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Wheat and barley increase phytate degradation and phosphorus digestibility in corn fed to pigs
Animal Feed Science and Technology 248 (2019) 77–84
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Wheat and barley increase phytate degradation and phosphorus digestibility in corn fed to pigs
T
⁎
H.D. Poulsena, , A.L. Voergaarda,b, A.B. Stratheb, K. Blaabjerga a b
Aarhus University, Foulum, Department of Animal Science, DK-8830 Tjele, Denmark University of Copenhagen, Department of Veterinary and Animal Sciences, DK-1870 Frederiksberg C, Denmark
A R T IC LE I N F O
ABS TRA CT
Keywords: Additivity Liquid feeding Phosphorus Phytase Phytate Pig
Two experiments evaluated the additivity of phytate content and coefficient of total tract apparent digestibility (CTTAD) of phosphorus (P) in wheat-barley (WB) and corn (C) when soaked (exp. 1) or fed dry to pigs (exp. 2) in combination with or without microbial phytase (1250 FTU/ kg as-fed). In exp. 1, WB (1:1, w/w) and C were soaked separately or together (W:B:C = 1:1:2, w/ w/w) without (WBC) or with microbial phytase (WBC + phy). Phytate content was affected by the interaction between diet and soaking time (P < 0.0001) as phytate decreased with time. The decrease was greatest for WBC + phy followed by WBC, WB and C resulting in a half-life of phytate (K) of 8.7, 15.8, 24.4 and 303.4 h, respectively. Soaking of WB and C together showed non-additive effects as the estimated (assuming additivity) phytate content at 2, 4, 8 and 24 h of soaking was 0.17, 0.34, 0.42 and 0.5 g/kg DM higher (P < 0.001) than the experimentally determined phytate content in WBC. Actually, phytate degradation in C was estimated to be 0.15, 0.30, 0.42 and 0.49 higher at 2, 4, 8 and 24 h, respectively, when soaked together with WB than when soaked alone. Accordingly, the estimated (assuming additivity) K-value for WBC was 148 h longer (P = 0.08) than the K-value derived from the experimentally determined phytate degradation in WBC. Exp. 2 comprised 24 pigs of 47 ± 2 kg from 6 litters. Pigs were housed in metabolism crates and fed one of four diets (WB, C, WBC or WBC + phy) for 12 days; 5 days adaptation and 7 days total collection of feces and urine. The CTTAD of P was greatest for WBC + phy followed by WB, WBC and C (P < 0.0001). Feeding WB and C in combination tended to show non-additive effects as the estimated (assuming additivity) CTTAD of P in WBC was 7.7 percentage units lower (P = 0.09) than the experimentally determined CTTAD of P in WBC. In fact, CTTAD of P in C was estimated to be 14.6 percentage units higher when fed together with WB than alone. In conclusion, phytases present in W and B are non-specific and catalyze the degradation of phytate in C when soaked or fed dry together (ratio 1:1:2). Thus, when phytate rich cereals very different in plant phytase activity, are mixed, additivity cannot be assumed.
Abbreviations: B, barley; C, corn; Ca, calcium; CTTAD, coefficient of total tract apparent digestibility; CSTTD, coefficient of standardized total tract digestibility; DM, dry matter exp experiment; F0, the relative instantaneously degradable fraction of phytate; FTU, phytase activity expressed in units; GMM, Generalised Michaelis Menten; K, the half-life of phytate phosphorus in hours; LS means, least square means; n, description of the shape of the phytate degradation curve; ND, not detectable; SEM, standard error of mean; P, phosphorus; SD, standard deviation; W, wheat; WB, wheatbarley; WBC, wheat-barley-corn ⁎ Corresponding author. E-mail address: [email protected] (H.D. Poulsen). https://doi.org/10.1016/j.anifeedsci.2018.12.006 Received 31 May 2018; Received in revised form 17 August 2018; Accepted 22 December 2018 0377-8401/ © 2019 Elsevier B.V. All rights reserved.
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1. Introduction Phytate, the main storage form of P in plant feedstuffs, is poorly digested by pigs, unless phytase is present. Thus, microbial phytase is widely used in pig diets to increase the digestibility of phytate P and to reduce the excretion of P to the environment. Inclusion of cereals like wheat (W), barley (B) and triticale in diets for pigs also increases the digestibility of phytate P as these cereals display considerable plant phytase activity (Pointillart et al., 1984, 1987; Weremko et al., 2001). In contrast, corn (C), soybean meal and rapeseed cake/meal exhibit negligible or no phytase activity (Eeckhout and De Paepe, 1994; Viveros et al., 2000). The phytate content in a mixture of W and soybean meal or W and rapeseed cake was shown to be 0.55 and 0.16 lower after 24 h of soaking in water (20 °C) compared with the sum of phytate determined from the separately soaked single feedstuffs (Blaabjerg et al., 2010). This shows that phytases from W catalyzed the degradation of phytate present in the soybean meal or rapeseed cake when soaked together. Consequently, the amounts of non-degraded phytate in the individual feedstuffs cannot just be added (non-additive; Blaabjerg et al., 2010). Likewise, the P digestibility values of individual feedstuffs containing phytate and different phytase activities may not always be additive when fed together. However, there is a lack of knowledge about the additivity of P digestibility values despite the fact that to ensure efficient utilization of P, the additivity of P digestibility values is a crucial factor in the formulation of diets for pigs. Therefore, the objectives of the two experiments were to test the hypotheses: phytate degradation and CTTAD of P in wheat-barley (WB) and C are non-additive when soaked (exp. 1) or fed to pigs (exp. 2) in combination (WBC). 2. Material and methods 2.1. Experimental diets The feedstuffs W, B and C used in exp. 1 and 2 originated from the same lot, respectively. All the feedstuffs and diets used in the experiments were ground by a roller mill (3 mm between the rolls). The WB and C were soaked (exp. 1) or fed (exp. 2) separately or in combination at a ratio of 1:1:2 (w/w/w) and supplemented with microbial phytase (granulate, Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany) at 0 or 1250 FTU/kg as-fed (Table 1). Dietary P was solely of plant origin as no feed phosphate was added. The diets based solely on cereals were not optimized regarding other nutrients. 2.2. Experiment 1 The feedstuffs (200 g) were soaked in preheated water (20 °C) at a ratio of 1:2.75 (w/w) in 1-L closed containers with a continuously magnetic stirring as described by Carlson and Poulsen (2003). No attempt was made to regulate pH. The containers were placed in an incubator (New Brunswick Scientific Model G25 Controlled Environment Incubator Shaker, GST Technical Sales, Edmonton, Alberta, Canada) to maintain a constant temperature of 20 °C. Samples of about 60 mL were collected at 2, 4, 8 and 24 h and immediately frozen by placing the samples in a freezer (−20 °C) to stop further degradation. Samples of the non-soaked feedstuffs (0 h) were also collected for analysis. Four replicates per treatment were conducted. 2.3. Experiment 2 The experimental protocol was approved by the Danish Animal Experiments Inspectorate, the Danish Ministry of Justice, Copenhagen, Denmark. The experiment comprised 24 female pigs (Landrace × Yorkshire × Duroc) from 6 litters weighing Table 1 Experimental diets and analyzed chemical composition of the experimental diets of exp. 1 and 2 (n = 4). Mean ± SD. Dietsu WB Ingredient composition (proportion of ingredient) Wheat Barley Corn Microbial phytase, FTU/kg as-fed Chemical composition DM, % Ca, g/kg DM Total P, g/kg DM Phytate P, g/kg DM Phytase, FTU/kg DM
C
WBC
WBC + phy
0
1.00 0
0.25 0.25 0.50 0
0.25 0.25 0.50 1250
88 ± 0.36 0.5 ± 0.2 3.0 ± 0.3 1.9 ± 0.06 770 ± 15
88 ± 0.29 0.1 ± 0.0 3.0 ± 0.3 2.3 ± 0.1 NDv
88 ± 0.23 0.3 ± 0.1 2.7 ± 0.1 2.1 ± 0.05 446 ± 54
88 ± 0.23 0.3 ± 0.0 2.8 ± 0.2 2.1 ± 0.15 2140 ± 130
0.50 0.50
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v ND = not detectable (below 50 FTU/kg DM).
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47 ± 2 kg. From each litter, the pigs were assigned randomly to 4 diets. The pigs were housed in stainless steel metabolism crates and were fed 800 g twice daily (0800 and 1400) for 12 days; 5 days for adaptation and 7 days for total collection of urine and feces. On day 5, the pigs were fitted with urine bladder catheters for separate collection of urine and feces. 2.4. Chemical analyses In exp. 1, the pH was measured by a pH meter (PHM210, MeterLab, Radiometer, Copenhagen) in connection with sample collection. Dry matter (DM) was determined by freeze drying with the following parameters: cold trap: −85 °C, time: 72 h, final product temperature: 30 °C, and pressure: 0.05 mbar. Total P was assayed by the colorimetric vanadomolybdate procedure (Stuffins, 1967). Phytate was analysed according to the method by Haug and Lantzsch (1983). Determination of phytase activity followed the procedure of Engelen et al. (1994) where one FTU is the amount of enzyme, which liberates 1 μmol inorganic orthophosphate per minute from 0.0051 mol/L sodium phytate at pH 5.5 and 37 °C. In exp. 2, all the experimental diets were analyzed for phytase activity and phytate as described above. Additionally, diets, feces, and urine were analyzed for P (described above) and for Ca according to the AOAC procedure (method 975.03; AOAC, 2000) with the following modifications: dry ashing was performed at 450 °C for 3 h in step 1 and 1 h in step 2. Dry matter in diets and feces was determined by oven drying at 103 °C for 20 h. 2.5. Calculations and statistical analyses The results were analyzed by use of the MIXED procedure of SAS, 2018. The results are least squares means (LS means ± SEM) and standard error of mean (SEM). Statistical significance was accepted at P < 0.05 and tendencies were declared at 0.05 ≤ P ≤ 0.10. Treatment differences were separated by the PDIFF option of SAS. Results of exp. 1 were analysed according to the following model: Yijke = μ + ai + bj + abij + Uk + εijke where Yijke is the dependent variable; μ the overall mean; ai the fixed effect of diet, i = WB, C, WBC, WBC + phy; bj the fixed effect of soaking time, j = 0, 2, 4, 8, 24; abij the diet and soaking time interaction effect; Uk the random effect of container; εijke the residual experimental error. It is assumed that Uk ˜ N(0, σU2) and εijke ˜ N(0, σe2). Treating container as a random effect generates a suitable correlation structure that accounts for measurements on the same container being correlated across time. Results of exp. 2 were analysed according to the following model: Yije = μ + ai + Uj + εije where Yije is the dependent variable; μ the overall mean; ai the fixed effect of diet, i = WB, C, WBC, WBC + phy; Uj the random effect of litter; εije the residual experimental error. It is assumed that Uj ˜ N(0, σU2) and εije ˜ N(0, σe2). 2.5.1. Additivity Exp. 1. Assuming additivity, the phytate content (g P/kg DM) in the mixture of WB and C at 2, 4, 8, and 24 h of soaking were estimated using the following equation: Phytate PWBC_t = phytate PWB × FCWB + phytate PC × FCC; where FCWB and FCC are the relative feedstuff contribution of WB and C to the WBC mixture (i.e. FCWB + FCC = 1); Phytate PWBC_t is the estimated (assuming additivity) phytate P content (g P/kg DM) at a given soaking time (t = 2, 4, 8, 24 h) in the mixture of WB and C while phytate PWB and phytate PC represent experimentally determined phytate P content (g P/kg DM) in WB and C soaked separately. Exp. 2. Assuming additivity, the CTTAD of P in the mixture of WB and C were estimated using the following equation: CTTAD of PWBC = CTTAD of PWB × PCWB + CTTAD of PC × PCC; where PCWB and PCC are the relative P contribution of WB and C to the WBC mixture (i.e. PCWB + PCC = 1); CTTAD of PWBC is the estimated (assuming additivity) CTTAD of P in the mixture of WB and C while CTTAD of PWB and CTTAD of PC represent experimentally determined CTTAD of P in WB and C, separately. Assuming additivity, the CTTAD of DM was estimated in the same way as shown for CTTAD of P. To test for additivity of phytate P content when soaking WB and C in combination (exp. 1), the experimentally determined phytate P content for WBC was regressed on the estimated phytate P content for WBC, while forcing the intercept through zero. A slope, deviating significantly from 1, would indicate that the assumption of additivity is not met. The Reg procedure of SAS was used for the analysis. The same statistical analysis was conducted to test for additivity of CTTAD of P and DM, respectively, when feeding WB and C in combination (exp. 2). 2.5.2. Fit of Generalised Michaelis Menten function In exp. 1, a Generalised Michaelis Menten (GMM) function was fitted to the results on phytate content as described in details by 79
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Blaabjerg et al. (2012). This function was parameterized to derive the following parameters: F0, the relative instantaneously degradable fraction of phytate; K, the half-life of phytate P in hours; n, describes the phytate degradation curve. n ≤ 1 indicates that the phytate degradation curve decreases continuously, whereas n > 1 displays a sigmoid shaped degradation curve. A K-value was estimated for each of the four diets. It was not possible to estimate (i.e. convergence issues for the nonlinear mixed model routine) an individual F0 and n-value for the different diets due to the structure of the data and number of replicates. Assuming additivity, the Kvalue for the mixture of WB and C was estimated using the following equation: KWBC = KWB × FCWB + KC × FCC; where FCWB and FCC are the relative feedstuff contribution of WB and C to the WBC mixture (i.e. FCWB + FCC); KWBC is the estimated (assuming additivity) half-life of the phytate content in hours in the mixture of WB and C while KWB and KC represent the half-life of the phytate content derived via the GMM function from the experimentally determined phytate P content in WB and C soaked separately. The difference between the estimated K-value and the K-value derived via the GMM function in the WBC diet was tested using the contrast analysis procedure in SAS. 3. Results The analyzed chemical composition of the diets used in exp. 1 and 2 is shown in Table 1. The WB showed considerable plant phytase activity, whereas the phytase activity of C was below the detection level. The total P content was almost similar for the four diets, while the Ca content differed. Phytate P constituted from 63 to 78% of total P in the diets. 3.1. Experiment 1 The phytate content during soaking was affected by the interaction between diet and soaking time (P < 0.0001; Table 2). This was due to the fact that the amount of degraded phytate increased with time, and this increase was greatest for WBC + phy followed by WBC, WB and C (Table 2). In accordance, K was lowest for WBC + phy (8.7 h) followed by WBC (15.8 h), WB (24.4 h), and C (303.4 h). Assuming additivity, the estimated content of non-degraded phytate in WBC was 0.17, 0.34, 0.42 and 0.5 g phytate P/kg DM greater (P < 0.0001) at 2, 4, 8, and 24 h of soaking compared with the experimentally determined content of non-degraded phytate in WBC. Moreover, the estimated K-value for WBC was 164 h which was 148 h longer (P = 0.08) than the K-value derived via the GMM function from the experimentally determined phytate degradation in WBC (15.8 h). These calculations show that when soaking WB and C in combination (W:B:C = 1:1:2), the degradation of phytate was not additive. The degradation of phytate in C was estimated to be 0.15, 0.30, 0.42 and 0.49 higher at 2, 4, 8 and 24 h, respectively, when soaked in combination with WB than when soaked alone. The phytase activity differed as planned between diets (P < 0.0001) and tended to decrease (P = 0.08) during the soaking period (Table 2). The pH was affected by the soaking time (P < 0.0001) primarily due to a drop from 8 to 24 h. 3.2. Experiment 2 One pig fed the WB diet, two pigs fed the C diet and one pig fed the WBC diet were omitted from the experiment due to large feed refusals. The determined CTTAD of DM tended (P = 0.07) to be affected by diet as CTTAD of DM for WB was lower (P = 0.04) compared with CTTAD of DM for C, WBC or WBC + phy (Table 3). The determined CTTAD of P was affected by diet (P < 0.0001). The CTTAD of P in C was 27 percentage units lower (P < 0.001) than the CTTAD of P in WB, and addition of microbial phytase to WBC increased (P = 0.0004) the CTTAD of P by 13 percentage units (WBC vs. WBC + phy; Table 3). Assuming additivity, the CTTAD of DM and P for WBC were estimated to be 0.7 and 7.7 percentage units lower (P = 0.35; P = 0.09), respectively, than the experimentally determined values. This shows that CTTAD of DM for WB and C were additive when WB and C were fed in combination (W:B:C = 1:1:2) whereas CTTAD of P for WB and C tended strongly to be non-additive despite the limited number of observations. The CTTAD of P in C was estimated to be 14.6 percentage units higher when fed together with phytase containing W and B than alone. The Ca intake differed between the diets (P < 0.0001; Table 3) reflecting the different Ca content of the diets. The urinary P excretion increased (P = 0.02), and the P retention decreased (P = 0.0005) concurrently with a reduced Ca intake (P < 0.0001) or an enhanced intake of digestible P (P < 0.0001). Likewise, the Ca retention increased (P < 0.0001) along with an increased intake of Ca and/or digestible P (P < 0.0001). 4. Discussion The lack of detectable phytase activity in C agrees with previous findings (Eeckhout and De Paepe, 1994; Viveros et al., 2000) and explains the very limited phytate degradation during soaking for 24 h and the high K-value (303.4 h). The pH development and the tending decrease in phytase activity of the present diets during 24 h of soaking correspond with similar soaking studies conducted at 20 °C (Carlson and Poulsen, 2003; Blaabjerg et al., 2010, 2012). Moreover, Esmaeilipour et al. (2012) reported a greater stability of the activity of phytase in W compared with B. Blaabjerg et al. (2012) observed that K-values for phytate P in soaked non-heat-treated W and B, derived from the GMM function, averaged 23 and 44 h, respectively. Using these K-values, a K-value for a mixture of W and B (ratio 1:1) was estimated to 34 h 80
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Table 2 Effects of diets and 0, 2, 4, 8 and 24 h of soaking at 20 °C on phytate P content, proportion of non-degraded phytate P in brackets, estimated phytate P content in WBC (assuming additivity), phytase activity and pH in exp. 1. LS means ± SEM. Dietsu
Observations
WB
C
WBC
WBC + phy
4
4
4
4
P, g/kg DM 2.26bA (1.00) 2.21bAC (0.98) 2.24bAB (0.99) 2.03bB (0.90) 2.04bBC (0.90)
P-valuesv SEM
Diet
Time
DxT
0.08
< 0.0001
< 0.0001
< 0.0001
Hours 0 2 4 8 24
Determined phytate 1.87aA (1.00) 1.74acA (0.93) 1.44aB (0.77) 1.36aB (0.73) 0.90acC (0.48)
Hours 2 4 8 24
Estimated phytate P, g/kg DM (assuming additivity) 1.98 1.84 1.69 1.47
Hours 0 2 4 8 24
Phytase activity, FTU/kg DM NDb, y 770a 773a NDb 786a NDb 756a NDb 641a NDb
446c 444c 431c 381c 350c
2140dA -z 1810dB 1880dBC 1970dC
55
< 0.0001
0.08
0.10
Hours 0 2 4 8 24
pH 6.1A 6.0A 6.0A 6.1A 5.3abB
6.2A 6.1A 6.1A 6.2A 5.7bB
6.2A 6.1A 6.0A 6.1A 5.3abB
0.15
0.27
< 0.0001
0.83
6.2A 6.2A 6.2A 6.3A 5.2aB
2.10bA (1.00) 1.81aB (0.86) 1.50aC (0.71) 1.26acD (0.60) 0.97aE (0.46)
2.13bA (1.00) 1.57cB (0.74) 1.45aB (0.68) 1.06cC (0.50) 0.71cD (0.33)
< 0.0001x
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v Within a row, means without a common lowercase superscript (a, b, c, d) differ (P < 0.05). Within a column, means without a common uppercase superscript (A, B, C, D, E) differ (P < 0.05). x Using the REG procedure in SAS it was shown that estimated phytate P content (assuming additivity) was different from the experimentally determined phytate P content in WBC. y ND = not detectable (below 50 FTU/kg DM). z The phytase activity at 2 h for WBC + phy dropped to 1435 FTU/kg DM. This resulted in an interaction between diet and soaking time (P < 0.0001). However, this drop cannot be true as the activity at the following time points (4, 8 and 24 h) was considerably higher. Therefore, the phytase activity at 2 h for WBC + phy was excluded from the statistical analysis (shown in Table 2).
(assuming additivity). This estimated half-life, derived from a previous study (Blaabjerg et al., 2012), is 11 h longer (34 vs. 23 h) than the present K-value observed for WB (ratio 1:1) suggesting that K-values for W and B are not additive. This could be due to the use of different W and B cultivars in the study by Blaabjerg et al. (2012) and the present study. On the other hand, it could also be due to that phytases from W and B complement each other resulting in faster and greater phytate degradation. The present study showed that phytate degradation in WBC was not additive demonstrating that W- and B-phytases are non-specific and also degrade C-phytate. Similarly, studies showed a non-additive phytate degradation when W or W-bran rich in phytase were soaked together with feedstuffs like C, soybean meal, rapeseed meal or rapeseed cake because the W-phytases stimulate the phytate degradation in these feedstuffs (Stone et al., 1984; Zhu et al., 1990; Blaabjerg et al., 2010). The present degradation of C-phytate by W- and B-phytases demonstrates that substrate and enzymes were able to get in contact. Despite a lower phytase activity, WBC showed a smaller K-value and a relatively greater phytate degradation at 2, 4 and 8 h of soaking compared with WB. This may be explained by the higher phytate content in WBC due to inclusion of phytate rich C and by differences in the storage site of phytate in W, B, and C. About 0.9 of the phytate in W and B is deposited in the aleurone layer, whereas 0.9 of the phytate in C is situated in the germ (O’Dell et al., 1972; Raboy, 1997, 2003). These differences in the storage site of phytate seem to influence the access of phytase to phytate (Adeola et al., 2004; Blaabjerg et al., 2010, 2012). Thus, the relatively greater phytate degradation within the first 8 h of soaking in WBC than in WB could be due to a better contact between the enzymes and the phytate in C compared with W and B. Furthermore, addition of microbial phytase to WBC decreased K from 15.8 to 8.7 h and increased the proportion of degraded phytate with 0.13, 0.03, 0.16 and 0.26 at 2, 4, 8 and 24 h, respectively, compared with WBC soaked without microbial phytase. This was most likely due to a degradation of C-phytate as previous soaking studies show that microbial phytase did not contribute to further degradation of phytate in W and B (Blaabjerg et al., 2010, 2012). 81
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Table 3 Effects of diets on coefficient of total tract apparent digestibility (CTTAD), excretion, and retention of P and Ca and digestible P and the estimated CTTAD of DM and P in WBC in exp. 2. LS means ± SEM. Dietsu
P-valuev
WB
C
WBC
WBC + phy
SEM
Diets
Observations DM intake, g/day CTTAD of DM
5 1268 0.866
4 1063 0.880
5 1127 0.880
6 1328 0.878
103 0.004
0.20 0.07
P intake, g/day P retention, g/day P in feces, g/day P in urine, g/day P net absorption, g/day CTTAD of P Proportion of utilized P Digestible P, g/kg DM
3.74 0.56a 1.68a 1.52a 2.05a 0.555a 0.138a 1.64a
3.24 −0.87b 2.36b 1.81a 0.88b 0.285b −0.288b 0.87b
3.06 −0.22c 1.55a 1.74a 1.50c 0.493c −0.096c 1.34c
3.68 0.03c 1.39a 2.26b 2.29d 0.621d 0.006ac 1.72a
0.29 0.18 0.17 0.16 0.16 0.022 0.062 0.06
0.20 0.0005 0.01 0.02 < 0.0001 < 0.0001 0.001 < 0.0001
Ca Ca Ca Ca
0.68a −0.06a 0.68a 0.06
0.15b −0.99b 1.14b 0.01
0.31c −0.55c 0.74a 0.12
0.37c −0.34d 0.60a 0.11
0.03 0.06 0.06 0.05
< 0.0001 < 0.0001 < 0.0001 0.46
intake, g/day retention, g/day in feces, g/day in urine, g/day
Experimentally determined CTTAD of DM Estimated CTTAD of DM (assuming additivity)
0.880 0.873
0.35x
Experimentally determined CTTAD of P Estimated CTTAD of P (assuming additivity)
0.493 0.416
0.09y
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v Within a row, means without a common lowercase superscript (a, b, c, d) differ (P < 0.05). x Using the REG procedure in SAS it was shown that estimated CATTD of DM (assuming additivity) did not differ from the experimentally determined CATTD of DM in WBC. y Using the REG procedure in SAS it was shown that estimated CATTD of P (assuming additivity) tended to differ from the experimentally determined CATTD of P in WBC.
The CTTAD of P was highest in WBC + phy due to the addition of microbial phytase, whereas it was lowest in C reflecting the lack of detectable cereal phytase activity. CTTAD of P in C varies considerably among studies, ranging from −0.41 to 0.41 (Jongbloed and Kemme, 1990; Weremko et al., 2001; Shen et al., 2002; Zhai and Adeola, 2013). The large variation is primarily due to the use of different dietary P levels and methods (direct or regression) as discussed by Zhai and Adeola (2013). The considerably higher CTTAD of P in WB compared with WBC was owing to the higher plant phytase activity and the lower phytate P content in WB. In line with the in vitro results on phytate degradation in exp. 1, CTTAD of P in WB and C tended to be non-additive as the W- and B-phytases increased CTTAD of P in C by 14.6 percentage units when WB and C were fed in combination (WBC). On the other hand, Rodehutscord et al. (1996) found that CTTAD of P in W and soybean meal was additive when fed in combination in a ratio of 3:1. In the study by Rodehutscord et al. (1996), W and soybean meal contributed with phytate in a ratio of 1:0.7, whereas in the present study, WB and C contributed with 1:1.2. Furthermore, about 0.15 and 0.23 of the phytate in C and soybean meal remained undegraded after 4 h of soaking with microbial phytase at 38 °C (Ton Nu et al., 2014). The more rapid degradation of phytate in C compared with soybean meal may be explained by a better contact between phytate and phytase in corn due to differences in storage site of phytate. Moreover, 0.17 of the phytate intake in pigs fed a wheat-barley diet with microbial phytase leaves the stomach within the very first hour after feeding without being degraded, illustrating that the time for phytate degradation in the stomach is too short for a considerable part of the phytate (Blaabjerg et al., 2011). Thus, it can be speculated that W-phytases had limited time to degrade phytate from soybean meal in the stomach of the pigs in the study by Rodehutscord et al. (1996), whereas the W-and B-phytases in the present study succeeded to degrade some of the phytate from C, probably because phytate in C is more accessible for phytases than phytate in soybean meal. Therefore, the choice of feedstuffs and the ratio between feedstuffs may explain why CTTAD of P in W and soybean meal were additive in the study by Rodehutscord et al. (1996) and why CTTAD of P in WB and C tended to be nonadditive in the present study. Another explanation could be that Rodehutscord et al. (1996) used the difference method showing higher variations in CTTAD of P values compared to CTTAD of P values determined in the present study by the direct method. Using the regression method, Zhai and Adeola (2013) found that the true total tract digestibility of P in C and soybean meal were additive when fed in combination due to minor or no phytase activity in C and soybean meal. This emphasizes that the assumption of additivity regarding the CTTAD of P is problematic when cereals with high phytase activity are mixed with cereals with no or low detectable phytase activity or processed protein sources with no phytase activity. This is particularly important when microbial phytase is not applied to the compound diets. Some studies determine the coefficient of standardized total tract digestibility (CSTTD) of P, which requires data on the endogenous P losses (EPL) in order to correct the CTTAD of P for basal EPL (She et al., 2017; NRC, 2012). However, EPL was not determined in the present experiment as determination of EPL requires a P free diet as an additional 82
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experimental treatment. According to NRC (2012) the basal EPL is constant, and CSTTD of P can therefore be calculated for all feedstuffs with known CTTAD of P. The homeostasis of P and Ca is tightly regulated (Fernandez, 1995), and the major part of both minerals is present in the skeleton as hydroxyapatite (Suttle, 2010). Consequently, absorbed P can only be utilized for skeleton growth and maintenance if sufficient dietary Ca is available and vice versa (Fernandez, 1995). Thus, the low or negative P retention for all diets was due to the deficient Ca intake, resulting in increased excretion of P in urine. The negative Ca retention was also mainly due to the low Ca intake. However, the improvement of the Ca retention by supplementing WBC with microbial phytase also indicates that the negative Ca retention was a result of deficient intake of digestible P. 5. Conclusion In conclusion, addition of microbial phytase increased phytate degradation in soaked WBC and CTTAD of P in dry fed WBC. Moreover, phytases present in W and B are non-specific and catalyze the degradation of phytate in C when soaked or fed dry together in a ratio of 1:1:2. As such, the phytate degradation determined in separately soaked WB and C were not additive when mixed. At the same time, a strong tendency to non-additivity in CTTAD of P was observed when the same combination was fed dry to pigs. Consequently, when phytate containing feedstuffs very different in plant phytase activity are soaked or fed together, additivity cannot always be assumed. Thus, non-additivity of P digestibility values needs to be taken into account when formulating diets for pigs to ensure efficient utilization of P. However, at present this is difficult due to the lack of knowledge about the additivity or nonadditivity of P digestibility values in diets for pigs. The present in vitro results on phytate degradation suggest that the soaking method can be used to give a hint about whether P digestibility values determined in individual feedstuffs are additive or not when fed together. The combinations of feedstuffs showing non-additivity during soaking should be followed up by digestibility studies clarifying the non-additivity of the P digestibility values of the feedstuffs when fed to pigs in combinations. Finally, endogenous plant phytase activity should be analyzed and reported in future phytase work as the presence of plant phytases may skew the expected results. Acknowledgement The project was granted by The Danish Food Industry Agency, The Ministry of Food, Agriculture and Fisheries, Copenhagen and Aarhus University. References Adeola, O., Sands, J.S., Simmins, P.H., Schulze, H., 2004. The efficacy of an Escherichia coli-derived phytase preparation. J. Anim. Sci. 82, 2657–2666. AOAC, 2000. Official methods of analysis. In: Assoc. Off. Anal. Chem. Arlington, VA, 17th ed. . Blaabjerg, K., Carlsson, N.G., Hansen-Møller, J., Poulsen, H.D., 2010. Effect of heat-treatment, phytase, xylanase and soaking time on inositol phosphate degradation in vitro in wheat, soybean meal and rapeseed cake. Anim. Feed Sci. Technol. 162, 123–134. Blaabjerg, K., Jørgensen, H., Tauson, A.H., Poulsen, H.D., 2011. The presence of inositol phosphates in gastric pig digesta is affected by time after feeding a nonfermented or fermented liquid wheat-barley based diet. J. Anim. Sci. 89, 3153–3162. Blaabjerg, K., Strathe, A.B., Poulsen, H.D., 2012. Modelling phytate degradation kinetics in soaked wheat and barley. Anim. Feed Sci. Technol. 175, 48–56. Carlson, D., Poulsen, H.D., 2003. Phytate degradation in soaked and fermented liquid feed - effect of diet, time of soaking, heat treatment, phytase activity, pH and temperature. Anim. Feed Sci. Technol. 103, 141–154. Eeckhout, W., De Paepe, M., 1994. Total phosphorus, phytate-phosphorus and phytase activity in plant feedstuffs. Anim. Feed Sci. Technol. 47, 19–29. Engelen, A.J., Vanderheeft, F.C., Randsdorp, P.H.G., Smit, Ed, L.C., 1994. Simple and rapid determination of phytase activity. J. AOAC Int. 77, 760–764. Esmaeilipour, O., Van Krimpen, M.M., Jongbloed, A.W., De Jonge, L.H., Bikker, P., 2012. Effects of temperature, pH, incubation time and pepsin concentration on the in vitro stability of intrinsic phytase of wheat, barley and rye. Anim. Feed Sci. Technol. 175, 168–174. Fernandez, J.A., 1995. Calcium and phosphorus metabolism in growing pigs .1. Absorption and balance studies. Livest. Prod. Sci. 41, 233–241. Haug, W., Lantzsch, H.J., 1983. Sensitive method for the rapid determination of phytate in cereals and cereal products. J. Sci. Food Agric. 34, 1423–1426. Jongbloed, A.W., Kemme, P.A., 1990. Apparent digestible phosphorus in the feeding of pigs in relation to availability, requirement and environment. I. Digestible phosphorus in feedstuffs from plant and animal origin. Netherlands J. Agric. Sci. 38, 567–575. NRC, 2012. Nutrient Requirements of Swine. The National Academies Press, Washington, DC, USA, pp. 400. O’Dell, B., De Boland, A.R., Koirtyohann, S.R., 1972. Distribution of Phytate and Nutritionally Important Elements among the Morphological Components of Cereals Grains. J. Agric. Food Chem. 20, 718–721. Pointillart, A., Fontaine, N., Thomasset, M., 1984. Phytate phosphorus utilization and intestinal phosphatases in pigs fed low phosphorus: Wheat or corn diets. Nutr. Rep. Int. 29, 473–483. Pointillart, A., Fourdin, A., Fontaine, N., 1987. Importance of cereal phytase activity for phytate phosphorus utilization by growing pigs fed diets containing triticale or corn. J. Nutr. 17, 907–913. Raboy, V., 1997. Accumulation and storage of phosphate and minerals. In: Larkins, B.A., Vasil, I.K. (Eds.), Cellular and Molecular Biology of Plant Seed Development. Kluwer Academic Publisher, Dordrecht, Boston, London, pp. 441–477. Raboy, V., 2003. Myo-Inositol-1,2,3,4,5,6-hexakisphosphate. Phytochemistry 64, 1033–1043. Rodehutscord, M., Faust, M., Lorenz, H., 1996. Digestibility of phosphorus contained in soybean meal, barley, and different varieties of wheat, without and with supplemental phytase fed to pigs and additivity of digestibility in a wheat-soybean-meal diet. J. Anim. Physiol. Anim. Nutr. 75, 40–48. SAS Institute Inc., Cary, NC, USA, version 9.4, 2018. She, Y., Li, D., Zhang, S., 2017. Methological aspects of determining phosphorus digestibility in swine: a review. Anim. Nutr. Feed Technol. 3 97–10. Shen, Y.G., Fan, M.Z., Ajakaiye, A., Archbold, T., 2002. Use of the regression analysis technique to determine the true phosphorus digestibility and the endogenous phosphorus output associated with corn in growing pigs. J. Nutr. 132, 1199–1206. Stone, F.E., Hardy, R.W., Spinelli, J., 1984. Autolysis of phytic acid and protein in canola-meal (Brassica SPP), wheat bran (Triticum SPP) and fish silage blends. J. Sci. Food Agric. 35, 513–519. Stuffins, C.B., 1967. The determination of phosphate and calcium in feeding stuffs. Analyst 92, 107–111. Suttle, N.F., 2010. Calcium. In: Suttle, N.F. (Ed.), Mineral Nutrition of Livestock. CABI, Cambridge, USA, pp. 54–91.
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Ton Nu, M.A., Blaabjerg, K., Poulsen, H.D., 2014. Effect of screen size and phytase on phytate degradation in incubated corn and soybean meal. Animal 8, 534–541. Viveros, A., Centeno, C., Brenes, A., Canales, R., Lozano, A., 2000. Phytase and acid phosphatase activities in plant feedstuffs. J. Agric. Food Chem. 48, 4009–4013. Weremko, D., Fandrejewski, H., Raj, St., Skiba, G., 2001. Enzymatic efficiency of plant and microbial phytase in cereal-rapeseed diets for growing pigs. J. Anim. Feed Sci. 10, 649–660. Zhai, H., Adeola, O., 2013. True total-tract digestibility of phosphorus in corn and soybean meal for fifteen-kilogram pigs are additive in corn-soybean meal diet. J. Anim. Sci. 91, 219–224. Zhu, X.S., Seib, P.A., Allee, G.L., Liang, Y.T., 1990. Preparation of low-phytate feed mixture and bioavailability of its phosphorus in chicks. Anim. Feed Sci. Technol. 27, 341–351.
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Wheat and barley increase phytate degradation and phosphorus digestibility in corn fed to pigs
T
⁎
H.D. Poulsena, , A.L. Voergaarda,b, A.B. Stratheb, K. Blaabjerga a b
Aarhus University, Foulum, Department of Animal Science, DK-8830 Tjele, Denmark University of Copenhagen, Department of Veterinary and Animal Sciences, DK-1870 Frederiksberg C, Denmark
A R T IC LE I N F O
ABS TRA CT
Keywords: Additivity Liquid feeding Phosphorus Phytase Phytate Pig
Two experiments evaluated the additivity of phytate content and coefficient of total tract apparent digestibility (CTTAD) of phosphorus (P) in wheat-barley (WB) and corn (C) when soaked (exp. 1) or fed dry to pigs (exp. 2) in combination with or without microbial phytase (1250 FTU/ kg as-fed). In exp. 1, WB (1:1, w/w) and C were soaked separately or together (W:B:C = 1:1:2, w/ w/w) without (WBC) or with microbial phytase (WBC + phy). Phytate content was affected by the interaction between diet and soaking time (P < 0.0001) as phytate decreased with time. The decrease was greatest for WBC + phy followed by WBC, WB and C resulting in a half-life of phytate (K) of 8.7, 15.8, 24.4 and 303.4 h, respectively. Soaking of WB and C together showed non-additive effects as the estimated (assuming additivity) phytate content at 2, 4, 8 and 24 h of soaking was 0.17, 0.34, 0.42 and 0.5 g/kg DM higher (P < 0.001) than the experimentally determined phytate content in WBC. Actually, phytate degradation in C was estimated to be 0.15, 0.30, 0.42 and 0.49 higher at 2, 4, 8 and 24 h, respectively, when soaked together with WB than when soaked alone. Accordingly, the estimated (assuming additivity) K-value for WBC was 148 h longer (P = 0.08) than the K-value derived from the experimentally determined phytate degradation in WBC. Exp. 2 comprised 24 pigs of 47 ± 2 kg from 6 litters. Pigs were housed in metabolism crates and fed one of four diets (WB, C, WBC or WBC + phy) for 12 days; 5 days adaptation and 7 days total collection of feces and urine. The CTTAD of P was greatest for WBC + phy followed by WB, WBC and C (P < 0.0001). Feeding WB and C in combination tended to show non-additive effects as the estimated (assuming additivity) CTTAD of P in WBC was 7.7 percentage units lower (P = 0.09) than the experimentally determined CTTAD of P in WBC. In fact, CTTAD of P in C was estimated to be 14.6 percentage units higher when fed together with WB than alone. In conclusion, phytases present in W and B are non-specific and catalyze the degradation of phytate in C when soaked or fed dry together (ratio 1:1:2). Thus, when phytate rich cereals very different in plant phytase activity, are mixed, additivity cannot be assumed.
Abbreviations: B, barley; C, corn; Ca, calcium; CTTAD, coefficient of total tract apparent digestibility; CSTTD, coefficient of standardized total tract digestibility; DM, dry matter exp experiment; F0, the relative instantaneously degradable fraction of phytate; FTU, phytase activity expressed in units; GMM, Generalised Michaelis Menten; K, the half-life of phytate phosphorus in hours; LS means, least square means; n, description of the shape of the phytate degradation curve; ND, not detectable; SEM, standard error of mean; P, phosphorus; SD, standard deviation; W, wheat; WB, wheatbarley; WBC, wheat-barley-corn ⁎ Corresponding author. E-mail address: [email protected] (H.D. Poulsen). https://doi.org/10.1016/j.anifeedsci.2018.12.006 Received 31 May 2018; Received in revised form 17 August 2018; Accepted 22 December 2018 0377-8401/ © 2019 Elsevier B.V. All rights reserved.
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1. Introduction Phytate, the main storage form of P in plant feedstuffs, is poorly digested by pigs, unless phytase is present. Thus, microbial phytase is widely used in pig diets to increase the digestibility of phytate P and to reduce the excretion of P to the environment. Inclusion of cereals like wheat (W), barley (B) and triticale in diets for pigs also increases the digestibility of phytate P as these cereals display considerable plant phytase activity (Pointillart et al., 1984, 1987; Weremko et al., 2001). In contrast, corn (C), soybean meal and rapeseed cake/meal exhibit negligible or no phytase activity (Eeckhout and De Paepe, 1994; Viveros et al., 2000). The phytate content in a mixture of W and soybean meal or W and rapeseed cake was shown to be 0.55 and 0.16 lower after 24 h of soaking in water (20 °C) compared with the sum of phytate determined from the separately soaked single feedstuffs (Blaabjerg et al., 2010). This shows that phytases from W catalyzed the degradation of phytate present in the soybean meal or rapeseed cake when soaked together. Consequently, the amounts of non-degraded phytate in the individual feedstuffs cannot just be added (non-additive; Blaabjerg et al., 2010). Likewise, the P digestibility values of individual feedstuffs containing phytate and different phytase activities may not always be additive when fed together. However, there is a lack of knowledge about the additivity of P digestibility values despite the fact that to ensure efficient utilization of P, the additivity of P digestibility values is a crucial factor in the formulation of diets for pigs. Therefore, the objectives of the two experiments were to test the hypotheses: phytate degradation and CTTAD of P in wheat-barley (WB) and C are non-additive when soaked (exp. 1) or fed to pigs (exp. 2) in combination (WBC). 2. Material and methods 2.1. Experimental diets The feedstuffs W, B and C used in exp. 1 and 2 originated from the same lot, respectively. All the feedstuffs and diets used in the experiments were ground by a roller mill (3 mm between the rolls). The WB and C were soaked (exp. 1) or fed (exp. 2) separately or in combination at a ratio of 1:1:2 (w/w/w) and supplemented with microbial phytase (granulate, Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany) at 0 or 1250 FTU/kg as-fed (Table 1). Dietary P was solely of plant origin as no feed phosphate was added. The diets based solely on cereals were not optimized regarding other nutrients. 2.2. Experiment 1 The feedstuffs (200 g) were soaked in preheated water (20 °C) at a ratio of 1:2.75 (w/w) in 1-L closed containers with a continuously magnetic stirring as described by Carlson and Poulsen (2003). No attempt was made to regulate pH. The containers were placed in an incubator (New Brunswick Scientific Model G25 Controlled Environment Incubator Shaker, GST Technical Sales, Edmonton, Alberta, Canada) to maintain a constant temperature of 20 °C. Samples of about 60 mL were collected at 2, 4, 8 and 24 h and immediately frozen by placing the samples in a freezer (−20 °C) to stop further degradation. Samples of the non-soaked feedstuffs (0 h) were also collected for analysis. Four replicates per treatment were conducted. 2.3. Experiment 2 The experimental protocol was approved by the Danish Animal Experiments Inspectorate, the Danish Ministry of Justice, Copenhagen, Denmark. The experiment comprised 24 female pigs (Landrace × Yorkshire × Duroc) from 6 litters weighing Table 1 Experimental diets and analyzed chemical composition of the experimental diets of exp. 1 and 2 (n = 4). Mean ± SD. Dietsu WB Ingredient composition (proportion of ingredient) Wheat Barley Corn Microbial phytase, FTU/kg as-fed Chemical composition DM, % Ca, g/kg DM Total P, g/kg DM Phytate P, g/kg DM Phytase, FTU/kg DM
C
WBC
WBC + phy
0
1.00 0
0.25 0.25 0.50 0
0.25 0.25 0.50 1250
88 ± 0.36 0.5 ± 0.2 3.0 ± 0.3 1.9 ± 0.06 770 ± 15
88 ± 0.29 0.1 ± 0.0 3.0 ± 0.3 2.3 ± 0.1 NDv
88 ± 0.23 0.3 ± 0.1 2.7 ± 0.1 2.1 ± 0.05 446 ± 54
88 ± 0.23 0.3 ± 0.0 2.8 ± 0.2 2.1 ± 0.15 2140 ± 130
0.50 0.50
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v ND = not detectable (below 50 FTU/kg DM).
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47 ± 2 kg. From each litter, the pigs were assigned randomly to 4 diets. The pigs were housed in stainless steel metabolism crates and were fed 800 g twice daily (0800 and 1400) for 12 days; 5 days for adaptation and 7 days for total collection of urine and feces. On day 5, the pigs were fitted with urine bladder catheters for separate collection of urine and feces. 2.4. Chemical analyses In exp. 1, the pH was measured by a pH meter (PHM210, MeterLab, Radiometer, Copenhagen) in connection with sample collection. Dry matter (DM) was determined by freeze drying with the following parameters: cold trap: −85 °C, time: 72 h, final product temperature: 30 °C, and pressure: 0.05 mbar. Total P was assayed by the colorimetric vanadomolybdate procedure (Stuffins, 1967). Phytate was analysed according to the method by Haug and Lantzsch (1983). Determination of phytase activity followed the procedure of Engelen et al. (1994) where one FTU is the amount of enzyme, which liberates 1 μmol inorganic orthophosphate per minute from 0.0051 mol/L sodium phytate at pH 5.5 and 37 °C. In exp. 2, all the experimental diets were analyzed for phytase activity and phytate as described above. Additionally, diets, feces, and urine were analyzed for P (described above) and for Ca according to the AOAC procedure (method 975.03; AOAC, 2000) with the following modifications: dry ashing was performed at 450 °C for 3 h in step 1 and 1 h in step 2. Dry matter in diets and feces was determined by oven drying at 103 °C for 20 h. 2.5. Calculations and statistical analyses The results were analyzed by use of the MIXED procedure of SAS, 2018. The results are least squares means (LS means ± SEM) and standard error of mean (SEM). Statistical significance was accepted at P < 0.05 and tendencies were declared at 0.05 ≤ P ≤ 0.10. Treatment differences were separated by the PDIFF option of SAS. Results of exp. 1 were analysed according to the following model: Yijke = μ + ai + bj + abij + Uk + εijke where Yijke is the dependent variable; μ the overall mean; ai the fixed effect of diet, i = WB, C, WBC, WBC + phy; bj the fixed effect of soaking time, j = 0, 2, 4, 8, 24; abij the diet and soaking time interaction effect; Uk the random effect of container; εijke the residual experimental error. It is assumed that Uk ˜ N(0, σU2) and εijke ˜ N(0, σe2). Treating container as a random effect generates a suitable correlation structure that accounts for measurements on the same container being correlated across time. Results of exp. 2 were analysed according to the following model: Yije = μ + ai + Uj + εije where Yije is the dependent variable; μ the overall mean; ai the fixed effect of diet, i = WB, C, WBC, WBC + phy; Uj the random effect of litter; εije the residual experimental error. It is assumed that Uj ˜ N(0, σU2) and εije ˜ N(0, σe2). 2.5.1. Additivity Exp. 1. Assuming additivity, the phytate content (g P/kg DM) in the mixture of WB and C at 2, 4, 8, and 24 h of soaking were estimated using the following equation: Phytate PWBC_t = phytate PWB × FCWB + phytate PC × FCC; where FCWB and FCC are the relative feedstuff contribution of WB and C to the WBC mixture (i.e. FCWB + FCC = 1); Phytate PWBC_t is the estimated (assuming additivity) phytate P content (g P/kg DM) at a given soaking time (t = 2, 4, 8, 24 h) in the mixture of WB and C while phytate PWB and phytate PC represent experimentally determined phytate P content (g P/kg DM) in WB and C soaked separately. Exp. 2. Assuming additivity, the CTTAD of P in the mixture of WB and C were estimated using the following equation: CTTAD of PWBC = CTTAD of PWB × PCWB + CTTAD of PC × PCC; where PCWB and PCC are the relative P contribution of WB and C to the WBC mixture (i.e. PCWB + PCC = 1); CTTAD of PWBC is the estimated (assuming additivity) CTTAD of P in the mixture of WB and C while CTTAD of PWB and CTTAD of PC represent experimentally determined CTTAD of P in WB and C, separately. Assuming additivity, the CTTAD of DM was estimated in the same way as shown for CTTAD of P. To test for additivity of phytate P content when soaking WB and C in combination (exp. 1), the experimentally determined phytate P content for WBC was regressed on the estimated phytate P content for WBC, while forcing the intercept through zero. A slope, deviating significantly from 1, would indicate that the assumption of additivity is not met. The Reg procedure of SAS was used for the analysis. The same statistical analysis was conducted to test for additivity of CTTAD of P and DM, respectively, when feeding WB and C in combination (exp. 2). 2.5.2. Fit of Generalised Michaelis Menten function In exp. 1, a Generalised Michaelis Menten (GMM) function was fitted to the results on phytate content as described in details by 79
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Blaabjerg et al. (2012). This function was parameterized to derive the following parameters: F0, the relative instantaneously degradable fraction of phytate; K, the half-life of phytate P in hours; n, describes the phytate degradation curve. n ≤ 1 indicates that the phytate degradation curve decreases continuously, whereas n > 1 displays a sigmoid shaped degradation curve. A K-value was estimated for each of the four diets. It was not possible to estimate (i.e. convergence issues for the nonlinear mixed model routine) an individual F0 and n-value for the different diets due to the structure of the data and number of replicates. Assuming additivity, the Kvalue for the mixture of WB and C was estimated using the following equation: KWBC = KWB × FCWB + KC × FCC; where FCWB and FCC are the relative feedstuff contribution of WB and C to the WBC mixture (i.e. FCWB + FCC); KWBC is the estimated (assuming additivity) half-life of the phytate content in hours in the mixture of WB and C while KWB and KC represent the half-life of the phytate content derived via the GMM function from the experimentally determined phytate P content in WB and C soaked separately. The difference between the estimated K-value and the K-value derived via the GMM function in the WBC diet was tested using the contrast analysis procedure in SAS. 3. Results The analyzed chemical composition of the diets used in exp. 1 and 2 is shown in Table 1. The WB showed considerable plant phytase activity, whereas the phytase activity of C was below the detection level. The total P content was almost similar for the four diets, while the Ca content differed. Phytate P constituted from 63 to 78% of total P in the diets. 3.1. Experiment 1 The phytate content during soaking was affected by the interaction between diet and soaking time (P < 0.0001; Table 2). This was due to the fact that the amount of degraded phytate increased with time, and this increase was greatest for WBC + phy followed by WBC, WB and C (Table 2). In accordance, K was lowest for WBC + phy (8.7 h) followed by WBC (15.8 h), WB (24.4 h), and C (303.4 h). Assuming additivity, the estimated content of non-degraded phytate in WBC was 0.17, 0.34, 0.42 and 0.5 g phytate P/kg DM greater (P < 0.0001) at 2, 4, 8, and 24 h of soaking compared with the experimentally determined content of non-degraded phytate in WBC. Moreover, the estimated K-value for WBC was 164 h which was 148 h longer (P = 0.08) than the K-value derived via the GMM function from the experimentally determined phytate degradation in WBC (15.8 h). These calculations show that when soaking WB and C in combination (W:B:C = 1:1:2), the degradation of phytate was not additive. The degradation of phytate in C was estimated to be 0.15, 0.30, 0.42 and 0.49 higher at 2, 4, 8 and 24 h, respectively, when soaked in combination with WB than when soaked alone. The phytase activity differed as planned between diets (P < 0.0001) and tended to decrease (P = 0.08) during the soaking period (Table 2). The pH was affected by the soaking time (P < 0.0001) primarily due to a drop from 8 to 24 h. 3.2. Experiment 2 One pig fed the WB diet, two pigs fed the C diet and one pig fed the WBC diet were omitted from the experiment due to large feed refusals. The determined CTTAD of DM tended (P = 0.07) to be affected by diet as CTTAD of DM for WB was lower (P = 0.04) compared with CTTAD of DM for C, WBC or WBC + phy (Table 3). The determined CTTAD of P was affected by diet (P < 0.0001). The CTTAD of P in C was 27 percentage units lower (P < 0.001) than the CTTAD of P in WB, and addition of microbial phytase to WBC increased (P = 0.0004) the CTTAD of P by 13 percentage units (WBC vs. WBC + phy; Table 3). Assuming additivity, the CTTAD of DM and P for WBC were estimated to be 0.7 and 7.7 percentage units lower (P = 0.35; P = 0.09), respectively, than the experimentally determined values. This shows that CTTAD of DM for WB and C were additive when WB and C were fed in combination (W:B:C = 1:1:2) whereas CTTAD of P for WB and C tended strongly to be non-additive despite the limited number of observations. The CTTAD of P in C was estimated to be 14.6 percentage units higher when fed together with phytase containing W and B than alone. The Ca intake differed between the diets (P < 0.0001; Table 3) reflecting the different Ca content of the diets. The urinary P excretion increased (P = 0.02), and the P retention decreased (P = 0.0005) concurrently with a reduced Ca intake (P < 0.0001) or an enhanced intake of digestible P (P < 0.0001). Likewise, the Ca retention increased (P < 0.0001) along with an increased intake of Ca and/or digestible P (P < 0.0001). 4. Discussion The lack of detectable phytase activity in C agrees with previous findings (Eeckhout and De Paepe, 1994; Viveros et al., 2000) and explains the very limited phytate degradation during soaking for 24 h and the high K-value (303.4 h). The pH development and the tending decrease in phytase activity of the present diets during 24 h of soaking correspond with similar soaking studies conducted at 20 °C (Carlson and Poulsen, 2003; Blaabjerg et al., 2010, 2012). Moreover, Esmaeilipour et al. (2012) reported a greater stability of the activity of phytase in W compared with B. Blaabjerg et al. (2012) observed that K-values for phytate P in soaked non-heat-treated W and B, derived from the GMM function, averaged 23 and 44 h, respectively. Using these K-values, a K-value for a mixture of W and B (ratio 1:1) was estimated to 34 h 80
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Table 2 Effects of diets and 0, 2, 4, 8 and 24 h of soaking at 20 °C on phytate P content, proportion of non-degraded phytate P in brackets, estimated phytate P content in WBC (assuming additivity), phytase activity and pH in exp. 1. LS means ± SEM. Dietsu
Observations
WB
C
WBC
WBC + phy
4
4
4
4
P, g/kg DM 2.26bA (1.00) 2.21bAC (0.98) 2.24bAB (0.99) 2.03bB (0.90) 2.04bBC (0.90)
P-valuesv SEM
Diet
Time
DxT
0.08
< 0.0001
< 0.0001
< 0.0001
Hours 0 2 4 8 24
Determined phytate 1.87aA (1.00) 1.74acA (0.93) 1.44aB (0.77) 1.36aB (0.73) 0.90acC (0.48)
Hours 2 4 8 24
Estimated phytate P, g/kg DM (assuming additivity) 1.98 1.84 1.69 1.47
Hours 0 2 4 8 24
Phytase activity, FTU/kg DM NDb, y 770a 773a NDb 786a NDb 756a NDb 641a NDb
446c 444c 431c 381c 350c
2140dA -z 1810dB 1880dBC 1970dC
55
< 0.0001
0.08
0.10
Hours 0 2 4 8 24
pH 6.1A 6.0A 6.0A 6.1A 5.3abB
6.2A 6.1A 6.1A 6.2A 5.7bB
6.2A 6.1A 6.0A 6.1A 5.3abB
0.15
0.27
< 0.0001
0.83
6.2A 6.2A 6.2A 6.3A 5.2aB
2.10bA (1.00) 1.81aB (0.86) 1.50aC (0.71) 1.26acD (0.60) 0.97aE (0.46)
2.13bA (1.00) 1.57cB (0.74) 1.45aB (0.68) 1.06cC (0.50) 0.71cD (0.33)
< 0.0001x
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v Within a row, means without a common lowercase superscript (a, b, c, d) differ (P < 0.05). Within a column, means without a common uppercase superscript (A, B, C, D, E) differ (P < 0.05). x Using the REG procedure in SAS it was shown that estimated phytate P content (assuming additivity) was different from the experimentally determined phytate P content in WBC. y ND = not detectable (below 50 FTU/kg DM). z The phytase activity at 2 h for WBC + phy dropped to 1435 FTU/kg DM. This resulted in an interaction between diet and soaking time (P < 0.0001). However, this drop cannot be true as the activity at the following time points (4, 8 and 24 h) was considerably higher. Therefore, the phytase activity at 2 h for WBC + phy was excluded from the statistical analysis (shown in Table 2).
(assuming additivity). This estimated half-life, derived from a previous study (Blaabjerg et al., 2012), is 11 h longer (34 vs. 23 h) than the present K-value observed for WB (ratio 1:1) suggesting that K-values for W and B are not additive. This could be due to the use of different W and B cultivars in the study by Blaabjerg et al. (2012) and the present study. On the other hand, it could also be due to that phytases from W and B complement each other resulting in faster and greater phytate degradation. The present study showed that phytate degradation in WBC was not additive demonstrating that W- and B-phytases are non-specific and also degrade C-phytate. Similarly, studies showed a non-additive phytate degradation when W or W-bran rich in phytase were soaked together with feedstuffs like C, soybean meal, rapeseed meal or rapeseed cake because the W-phytases stimulate the phytate degradation in these feedstuffs (Stone et al., 1984; Zhu et al., 1990; Blaabjerg et al., 2010). The present degradation of C-phytate by W- and B-phytases demonstrates that substrate and enzymes were able to get in contact. Despite a lower phytase activity, WBC showed a smaller K-value and a relatively greater phytate degradation at 2, 4 and 8 h of soaking compared with WB. This may be explained by the higher phytate content in WBC due to inclusion of phytate rich C and by differences in the storage site of phytate in W, B, and C. About 0.9 of the phytate in W and B is deposited in the aleurone layer, whereas 0.9 of the phytate in C is situated in the germ (O’Dell et al., 1972; Raboy, 1997, 2003). These differences in the storage site of phytate seem to influence the access of phytase to phytate (Adeola et al., 2004; Blaabjerg et al., 2010, 2012). Thus, the relatively greater phytate degradation within the first 8 h of soaking in WBC than in WB could be due to a better contact between the enzymes and the phytate in C compared with W and B. Furthermore, addition of microbial phytase to WBC decreased K from 15.8 to 8.7 h and increased the proportion of degraded phytate with 0.13, 0.03, 0.16 and 0.26 at 2, 4, 8 and 24 h, respectively, compared with WBC soaked without microbial phytase. This was most likely due to a degradation of C-phytate as previous soaking studies show that microbial phytase did not contribute to further degradation of phytate in W and B (Blaabjerg et al., 2010, 2012). 81
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Table 3 Effects of diets on coefficient of total tract apparent digestibility (CTTAD), excretion, and retention of P and Ca and digestible P and the estimated CTTAD of DM and P in WBC in exp. 2. LS means ± SEM. Dietsu
P-valuev
WB
C
WBC
WBC + phy
SEM
Diets
Observations DM intake, g/day CTTAD of DM
5 1268 0.866
4 1063 0.880
5 1127 0.880
6 1328 0.878
103 0.004
0.20 0.07
P intake, g/day P retention, g/day P in feces, g/day P in urine, g/day P net absorption, g/day CTTAD of P Proportion of utilized P Digestible P, g/kg DM
3.74 0.56a 1.68a 1.52a 2.05a 0.555a 0.138a 1.64a
3.24 −0.87b 2.36b 1.81a 0.88b 0.285b −0.288b 0.87b
3.06 −0.22c 1.55a 1.74a 1.50c 0.493c −0.096c 1.34c
3.68 0.03c 1.39a 2.26b 2.29d 0.621d 0.006ac 1.72a
0.29 0.18 0.17 0.16 0.16 0.022 0.062 0.06
0.20 0.0005 0.01 0.02 < 0.0001 < 0.0001 0.001 < 0.0001
Ca Ca Ca Ca
0.68a −0.06a 0.68a 0.06
0.15b −0.99b 1.14b 0.01
0.31c −0.55c 0.74a 0.12
0.37c −0.34d 0.60a 0.11
0.03 0.06 0.06 0.05
< 0.0001 < 0.0001 < 0.0001 0.46
intake, g/day retention, g/day in feces, g/day in urine, g/day
Experimentally determined CTTAD of DM Estimated CTTAD of DM (assuming additivity)
0.880 0.873
0.35x
Experimentally determined CTTAD of P Estimated CTTAD of P (assuming additivity)
0.493 0.416
0.09y
u WB = wheat and barley mixed in a ratio of 1:1; C = corn; WBC = wheat, barley and corn mixed in a ratio of 1:1:2; WBC + phy = WBC with microbial phytase [1250 phytase units of phytase (Aspergillus niger, Natuphos 5000 G, BASF Animal Nutrition, Ludwigshafen, Germany)/kg, as fedbasis]. v Within a row, means without a common lowercase superscript (a, b, c, d) differ (P < 0.05). x Using the REG procedure in SAS it was shown that estimated CATTD of DM (assuming additivity) did not differ from the experimentally determined CATTD of DM in WBC. y Using the REG procedure in SAS it was shown that estimated CATTD of P (assuming additivity) tended to differ from the experimentally determined CATTD of P in WBC.
The CTTAD of P was highest in WBC + phy due to the addition of microbial phytase, whereas it was lowest in C reflecting the lack of detectable cereal phytase activity. CTTAD of P in C varies considerably among studies, ranging from −0.41 to 0.41 (Jongbloed and Kemme, 1990; Weremko et al., 2001; Shen et al., 2002; Zhai and Adeola, 2013). The large variation is primarily due to the use of different dietary P levels and methods (direct or regression) as discussed by Zhai and Adeola (2013). The considerably higher CTTAD of P in WB compared with WBC was owing to the higher plant phytase activity and the lower phytate P content in WB. In line with the in vitro results on phytate degradation in exp. 1, CTTAD of P in WB and C tended to be non-additive as the W- and B-phytases increased CTTAD of P in C by 14.6 percentage units when WB and C were fed in combination (WBC). On the other hand, Rodehutscord et al. (1996) found that CTTAD of P in W and soybean meal was additive when fed in combination in a ratio of 3:1. In the study by Rodehutscord et al. (1996), W and soybean meal contributed with phytate in a ratio of 1:0.7, whereas in the present study, WB and C contributed with 1:1.2. Furthermore, about 0.15 and 0.23 of the phytate in C and soybean meal remained undegraded after 4 h of soaking with microbial phytase at 38 °C (Ton Nu et al., 2014). The more rapid degradation of phytate in C compared with soybean meal may be explained by a better contact between phytate and phytase in corn due to differences in storage site of phytate. Moreover, 0.17 of the phytate intake in pigs fed a wheat-barley diet with microbial phytase leaves the stomach within the very first hour after feeding without being degraded, illustrating that the time for phytate degradation in the stomach is too short for a considerable part of the phytate (Blaabjerg et al., 2011). Thus, it can be speculated that W-phytases had limited time to degrade phytate from soybean meal in the stomach of the pigs in the study by Rodehutscord et al. (1996), whereas the W-and B-phytases in the present study succeeded to degrade some of the phytate from C, probably because phytate in C is more accessible for phytases than phytate in soybean meal. Therefore, the choice of feedstuffs and the ratio between feedstuffs may explain why CTTAD of P in W and soybean meal were additive in the study by Rodehutscord et al. (1996) and why CTTAD of P in WB and C tended to be nonadditive in the present study. Another explanation could be that Rodehutscord et al. (1996) used the difference method showing higher variations in CTTAD of P values compared to CTTAD of P values determined in the present study by the direct method. Using the regression method, Zhai and Adeola (2013) found that the true total tract digestibility of P in C and soybean meal were additive when fed in combination due to minor or no phytase activity in C and soybean meal. This emphasizes that the assumption of additivity regarding the CTTAD of P is problematic when cereals with high phytase activity are mixed with cereals with no or low detectable phytase activity or processed protein sources with no phytase activity. This is particularly important when microbial phytase is not applied to the compound diets. Some studies determine the coefficient of standardized total tract digestibility (CSTTD) of P, which requires data on the endogenous P losses (EPL) in order to correct the CTTAD of P for basal EPL (She et al., 2017; NRC, 2012). However, EPL was not determined in the present experiment as determination of EPL requires a P free diet as an additional 82
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experimental treatment. According to NRC (2012) the basal EPL is constant, and CSTTD of P can therefore be calculated for all feedstuffs with known CTTAD of P. The homeostasis of P and Ca is tightly regulated (Fernandez, 1995), and the major part of both minerals is present in the skeleton as hydroxyapatite (Suttle, 2010). Consequently, absorbed P can only be utilized for skeleton growth and maintenance if sufficient dietary Ca is available and vice versa (Fernandez, 1995). Thus, the low or negative P retention for all diets was due to the deficient Ca intake, resulting in increased excretion of P in urine. The negative Ca retention was also mainly due to the low Ca intake. However, the improvement of the Ca retention by supplementing WBC with microbial phytase also indicates that the negative Ca retention was a result of deficient intake of digestible P. 5. Conclusion In conclusion, addition of microbial phytase increased phytate degradation in soaked WBC and CTTAD of P in dry fed WBC. Moreover, phytases present in W and B are non-specific and catalyze the degradation of phytate in C when soaked or fed dry together in a ratio of 1:1:2. As such, the phytate degradation determined in separately soaked WB and C were not additive when mixed. At the same time, a strong tendency to non-additivity in CTTAD of P was observed when the same combination was fed dry to pigs. Consequently, when phytate containing feedstuffs very different in plant phytase activity are soaked or fed together, additivity cannot always be assumed. Thus, non-additivity of P digestibility values needs to be taken into account when formulating diets for pigs to ensure efficient utilization of P. However, at present this is difficult due to the lack of knowledge about the additivity or nonadditivity of P digestibility values in diets for pigs. The present in vitro results on phytate degradation suggest that the soaking method can be used to give a hint about whether P digestibility values determined in individual feedstuffs are additive or not when fed together. The combinations of feedstuffs showing non-additivity during soaking should be followed up by digestibility studies clarifying the non-additivity of the P digestibility values of the feedstuffs when fed to pigs in combinations. Finally, endogenous plant phytase activity should be analyzed and reported in future phytase work as the presence of plant phytases may skew the expected results. Acknowledgement The project was granted by The Danish Food Industry Agency, The Ministry of Food, Agriculture and Fisheries, Copenhagen and Aarhus University. References Adeola, O., Sands, J.S., Simmins, P.H., Schulze, H., 2004. The efficacy of an Escherichia coli-derived phytase preparation. J. Anim. Sci. 82, 2657–2666. AOAC, 2000. Official methods of analysis. In: Assoc. Off. 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