEZEARALENONE

D.O.N.D.O.N.

dox-al subsidiaries

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