EUROTIOX

Feed Ingredients Vitamins, pigments Fat, oils (energy) OXIDATION Feed Ingredients Vitamins, pigments Fat, oils (energy)

AUTOXIDATION SERIES OF DESTRUCTIVE CHEMICAL PROCESSES OCCURS IN ORGANIC MATERIALS CHANGES THE CHEMICAL NATURE OF AFFECTED MOLECULES INCREASINGLY FAST ROOM TEMPERATURE REACTION CAUSES RANCIDITY IN FATS, OILS Series of destructive chemical process, it is important to mention that it is also irreversible. This reaction occurs mainly in organic materials Due to the reaction, the chemical nature of molecules is changed. Vitamins for example will loose their activity. The auto oxydation is an increasingly fast reaction, which is already present within a few hours after mixing. In untreated raw materials, often the pericarp protects the material from oxydation (no oxygen), once it is grinded, protection has dissappeared. The reaction of autooxydation is a reaction which takes place at room temperature, this in contradiction to many other chemical reactions. Of coarse higher temperatures will increase the speed of the reaction. BIOTIN

Oxidation influenced by: Moisture: free water in raw material and feed Catalysts: metals promoting free radical formation, Light: promotes oxidation Temperature: increase oxidation also during cooling Contact with air: improves peroxide formation Enzymes: lipase, lipoxygenase Time: storage time, production Complet reactions involved including all of the above Moisture is outside (humidity) also Aw, Humidity from steam during the process. Cataly mostly in premixes

Aldehydes from Fat Autoxidation Frankel, 1984

THE INFLUENCE OF OXIDISED FAT ON THE MICRO-FLORA IN BROILERS (11 DAYS)

TOXIC METABOLITES OF FAT OXIDATION Shorten small intestine villi length FROM: Poul. Sci. Abstracts (1994) Vol. 73, Suppl. 1, pg 47. Reduce nutrient adsorption

Autoxidation and Animal production Reduced Egg production in laying hens (72%) (Voreck & Kirehgessner, 1981) Autoxidation products are cancernogenic (Morita et al., 1983) Even low increase in Peroxide Value (>7 meq/kg) has a significant effect on Feed Conversion Ratio (Cabel et al., 1988) Rancid Feed has an impact on the gastro-intestinal Structure, Cell proliferation and intestinal Microflora (Shermer et al., 1995) Oxidised vegetable oil in Broiler feed reduces growth rate (5%) (Engberg et al 1996) Rancid fish oil induced anaemia in Mink, reduced iron absorption (Borsting et al., 1994)

Effect of peroxides on broiler performances (49 days) University of Arkansas + 4.78 % c a ab b - 6.29 % Cabel, M. C., P. W. Waldroup, W. D. SHermer and D. F. Calabotta (1989), Poultry Science 67: 1725-1730

Effect of raw material quality on broiler performances (56 days) INTA (Argentina) - 10.69 % a a a b a a + 6.43 % a b Schang et al. - 1987

The three stages of OXIDATION 1) Initiation phase RH -----> R° + H° Fat molecule Free radical 2) Propagation phase R° + O2 ----> ROO° ROO° + RH ----> ROOH + R° (peroxide) 3) Termination phase ROO = peroxide= faster oxidation Aim is to control the peroxide levels so want to stay in initial phase peroxide is only an intermediate molecule peroxide good indicator of oxidation /quality……. Snap shot only ROO° + ROO° ---> stable end products (aldehydes, ketones, short chain fatty acids)

Oxidation Process involving Oxygen & lipids temp. Catalysts Light Air Time FREE RADICALS Damage critical nutrients Lipids FREE RADICALS PEROXIDES + Oxygen forms Reaction with cell membranes Toxic for animals Destruction of fat soluble vitamins Reduction of performances PEROXIDES Aldehydes Ketons Rancidity Reduction of feed intake En-products absorbed in the body Start of in vivo oxidation

The three stages of OXIDATION Peroxides value (meq) Polimers, oxidised fat, acids, aldehides ketones Peroxides, radicals water Therefore need to treat early because everything lost cannot be reversed, easier to stop/control reaction when reaction is slow Induction When there is dis -association of fat molecules and then reaction with oxygen Oxidation Phase dis-association + direct reaction from peroxide on fat molecule Polimers, oxided fat, etc are all stable forms of lower energy value, also palatability issues Induction Oxidation Termination period

Different antioxidants Free radical scavengers: Ethoxyquin, BHA, BHT, Propyl Gallate Chelators: Citric acid, phosphoric acid Combination of 2 Free radical scavengers best (except in aquaculture) Oxidation scavengers to absorb oxygen from air not really revealent for feed Chelators To bind metals so not so availableto promote free radicals but not bound for nutrition. Only use citric acid as literature good chelator , we know that phosphoric acid sometimes works sometimes increases sometimes no effect (Endox Kemin uses phosphoric..is a cheeper ingredient)

Differences between antioxidants Ethoxyquin: very stable, only feed grade, raw materials Gallates: very stable, regenerates ethoxyquin or BHA activity BHA: heat stable, mainly food and petfood BHT: heat stability problem, repartition problem, readily broken down in premixes

Structures of commonly used synthetic antioxidants Butylated hydroxytoluene (BHT) Mol. Wt. 220 Tertiary butylhydroquinone (TBHQ) Mol. Wt. 166 Non allowed in UE Ethoxyquin Mol. Wt. 217 These all are relatively short chain compounds compared to fats and Vitamins Longer chain > reactivity and easier to oxidise (they are less stable) > number of bonds > reactivity and easier to oxidise (again less stable) When RH  RO - + H+ The fat chain then has a -ve charge which for reasons that we don’t really understand makes it easier for O2 to attach and oxidise the molecule leading to the reduction in nutrient value ransidity etc. To stop this reaction anti-oxidant molecules (which are shorter chain so more stable) donate a H+ to reverse the Initial phase reaction this only helps DELAY oxidation as the RH  RO - + H+ is a dynamic reaction. Propyl gallate Mol. Wt. 212 Butylated hydroxyanisole (BHA) Mol. Wt. 180

Triple Mode of Action EUROTIOX 1.Citric acid forms reversible bonds with metal pro-oxidants in the premix and feed to inhibit them from initiating oxidation 2. Ethoxyquin blocks oxidation by donating hydrogen ions to free and peroxy radicals formed during the onset and propagation stages 3. Propyl gallate restores and potentiates ethoxyquin activity up to 3 times by donating hydrogen ions from its 3 active sites to oxidised (spent) ethoxyquin molecules

Efficacy of synthetic antioxidants BHT <ETQ<BHA<gallate($30) for cost of material Gallate 20x more expensive than BHT Data above is independent work Slide to show why we use our combination of ETQ and Galate Days Stability test in mixed fat - Dr. M. Coelho (1995)

Formation of Ellagic Acid from Propyl Gallate

Antioxidant Activity of BHA, Propyl Gallate and Heterodimers T. Kurechi and A.Kunugi, J. American Oil Chem. Soc. 60:33 (1983)

Ethoxyquin/Propyl Gallate SYNERGY mechanism

Ethoxyquin/Propyl Gallate SYNERGY mechanism

Effect of citric acid on soya oil stability Metal chelating Effect of citric acid on soya oil stability Trail was done by adding metals to oil With citric reduced oxidation so less peroxide Mustakas et Al. (1984) 17

Inhibition of Initiation by Chelation of Copper OOC CH2 C COO OH O HO Cu++ + Cu++ HOCCOOH CH2COOH Citric Acid Cu Citrate complex with divalent cation, forming two five-membered rings

Eurotiox 32-Premix Comparative granulometry Physical characteristics VIP after getting the right ingredients and ratios because citric cannot use limestone as some other competitors are doing need small homog. Particles, cannot be re-active with other ingredients Particle size class ( Mesh, mm) - Sieve analysis by industrial lab, Belgium, July 1996

EUROTIOX unique antioxidant complex ETHOXYQUIN - high purity (% mono) - high stability PROPYL GALLATE -highly effective - EtQ regenarator Two very potent free radical scaven-gers Only proven synergism Very effective economical complex stable micro- pearled + CITRIC ACID -metal scavenger Maximum protection at low dosage + UNIQUE CARRIER - high absorption - free-flowing - impr. stability + ALUMINIUM bag -protected 23 20

EUROTIOX ANTIOXIDANT PROGRAM Vitamins, Pigments protection in premixes & feed Preventing rancidity in fat, oils, bone meals EUROTIOX 32-Premix EUROTIOX L-32

Dosage in feed and raw materials EUROTIOX Dosage in feed and raw materials

EUROTIOX 32 Premix in feed Eurotiox 32 Premix at 20 - 25 g/t in Feed with < 4% fat and < 1 month storage time + 5 g/t for each 2% fat increase + 5 g/t for each month storage increase

EUROTIOX 32 Premix in feed

EUROTIOX 32 Premix in premixes and concentrates Vitamin premix: 50g/ton Vitamin and traces premix: 100 g/ton Vitamin and traces and choline chloride: 200 g/ton Higher dosages are required in case proteins are present.

EUROTIOX L 32 for raw material protection

EUROTIOX L 32 for raw material protection

Dosage of EUROTIOX L 32 Raw material For one month protection For Two Months Protection For Three Months Protection For Four Months Protection Vegetable oil (crude) 75 150 200 300 Palm oil (crude) Rice Bran oil (crude) 125-150 250-300 375-450 500-600 Rice Bran oil (Refined) Sunflower oil (crude) 100 400 Copra oil (crude) Soya oil (crude) Fish oil (crude) Poultry fat Lard Tallow 35 70 135 Mixed fat MBM 30 60 90 120 Rice Bran (14-16% Fat) Full Fat Soy Bean Source: e-mail communication AVITECH

Vitamin recovery test against oxidation (Vitamin A in a vitamin mineral premix) HPLC assay - 6 months testing period March ‘95-Sep ‘95 EUROTIOX 32-Premix - 45 g/ton 45 g/t EUROTIOX 32 200, g/t ETQ-66% Control Verlies van 450 naar 300 is toch nog steeds hoog, vandaar dosering naar 100 g. Wel blijkt duidelijk dat 45 g/Eurotiox vergelijkbaar is met 200 g Ethoxyquin.

Vitamin recovery test against oxidation (Vitamin A and E in a Broiler premix) HPLC assay - 3 months testing period - Netherlands - Oct ‘96/ Feb ‘97 EUROTIOX 32-Premix - 20 g/ton 89.4% Vitamin E recovered after 3 months 94.5% Vitamin A recovered after 3 months Periode beperkt over 3 maanden, dit is in praktijk dikwijls te kort. No significant loss of Vitamin A & E during a 3 month period Vitamin A & E analytical detection test by HPLC performed by an independent laboratory (SERLABO) in the Netherlands.

Vitamin A Stability in Premix Eurotiox 32 premix EUROTIOX 32-Premix - 20 and 30 g/ton Periode van 1 maand, in praktijk te weinig Independant laboratory - HPLC analysis.

Vitamin A Stability comparative test Broiler feed - March 1997 EUROTIOX 32-Premix At 25 g/ton, EUROTIOX provides the best VITAMIN A protection of all products tested Project with large industrial company(Broiler integration) in Philippines. Vitamin detection analysis performed by an independent laboratory (SERLABO) in the Netherlands.

Vitamin recovery test (Vitamin A in a vitamin mineral premix) HPLC assay - Broiler premix from Thailand - Independent laboratory Holland - Sep 99 EUROTIOX 32-Premix - 20 g/ton Pdt N 125 g/ton EUROTIOX 20 g/ton Control Pdt K 125 g/ton Periode van 14 dagen, in praktijk te weinig

Trial results

EUROTIOX 32 PREMIX vs ENDOX TRIAL AO 001

Eurotiox 32 Premix in meat and bone meal (poultry origin) Trial set-up: A control sample (no anti-oxydant added) and a treated sample (150 ppm Eurotiox 32) was incubated in the OXIPRESS. The OXIPRESS was running until the control sample was completely oxidised. Both samples were analysed on their nutritional value and compared to a control sample which was not put in the OXIPRESS at all.

Eurotiox 32 Premix in meat and bone meal (poultry origin) CONTROL WITHOUT OXIPRESS CONTROL WITH OXIPRESS CONTROL WITH OXIPRESS + 150 PPM EUROTIOX 32 PREMIX Crude fat (g/kg) 121.2 105.4 115.6 Metaboliseble Energy (MJ/kg) 11.13 10.22 10.95

Eurotiox 32 Premix in meat and bone meal (poultry origin) -1.6% -8.1%

OXIPRESS RESULTS

Eurotiox 32 Premix in meat and bone meal (Poultry origin) Conclusions: Oxidation is responsible for loss of nutrients, mainly energy EUROTIOX 32 PREMIX is able to prevent nutrient losses due to oxidation.

Effect of EUROTIOX 32 PREMIX on energy values (kcal/kg) of MBM (ruminant origin) Source: Trial AO 002: INDIA

Eurotiox 32 Premix versus competitor in feed (8% fat) Competitor = ENDOX C