Repox50

1. Repox50 A novel, natural polyphenol partial replacement for vitamin E in feeding diets

2. Repox50 Presentation overview • The problem • Oxidative stress • The solution • Antioxidants • Our recommended solution • Repox50

3. Repox50 • The problem • Oxidative stress • The solution • Antioxidants • Our recommended solution • Repox50

4. Repox50 Reactive oxygen species • Reactive oxygen species (ROS) are chemically-reactive molecules containing oxygen. Examples include oxygen ions and peroxides. • Increased ROS can result in significant damage to cell structures. This cumulates into a situation known as oxidative stress. • Antioxidants are used to counteract the effect of these increased ROS levels often in combination with the addition of extra minerals and vitamins to the diet.

5. Repox50 Some causes of oxidative stress?

6. Repox50 Problems resulting from oxidative stress • Growth inhibition • Muscle degeneration, leading to inferior meat quality • Suppression of the immune system response • Loss of flavour, texture, appearance and in some cases nutritional value of the meat

7. Repox50 • The problem • Oxidative stress • The solution • Antioxidants • Our recommended solution • Repox50

8. Repox50 What are antioxidants? • Antioxidants are phytochemicals, vitamins and other nutrients that protect our cells from damage caused by free radicals. • In vitro en in vivo studies have shown that antioxidants help prevent the free radical damage that is associated with disease. • Antioxidants have the ability to neutralize free radicals without becoming a free radicals themselves, however, when the antioxidant neutralises a free radical it becomes inactive. Therefore we need a continuous supply of antioxidants.

9. Repox50 Antioxidants in action

10. Repox50 Some commonly used antioxidants • Vitamin E • A fat-soluble antioxidant that stops the production of reactive oxygen species formed when fat undergoes oxidation. • Vitamin C • Vitamin C or L-ascorbic acid or L-ascorbate is an essential nutrient for humans and certain other animal species, in which it functions as a vitamin. • Selenium • Selenium is necessary for cellular function in most, if not all, animals, forming the active centre of the enzymes glutathione peroxidase and thioredoxinreductase.

11. Repox50 • The problem • Oxidative stress • The solution • Antioxidants • Our recommended solution • Repox50

12. Repox50 Repox50 the natural choice • Super concentrated polyphenols extracted from specifically selected botanical sources. • Works in synergy with Vitamin E, able to replace up to 50% resulting the same or superior protective effect, thus creating more cost effective inclusion into in poultry diets. • Sustainably produced from natural renewable resources. • Safe and easy to handle.

13. Repox50 Repox50 Heat Stability OVERVIEW Two representative samples of Repox50 were received from Phytologica Ltd, batch number LAB/PHY01. This sample was separated into 2 samples for test purposes. Sample 1 and Sample 2. • Sample 1 was subjected to an increase in temperature for 60 minutes in an autoclave at a temperature of 120°C. • Sample 2 was untreated. • Sample 1 and Sample 2 were analysed for polyphenols using a cyclic voltammetry technique (CV) used routinely in the Citrox QC laboratory.

14. Repox50 Repox50 Heat Stability LAB/PHY01 - Sample 1 (120°C)

15. Repox50 Repox50 Heat Stability LAB/PHY02 - Sample 2 (untreated)

16. Repox50 Repox50 Heat Stability Conclusion • “there were no discernable differences in the voltammograms which can be thought of as “fingerprints” of the polyphenol activity” Testing carried out by Dr. David J. Lax BSc MRSC PhD Chemical Research Services

17. Repox50 Some scientific evidence • Sparing effect of procyanidins from Vitisvinifera on vitamin E: in vitro studies • Carini M, MaffeiFacino R, Aldini G, CalloniI MT, Bombardelli E, Morazzoni P, 1998 : The protection of polyunsaturated fatty acids in micellar systems against UVB-induced photo-oxidation by procyanidins from Vitisvinifera L., and the protective synergy with vitamin E. International J. of Cosmetic Science, Volume 20 Issue 4 Page 203-215,August 1998. • Select flavonoids and whole juice from purple grapes inhibit platelet function and enhance nitric oxide release • Freedman JE, Parker C 3rd, Li L, Perlman JA, Frei B, Ivanov V, Deak LR, Iafrati MD, Folts JD. , 2001. Select flavonoids and whole juice from purple grapes inhibit platelet function and enhance nitric oxide release. Circulation. 2001 Jun 12;103(23):2792-8. • Catechins and procyanidins in Mediterranean diets • AUGER Cyril, AL-AWWADI Najim, BORNET Aurélie, ROUANET Jean-Max, GASC Francis, CROS Gerard, TEISSEDRE Pierre-Louis : Catechins and procyanidins in Mediterranean diets Food research International 2004, vol. 37, no3, pages. 233-245 [13 page(s) (article)] (1 p.1/4)

18. Repox50 In vitro antioxidant trials Trial Objective To measure the antioxidant activity at 0%, 25%, and 50% of the vitamin E substitued by Repox50 and compare with control. • Growth Control: No antioxidant added • 100% Vitamin E: 0% substitution • 75% Vitamin E: 25% substition • 50% Vitamin E: 50% substitution Conclusion • 100% Vitamin E antioxidant activity is 250% higher than negative control • Replacement of 25% and 50% of Vitamin E by Repox50 the antioxidant activity is still identical to 100% Vitamin E Increase of antioxidant activity compared to no antioxidant addition 33.00% 34.00% 33.00%

19. Repox50 Poultry Applications 33.00% 34.00% 33.00%

20. Repox50 Poultry diet trials Effect of Repox50 and Vitamin E on Growth Performance, Nutrient Digestibility in Chickens • 120 one-day-old male broiler chicks were obtained from a commercial hatchery and housedin an environmentally controlled room over a period of 3 weeks. All diets were formulated to meet or exceed the minimum NRC (1994)requirements for broiler chickens. • At the end of the experimentalperiod, birds were weighed, and feed consumption was recorded • The addition of increasing concentration of Repox50 in the chickendiets did not impair growth performance (BW, feed consumption,and feed efficiency) compared with the birds fed the non supplementedand supplemented vitamin E diets • Antioxidant activity in vitamin E and Repox50 diets exhibited significantlyhigher scavenging free radical capacity than controls. • Similarly, the birds fed vitaminE and Repox50 diets exhibited significantly higher scavenging freeradical capacity in excreta than those fed control diets.

21. Repox50 BANGKOK ANIMAL RESEARCH CENTER CO., LTD. HEAD Office 487/1 Si Ayutthaya Road, KhwaengThanonPhayathai, KHET Ratchathewi, Bangkok 10400, Thailand Evaluation of Repox50 in Broilers under research station circumstancesTrial No. AB11238B

22. Repox50 Trial Detail Objective This trial was conducted to evaluate the effect of replacing vitamin E by Repox 50 on performance of broilers. Materials and methods The trial was conducted at BARC research facility during July 6 – August 10, 2011. Six hundred newly hatched male broiler chicks of commercial strain (AA Plus) were randomly allocated to 3 treatments with 8 replications in a randomized complete block design experiment, using 25 chicks per experimental unit. Practical corn-soybean diet with 100 g/t added vitamin E 50 was used as control diet and two levels of replacing of vitamin E 50 by Repox50. Treatment diets were presented as below. Diet 1 = Control diet: practical diet with 100 g/tone added Vitamin E 50 Diet 2 = Replacing 25% of added Vitamin E of diet 1 by Repox 50 Diet 3 =Replacing 50% of added Vitamin E of diet 1 by Repox 50

23. Repox50 Composition of starter and grower diets

24. Repox50 Nutrients of starter and grower diets

25. Repox50 Statistical Analysis Feed intake, body weight gain, feed conversion ratio, liability, FRAP value and TBA value were determined and were subjected to ANOVA as RCBD. Significant differences among treatment groups were detected by LSD tests (least square deviation).

26. Repox50 Results Table1 Evaluation of Repox 50in broilers1 under research station circumstances (0-18 days of age). 1 Male broilers (Arbor Acres Plus). 2 Feed conversion ratio corrected for mortality and culls. 3 Feed conversion ratio uncorrected for mortality and culls.

27. Repox50 Table 2 Evaluation of Repox 50in broilers1 under research station circumstances (18-35 days of age). 1 Male broilers (Arbor Acres Plus). 2 Feed conversion ratio corrected for mortality and culls. 3 Feed conversion ratio uncorrected for mortality and culls.

28. Repox50 Table 3 Evaluation of Repox 50in broilers1 under research station circumstances (0-35 days of age). 1 Male broilers (Arbor Acres Plus). 2 Feed conversion ratio corrected for mortality and culls. 3 Feed conversion ratio uncorrected for mortality and culls.

29. Repox50 Table 4 Evaluation of Repox 50 on fat oxidation abilityin broilers1 under research station circumstances. 1 Male broilers (Arbor Acres Plus). 2Thiobarbituric acid 3 Ferric reducing antioxidant power

30. Repox50 Fish Applications

31. Repox50 Repox50 will enhance colour and shelf life via replacing a portion of Vitamin E within the diet

32. Repox50 Polyphenols can improve body color and meat brilliance of cultured fishes, preventing decrease in astaxanthin levels in the fish. Polyphenols added by weight from 0.004% 40ppm to 0.04% 400ppm. Sakiura US 6,509,030, B2 Cultured fish carotenoid and polyphenol added feed for improving fish body color tone and fish meat brilliance 

33. Repox50 Meat Applications

34. Repox50 Some scientific evidence • Ahn, J., Grün, I.U., Fernando, L.N. 2002. Antioxidant properties of natural plant extracts containing polyphenolic compounds in cooked ground beef. J. Food Sci. 67(4), 1364-1369. • Ahn, J., Grün, I.U., Mustapha, A. 2007. Effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef. Food Micro. 24, 7-14 • Bekhit, A.E.D., Geesink, G.H., Ilian, M.A., Morton, J.D., Bickerstaffe, R. 2003. The effects of natural antioxidants on oxidative processes and metmyoglobin reducing activity in beef patties. Food Chem. 81(2), 175-187 • Brannan, R.G., Mah, E. 2007. Grapeseed extract inhibits lipid oxidation in muscle from different species during refrigerated and frozen storage and oxidation catalyzed by peroxynitrite and iron/ascorbate in a pyrogallol red meat model system. Meat Science. 77(4), 540-546. • Jayaprakasha, G.K., Singh, R.P., Sakariah, K.K. 2001. Antioxidant activity of grape seed (vitisvinifera) extracts on peroxidation models in vitro. Food Chem. 73(3), 285-290.

35. Repox50 Factors affecting meat colour • Vitamin E feeding of cattle • Prevents oxidation; retards conversion of myoglobin to metmyoglobin • Bacteria • Produce metmyoglobin, choleglobin, and sulfmyoglobin pigments • Curing • Nitrosylhemochromogen is the stable cured meat pigment

36. Repox50 Repox50 improves meat colour

37. Repox50 Factors affecting meat colour Exposure to long-term or short term stress • Effects glycogen content of muscle and ultimate pH of muscle • Long Term Stress: DFD (dark cutter)Transport, Hunger, Fear, Aggression • Ultimate pH above 5.9 (beef), 6.5 (pork) • Short Term Stress: PSE Usually only problematic in pork • Ultimate pH below 5, generally problem can be overcome with enhancement

38. Thank you for your time Any questions? Repox50