ADIMIX ® Sodium Butyrate For animal’s Health & Performance

1. ADIMIX® ADIMIX® Sodium Butyrate For animal’s Health & Performance Improves Feed Conversion Stimulates Early Feed Intake Enhances Intestinal Health Natural Attractant

2. ADIMIX® Active substance butyric acid an organic acid with specific characteristics a short chain fatty acid (SCFA) a viscous liquid miscible with water, ethanol and ether (lipids) (unlike other SCFA’s, like propionic acid / acetic acid) the characteristic smell of rancid butter (when it is present as -very volatile-free fatty acid) CH3 - [CH2]2 - COOH

3. Water soluble Water & Fat soluble Physical and Chemical Characteristics of Short Chain Fatty Acids (SCFA) • Distribution in chloroform/water. Increasing number indicates higher solubility in lipids • Na-n-butyrate: white stable non-volatile powder up till 250°C

4. What is ADIMIX® ? N – Butyric acid sodium salt,or N – Butyrate, sodium salt, or Butanoic acid, sodium salt, or Sodium butanoate, or E 470 Spray-dried white powder Pure form contains 98% sodium butyrate or 78% butyric acid CH3 - [CH2]2 - COONa

5. Adimix®: dissociation pH CH3 - [CH2]2 – COOHCH3 - [CH2]2 – COO¯+ H+ n-butyric acid n-butyrate (Undissociated) (Dissociated) In the undissociated state, butyric acid is strongly lipophilic and can easily cross membranes (both bacterial and cellular membranes) The dissociation rate of organic acids is mainly influenced by the pKa value (pH at which 50% of the acid is in the dissociated state and 50% is in the undissociated state) and the pH of the intestinal liquid The pKa value of butyric acid is about 1 pH unit higher than ‘traditional’ organic acids like formic and lactic acid (see next slide). As a result, butyric acid has different characteristics with respect to its anti-microbial activity, but also the biological effect of butyric acid is linked with its high pKa value.

6. Only at 10 X normal dose Favours / does not harm / even stimulates Lactobacillus Antibacterial effect : G-&G+ Organic Acids • The combination of Organic Acids & Adimix® • Performs better than single approach (synergy) • Is the most economical application X G- bacteria G+ bacteria More complex membrane ( > FAT !! ) Butyric acid

7. ATP ADP + P H+ Antibacterial effect RCOOH (Organic Acid – Undissociated) pKa RCOO- H+ Bacterial Cell pH drop DNA

8. Antibacterial effect • Stop of DNA transcription (prevents growth and cell multiplication) • Bacterial Cell exhausts • No energy for multiplication  Bacterial Cell collapses Bacterial Cell

9. pKa value 4.82 3.75 3.79 Dissociation of n-Butyric acid: pH dependent Butyric acid travels further down the digestive tract pH • At higher pH values (5-6), more butyric acid is present in the undissociated form compared to formic or lactic acid (differences in pKa value) • This means there is a synergy between Butyric acid and Formic/Lactic acid in terms of their anti-microbial effect (this is the basis for the Nutri-Ad acidification program for pigs).

10. Short Chain Fatty Acids: metabolism Butyric acid, Acetic acid, Propionic acid metabolic end-products of carbohydrate digestion by bacteria in the rumen or large intestine of pigs, poultry, rabbits and ruminants • Low Fibre-fermentation capacity for young animals • Naturally present, . . . but insuffient ? • 1 - 2 % in milk fat

11. Metabolism of n-butyrate High levels in human : leaves body via sweat and breath  socially unacceptable Cellulose and/or Starch (from diet) Bacterial fermentation pigs, (poultry)  colon multigastrics  rumen n-butyrate Blood absorption 30% 70% Secretion via respiratory tract, skin, . . . Metabolisation by rumen and intestinal eptithelium cells (villi)

12. Cell metabolism Immune system Selective action on bacterial micro-flora ADIMIX® Biological Functions

13. Cell metabolism Energy source for intestinal mucosa (brush border) Differentiation of enterocytes +++ Contact surface of intestinal microvilli +++ Calcium aborption +++ Secretion of pancreatic enzymes +++ Secretion of intestinal digestive enzymes ++++ Differs it from any other acid ADIMIX®: Biological Functions 2.Immune system 3. Selective action on bacterial micro-flora

14. 1.Cell metabolism 2.Immune system Increased haemoglobin synthesis Stimulation of non-specific immunity(phagocytosisbymacrophages) Stimulation of local immunity in the intestine (Gut Associated Lymphoid Tissue) Increase in haptoglobine values (indicator for immune stimulation) Correlation with LPS (liposaccharides) from pathogenic bacteria in a way that it controls the nitric oxide production 3.Selective action on bacterial micro-flora ADIMIX®: Biological Functions

15. 1.Cell metabolism 2.Immune system Selective action on bacterial micro-flora Increase in Bifido-bacteria + Lactobacils Colonisation resistance against harmful/bad-natured bacteria, like E. Coli, Salmonella Direct anti-bacterial action (organic acid effect) ADIMIX®: Biological Functions

16. Prevention of inflammatory diseases in the gastro-intestinal tract Chrone disease Enterocolitis Necrotic enteritis Regression of colon and rectum cancers ‘A deficiency of butyric acid or other SCFA to the colon causes inflammation’ (Kien et al., 1999) Butyric acid in human gut pathology Inhibits growth of abnormal cells

17. New Spray Drying Tower installed Finalized early 2003

18. Trial Veterinary Faculty of the University of Gent – Belgium Anti-Salmonella / Anti-Dysenterie Effect

19. Adimix®C 30 Coated

20. "Protected"

21. LIPASE 1 "Slow release"

22. LIPASE 2 "Gradual release"

23. "Target release" LIPASE 3

24. Na-ButyrateversusCa-Butyrate • A small molecule penetrates the bacterial cell more easy • Calcium Butyrate has a lower anti-bacterial effect, as its molecular weight is 2 X higher than Sodium Butyrate ( resp. 216 and 111, resulting in less diffusion capacity) • Sodium Butyrate is perfectly water-soluble (1.0 kg in 1 kg water) • Calcium Butyrate’s water-solubility is poor (0.3 kg in 1 kg water) • the higher the solubility, the easier the reaction to salt vice versa ; otherwise it only stays as a crystal • Calcium Butyrates are not spray-dried ( 1 - 2% free, volatile But.-acid) • ADIMIX (Na-Butyrate) is spray-dried (< 0.1% free, volatile But.-acid) • ADIMIX : high concentration active ingredient • ADIMIX : captured and covered odeur

25. Na-ButyrateversusCa-Butyrate • Calcium Butyrate has a much stronger ‘typical’ odeur/smell than spray-dried Sodium Butyrate (= related to the level of ‘free buteric acid’, which is an extremely volatile molecule) • Calcium Butyrate have lower levels of active ingredients, and often contains a rather high level of (non-nutritional) anti-caking products • Dust level (measured by ITECH) is twice as high in Ca-Butyrate (>4%) compared to ADIMIX (2%) • ADIMIX (spray-dried) has a much more uniform particle size than Ca-Butyrate (dry mix)

26. Granulometry and particle size distribution Fines/dust ADIMIX More uniform particle size distribution Significantly less fine particlesless dusty product

27. ADIMIX Calcium-butyrate Spray-dried powder Uniform particles Low dust Dry mix (butyric acid+mineral support) Variable particle size High dust

28. Perfectly water soluble, no sedimentation • Higher bio-availability • Preferred source for all in-feed applications -specifically for milk replacers & liquid applications- • Poor water solubility • Risk of complex formation of calcium with other dietary components ADIMIX : superior solubility Adimix Butyrate 1000 g in 1000 ml of water Calcium Butyrate: 300 g in 1000 ml of water