CHOOSING THE RIGHT PHYTASE TO MAXIMIZE PHYTATE DEGRADATION

CHOOSING THE RIGHT PHYTASE TO MAXIMIZE PHYTATE DEGRADATION Wenting. Li, and Rafael. Durán Giménez-Rico Danisco Animal Nutrition, Marlborough, United Kingdom

OBJECTIVES ANTI-NUTRITIONAL IMPACTS OF PHYTATE EXTENDING OUR KNOWLEDGE BEYOND P MATRIX DEVELOPMENT

MORE THAN 50% NPP IS FROM INORGANIC P SOURCE Ingredient Starter Maize 58.71 SBM 34.89 Soybean Oil 1.77 Monocalcium Phosphate 1.65 Limestone 1.56 Salt 0.44 DL-methionine 0.33 L-Lysine HCl 0.25 Poultry Vits/TE's 0.25 L-threonine 0.07 Choline Chloride 60% 0.06 P 0.75 Phytate P 0.26 Non-phytate P 0.49 P from monocal 76% 0.37% (total 0.49%) npp from monocal ~$10 (600 $/MT) Replace of 0.1% npp by phytate P ~ $2.6 3

PHYTATE CONCENTRATION VARIES AMONG INGREDIENTS Ingredient Total P Phytate-P % P from Phytate Rice bran (14) Wheat midds (8) Min Mean Max SBM 0.71 0.38 53.5 Canola meal 1.08 0.65 60.2 Wheat middlings 0.98 0.61 62.2 Barley 0.35 0.22 62.9 Wheat 0.30 0.20 66.7 Corn 0.26 0.21 80.8 Sunflower meal 0.95 0.84 88.4 Canola (24) Cottonseed (6) SBM (56) Corn (56) Wheat (28) Sorghum (29) Barley (12) Corn DDGS (17) Rye (3) NRC 2012, Swine 0.00 2.00 4.00 6.00 8.00 % IP6 DuPont lab 4

PHYTASE SIGNIFICANTLY IMPROVES PHYTATE AVAILABILITY % 80 70 60 50 40 30 20 10 0 Ileal phytate degradation increased by 0.5-1.7X Leske and Coon, 1999 5

PHYTATE STRUCTURE IP6 IP5 IP4 IP3 IP2 IP1 Ins 6

HISTORY OF PHYTASE DEVELOPMENT The first commercial available phytase was a fungal phytase marketed in early 90s, with a main objective of reducing P excretion New generation, more effective E. coli phytases were marketed around 2000 Recently a Buttiauxella phytase is commercially available in the market - an unique phytase with high activity at low ph and wide ph range 7

INTERACTION BETWEEN PHYTATE AND NUTRIENTS IS PH DEPENDENT Starch Protein + Ca + CH 2 NH 3 + Zn + + - Protein H O H - O + Ca + Angel et al., 2010 O + Ca + - O O Fatty acid C O 8

CA REDUCES P AND PHYTATE DIGESTIBILITY 80 P digestion 90 Phytate digestion Digestibility, % 70 60 50 40 30 68 71 0 FTU/kg 500 FTU/kg 47 29 Digestibility, % 80 70 60 50 40 30 69 80 0 FTU/kg 500 FTU/kg 45 25 20 0.17% Ca 0.68% Ca * E.Coli phytase 20 0.17% Ca 0.68% Ca Tamin et al., 2004 9

IP6 HAS THE HIGHEST AFFINITY TOWARDS DIET MINERALS Proventriculus and gizzard Small intestine IP6 IP5 IP4 IP3 Persson et al., 1998 10

BUTTIAUXELLA SP. PHYTASE HAS MUCH HIGHER ACTIVITY AT UPPER GIT Butt.Phytase DuPont Lab 2016 11

BUTT. PHYTASE MORE THOROUGHLY DEGRADED IP6, IP5 IN THE INTESTINE (IN VITRO) SIMULATION Buttiauxella E coli 2 P lycii A. Niger Menezes-Blackburn et al., 2015 12

BUTT. PHYTASE IS MORE EFFECTIVE IN REVERTING CA- PHYTATE BINDING In vitro dose: 750 FTU/kg 50 Percent of Ca-PP precipitant 40 30 % 20 10 0 30 60 Time, min Buffer Buttiauxella sp. E.coli 2 E.coli 1 Citrobacter DuPont Lab 2016 13

MORE THAN 90% IP6 REDUCTION USING BUTT. PHYTASE IN UPPER GIT IP6 concentration in proventriculus and gizzard 0,4 500 FTU/kg 0,4 1000 FTU/kg % of digesta DM 0,3 0,2 0,1 0,0 a NC 90% c NC+ Buttiauxella sp. 67% b NC+ Citrobacter % of digesta DM 0,3 0,2 0,1 0,0 a NC 93% c NC+ Buttiauxella sp. 74% b NC+ Citrobacter Unpublished data 14

HIGHER IP6 REMOVAL IN UPPER GIT LEAD TO GREATER IP6 DIGESTIBILITY AT ILEUM 500 FTU/kg 1000 FTU/kg 80 70 15% 90 80 16% Digestibility, % 60 50 40 Digestibility, % 70 60 50 40 30 NC NC+ Buttiauxella sp. NC+ Citrobacter 30 NC NC+ Buttiauxella sp. NC+ Citrobacter Unpublished data 15

HIGH IP6 DEGRADATION IMPROVED BONE MINERALIZATION AND INCREASED BONE BREAKING STRENGTH Bone breaking strength, kgf, d 33 23 22 21 20 19 18 17 a ab Buttiauxella sp. R² = 0,8452 b b Citrobacter P <0.05 cd d 0 0,05 0,1 0,15 0,2 0,25 0,3 P + G IP6 concentration, % digesta DM, d 22 LSD test, BW as covariance 16

COMPLETE REDUCTION IN IP6 IN UPPER GIT IS CRITICAL TO OVERCOME THE DETRIMENTAL EFFECT OF CA Ileal P digestibility, broilers, 11 d old 80 NS % 60 40 a a b c 0.65% Ca 0.80% Ca 0.95% Ca b c 20 0 FTU/kg 500 FTU/kg 1000 FTU/kg * Buttiauxella sp. phytase Li et al., 2012 17

Protein forms binary and ternary complex with phytate Starch Protein + Ca + CH 2 NH 3 + Zn + + - Protein H O H - O + Ca + + Ca + - O O O Fatty acid C O 18

Turbidity (optical density) IP6 IS ALSO THE MOST POTENT ANTI-NUTRITIONAL FACTOR Aggregation of soy protein by IP esters at ph=2.5 0,5 0,4 0,3 44X 5X 0,2 0,1 0 Rapid breakdown of IP6 is essential to maximise efficacy of a phytase and reduce anti-nutritional potential of phytate 19

PHYTATE INHIBITS PEPSIN ACTIVITY AND ABILITY OF THE BIRD TO DIGEST PROTEIN AND AMINO ACIDS 120 Pepsin activity (% of added pepsin) 100 80 60 40 20 A diet with 0.20% phytate phosphorus 0 0 2 4 6 8 10 conc. of phytic acid / mm Adapted from Yu et al., 2012 20

THE INHIBITION CAN BE REVERSED SIMPLY BY REMOVING 1 P GROUP FROM PHYTATE (IP6 IP5) ph 2.5 at 37 C, soy protein, porcine pepsin, phytate 1,8 0,30 1,5 0,25 Pepsin activity 1,2 0,9 0,6 0,3 0,0 0 30 60 90 120 Time of Butt phytase hydrolysis (min) 0,20 0,15 0,10 0,05 0,00 soy protein turbidity IP6 IP5 Pepsin activity Phytate-Protein Yu et al., 2012 21

Summary PHYTATE DOSE NOT ONLY AFFECT P ABSORPTION, BUT ALSO OTHER NUTRIENT DIGESTIBILITY 100 Ileal digestibility (%) 90 80 70 60 50 40 Ileal amino acid digestibility R² = 0.68 Ileal P digestibility R² = 0.90 30 20 30 40 50 60 70 80 90 100 Phytate (IP6) degradation (%) Total AA digestibility Ileal P digestibility Amerah et al., 2012 22

IMPROVED NUTRIENT AVAILABILITY WITH BUTT. PHYTASE Positive Control Negative Control 250 FTU Axtra Butt. phy PHY 500 FTU Axtra Butt. PHY phy 1000 FTU Axtra Butt. phy PHY Aspartic acid 79.8 c 80.6 c 81.3 bc 83.3 ab 84.5 a Threonine 72.6 b 72.9 b 73.4 b 76.3 ab 78.6 a Serine 77.5 b 78.4 b 79.0 b 81.9 a 83.6 a Glutamine 87.5 c 87.8 c 88.4 bc 89.7 ab 90.4 a Proline 81.5 bc 79.5 c 80.8 bc 82.8 ab 84.0 a Glycine 77.0 c 77.2 c 78.3 bc 80.3 ab 82.0 a Alanine 81.8 c 81.6 c 83.0 bc 85.0 ab 85.9 a Valine 79.8 c 79.8 c 80.9 bc 82.8 ab 84.2 a Isoleucine 81.9 c 82.3 c 83.3 bc 85.1 ab 86.3 a Leucine 84.0 c 83.6 c 85.2 bc 86.8 ab 87.7 a Tyrosine 81.0 b 81.2 b 82.1 b 84.8 a 86.2 a Phenylalanine 83.9 b 83.8 b 85.2 b 87.2 a 88.0 a Histidine 80.4 b 79.5 b 80.4 b 82.4 ab 84.1 a Lysine 85.9 b 89.0 a 88.1 ab 88.9 a 89.8 a Arginine 88.3 c 89.1 bc 89.5 b 90.2 ab 91.0 a Cysteine 67.9 ab 62.8 c 65.7 bc 69.3 ab 70.6 a Methionine 91.4 c 91.2 c 91.7 bc 92.9 ab 93.9 a All amino acids 81.3 c 81.2 c 82.1 bc 84.1 ab 85.3 a PHY.NZ.B.07 Massey University ab Values without a common superscript are significantly different (P<0.05) 23

IMPROVED NUTRIENT AVAILABILITY WITH BUTT. PHYTASE 3,240 3,230 3,220 3,210 3,200 3,190 3,180 3,170 3,160 3,150 AMEn (kcal/ kg DM, 7-21 days) a a b b Positive control b 0 250 500 750 1000 Phyt ase dose (FTU/ kg feed) ab Values without a common superscript are significantly different (P<0.05) PHY.NZ.B.07 Massey University 24

HIGH DOSE BUTT. PHYTASE EXTRA-PHOSPHORIC EFFECT Extra phosphoric effect 84,0 Mean AID AA, % P linear < 0.0001 83.4a 83.6a 82,0 80,0 80.4e 80.8de 82.1b 79.6f 81.6bc 81.3cd 78,0 76,0 0 250 500 750 1000 NC + 0.6g P NC + 1.2g P NC + 1.8g P Buttiauxella phytase, FTU/kg Pooled data from two trials Ross broilers fed NC diet + Buttiauxella phytase from 5-21d AID measured at d21 Schothorst Feed Research, NL 25

INCREASE BUTT. PHYTASE DOSE IMPROVED ILEAL NA DIGESTIBILITY 10,0 0 250 500 750 1000 Phytase, FTU/kg NC + 0.6g P NC + 1.2g P NC + 1.8g P 0,0-10,0-20,0-15.8bc -9.7ab 5.7a -30,0-26.3bc -29.2d -29.9d -40,0-50,0-44.4d Ileal Na digestibility, % -45.9cd P < 0.0001 Increase MCP-P from 1.2 to 1.8 had no impact on ileal Na digestibility Schothorst Feed Research, NL 26

BUTT. PHYTASE AT HIGH DOSE CONTINUES TO IMPROVE PERFORMANCE 3.6 3.4 3.2 3.0 2.8 2.6 2.4 de f f a Ileal Digestible Phosphorus (g/ kg diet) e e de bc cd 0 250 500 750 1000 1250 1500 1750 2000 Phytase dose (FTU/kg) E. coli phytase Axtra PHY NC+0.6 g P* NC+1.2 g P* NC+1.8 g P* b de cd 188 186 184 182 180 cd Ileal Digestible Amino Acid (g/ kg diet) cd bcd abc ab bcd 178 d 176 0 250 500 750 1000 1250 1500 1750 2000 Phytase dose (FTU/kg feed) E. coli phytase Axtra PHY NC+0.6 g P* NC+1.2 g P* NC+1.8 g P* a abcd abc bcd bcd 27

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Summary Phytate negatively impacts growth both directly and indirectly WEAKER BONES DUE TO REDUCED BONE MINERALISATION INCREASED PHOSPHORUS EXCRETION DIVERTING ENERGY AWAY FROM GROWTH COMPOMISED NUTRIENT UPTAKE POOR AMINO ACID DIGESTIBILITY 29

PHYTATE REDUCES PERFORMANCE IN BROILERS 1,60 1,55 FCR, 0-28 d a 1000 980 BWG (g/b), 0-28 d a 1,50 b 960 940 b 1,45 920 1,40 0.2% PP 0.42% PP 900 0.2% PP 0.42% PP *10% rice bran added Liu et al., 2008 30

AXTRA PHY MATRIX DETERMINATION 31

WAYS TO DEVELOP MATRIX VALUES Method PROS CONS Estimation of nutrient contributions based on digestibility improvements seen in in vivo studies 1 Calculate matrix based on assumption of a log linear response for P Determine AvP relative to inorganic standard Species specific matrix values, based on actual animal responses Each nutrient separately modelled Accounts for varied responses between trials Quick to do Few studies required (as few as 1) Same values used for both pigs and poultry Few trials required (as few as 1 per species) Species specific values Large dataset is needed (~10 trials) Takes time (and $) to generate matrix Models used are out of date (1 st generation phytases) and were poultry only. Wrongly assumes the pattern for all nutrients follows P response Does not account for antinutritional effects phytate on Energy & AAs Results depend on inorganic standard that is used Results can be manipulated by changing the calcium levels in the diet Only covers P, not other nutrients 1 approach used by DuPont 32

METHODS USED BY DUPONT Based on dig P improvement values using a exponential curve Validation using inorganic P standard curve, based on bone ash 33

AXTRA PHY: SUPERIOR BIOEFFICACY TRANSLATES TO SUPERIOR MATRIX Meta analysis was performed to model Phosphorus Contribution from Axtra PHY: 10 broiler ileal digestibility trials conducted from 2008 to 2011 Ross 308 and Cobb 500 broilers Range of phytase dosing from 250 2000 FTU/kg feed Average phytate P level = 0.26% 296 data points in data set after removing positive controls and other phytase sources. Increments in digestible P vs. negative control diets calculated Modeling used non-linear regression 34

AXTRA PHY: SUPERIOR BIOEFFICACY TRANSLATES TO SUPERIOR MATRIX Available Phosphorus Contribution (g/ kg feed) 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 This model included all digestibility trials - 296 data points 1.12 0.9 1.46 1.2 1.74 1.5 1.81 1.83 500 FTU Axtra Phy = 836 FTU E.Coli Phytase relative bioefficacy = 836 500 =1.67:1 0 250 500 750 1000 1250 1500 1750 2000 Axtra Phy dose (FTU/ kg feed) Axtra PHY Phycheck TM 35

AXTRA PHY VERSUS PHYZYME XP CONTRIBUTIONS AT 500 FTU/KG FEED Phytase Enzyme Matrix Value Comparison AT 500 FTU/kg feed* Nutrient Phyzyme XP Axtra PHY % Change Available P (%) 0.120 0.146 122% Digestible P (%) 0.110 0.134 122% Calcium (%) 0.110 0.134 122% ME (kcal/kg) 63.0 67.6 107% ME (MJ/kg) 0.264 0.283 107% Sodium (%) 0.030 0.030 100% Digestible Crude Protein (%) 0.324 0.359 111% Digestible Lysine (%) 0.017 0.021 119% Digestible Methionine(%) 0.006 0.006 100% Digestible Cystine (%) 0.012 0.012 100% Digestible TSAA (%) 0.018 0.018 100% Digestible Threonine (%) 0.013 0.017 130% Digestible Tryptophan (%) 0.005 0.005 100% Digestible Leucine (%) 0.031 0.036 117% * Based on Broiler Starter diets w ith 0.26% Phytate Phosphorus 36

AXTRA PHY CONTRIBUTIONS, 250-2000 FTU/KG FEED AXTRA PHY DOSE (FTU/ KG FEED) Nutrient 250 500 750 1000 1500 2000 Available P (%) 0.112 0.146 0.164 0.174 0.181 0.183 Digestible P (%) 0.103 0.134 0.151 0.159 0.166 0.168 Calcium (%) 0.103 0.134 0.151 0.159 0.166 0.168 ME (kcal/kg) 52.1 67.6 72.4 73.9 74.5 74.6 ME (MJ/kg) 0.218 0.283 0.303 0.309 0.312 0.312 Sodium (%) 0.020 0.030 0.037 0.041 0.045 0.047 Digestible Crude Protein (%) 0.190 0.359 0.521 0.671 0.935 1.151 Digestible Lysine (%) 0.011 0.021 0.030 0.038 0.052 0.063 Digestible methionine(%) 0.003 0.006 0.009 0.011 0.015 0.018 Digestible Cystine (%) 0.006 0.012 0.017 0.021 0.029 0.034 Digestible Threonine (%) 0.009 0.017 0.024 0.031 0.043 0.053 Digestible Tryptophan (%) 0.002 0.005 0.007 0.009 0.012 0.015 Digestible Arginine (%) 0.008 0.016 0.023 0.030 0.041 0.050 Digestible Valine (%) 0.011 0.021 0.030 0.038 0.052 0.063 Digestible Leucine (%) 0.019 0.036 0.052 0.066 0.090 0.108 Digestible Serine (%) 0.012 0.022 0.032 0.041 0.056 0.068 Digestible Proline (%) 0.013 0.024 0.035 0.045 0.062 0.076 37

META-ANALYSIS DATA Data from 4 trials were collected Five treatments: PC (adequate in all nutrients) NC (reduced 0.2 MJ/kg ME, 0.17 % AvP, 0.16 % Ca vs PC) NC+ 250, 500 and 1000 FTU/kg either Buttiauxella or E. coli phytase. Diets based mainly on corn and soybean meal Birds were fed test diets from 0-42days Statistics conducted using the Fit Model Platform of JMP 11, trial code was included in the model as a random effect. Tukey s HSD test was used for means separation. 38

RESULTS: COMPARISON OF PHYTASE SOURCES, 0-42D ADG, g 72 70 68 66 64 62 60 bc d bc c ab ab a a Buttiauxella E coli PC Buttiauxella phytase at 1000 FTU/kg improved FCRc* by 4 points vs PC Buttiauxella phytase improved efficacy compared to E. coli phytase 58 0 250 500 1000 Phytase dose, FTU/kg * FCRc: body weight corrected feed conversion ratio, correction of 3 points for each 100g body weight difference from PC FCRc 1,85 1,8 1,75 1,7 1,65 1,6 1,55 a Buttiauxella E coli b PC bc bc bc bc c d 0 250 500 1000 39

TAKE HOME MESSAGE Must haves for good phytase High efficacy in P release Highly active in acidic part of the digestive tract Fast degradation of IP6 to IP5 Ability to degrade protein-phytate complexes Buttiauxella sp. phytase showed superior effect when compare to other commercial phytases in: Total P release IP6 and IP5 removal Breakdown protein-phytate complex Better growth performance 40

THE MIRACLE OF PHYTASE https://youtu.be/hvqk6xnd9z0 41

Buttiauxella sp. phytase breaks down phytate more quickly (as shown below) and more completely than any other phytase on the market Some form of similar image DuPont Laboratory 2015 42

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