Digestibility of nutrients in growing–finishing pigs is affected by Aspergillus niger phytase, phytate and lactic acid levels

Livestock Production Science 58 (1999) 107–117

Digestibility of nutrients in growing–finishing pigs is affected by Aspergillus niger phytase, phytate and lactic acid levels 1. Apparent ileal digestibility of amino acids a, a a a b Paul A. Kemme *, Age W. Jongbloed , Zdzisław Mroz , Jan Kogut , Anton C. Beynen b

a Institute for Animal Science and Health ( ID-DLO), P.O. Box 65, 8200 AB Lelystad, The Netherlands Department of Large Animal Medicine and Nutrition, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.152, 3508 TD Utrecht, The Netherlands

Received 18 March 1998; accepted 3 November 1998

Abstract In growing–finishing pigs, the effects of microbial phytase, lactic acid and phytate levels in a maize-soybean meal diet on the apparent ileal digestibility (AID) of N and amino acids were studied. The experimental design was a 2 3 2 3 2 factorial arrangement plus a positive control treatment. Six crossbred castrates of 37 kg initial BW, fitted with steered ileo-caecal valve cannulas, were used during six collection periods. The dietary treatments consisted of Aspergillus niger phytase (Natuphos  ; 0 or 900 FTU kg 21 ), Na phytate (0 or 1.5 g P kg 21 ) and lactic acid (0 or 30 g kg 21 ). The positive control diet was supplemented with 1.0 g P kg 21 from monocalcium phosphate monohydrate (MCP). Ileal digestible amino acids were supplied at 80% of the Dutch recommended allowances for a 60-kg growing pig. The feeding level was 2.3 times the maintenance requirement for energy (418 kJ ME BW 20.75 ). Estimates of AID were calculated using Cr 2 O 3 as a marker. Results showed that for almost all amino acids there was a significant interaction between Na phytate and phytase. Supplementing Na phytate to the diets without phytase increased the AID of N and amino acids. In general, phytase alone stimulated the AID of N and amino acids in the diets without Na phytate. Supplementing both Na phytate and phytase slightly decreased AID compared with the diets with only one supplement. Lactic acid stimulated the AID of N and amino acids, but a synergistic effect between phytase and lactic acid was not detected. Adding MCP had no effect on the AID of N and amino acids. It was concluded that when diets are supplemented with either phytase or lactic acid, AID of amino acids is improved. However, supplementing both did not result in a further increase in AID.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pigs; Phytase; Phytate; Acidification; Amino acids; Digestibility

1. Introduction *Corresponding author. Tel.: 1 31-320-237-324; fax: 1 31320-237-320. E-mail address: [email protected] (P.A. Kemme)

Plant ingredients used to formulate diets for pigs contain poorly soluble salts of phytic acid that are practically unavailable for the pig (Jongbloed, 1987;

0301-6226 / 99 / $ – see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-6226( 98 )00203-6

108

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

Cromwell, 1992). Phytic acid binds approximately two thirds of intrinsic phosphorus in vegetal feedstuffs (Cosgrove, 1980), and it may form insoluble complexes with protein and amino acids (O’Dell and De Boland, 1976; Knuckles et al., 1985) and inhibit proteolytic enzymes such as pepsin and trypsin (Camus and Laporte, 1976; Singh and Krikorian, 1982). In short, phytic acid can reduce the absorption of these important nutrients. The addition of phytase to pig diets does not only render phytate P available for absorption ¨ (Dungelhoef and Rodehutscord, 1995; Jongbloed et al., 1996), but also improves the digestibility of protein and amino acids (Mroz et al., 1994). Dietary acidification may also enhance ileal protein and amino acid digestibility (Mosenthin et al., 1992). A large portion of gastric digesta leaves the pig stomach shortly after feeding and has a pH that is too high for optimal microbial phytase action (Jongbloed et al., 1992). Feed acidification may reduce the rate of gastric emptying (Mayer, 1994), which could favour the action of phytase. It was thus hypothesized that dietary acidification and microbial phytase may have a synergistic effect on amino acid digestibility. To test this hypothesis, the apparent ileal digestibility (AID) of dietary protein and amino acids was determined in growing–finishing pigs, which were fed a maize-soybean meal diet with or without added microbial phytase, Na phytate and lactic acid.

2. Materials and methods

2.1. Animals, housing and experimental design Six crossbred castrated male pigs (Great Yorkshire 3 Finnish Landrace 3 Dutch Landrace) of approximately 37 kg initial BW, were fitted under inhalation anaesthesia with steered ileo-caecal valve (SICV) cannulas, according to Mroz et al. (1996). After a 14-d recovery period, during which they were gradually adapted to the experimental diets, the pigs were housed individually in metabolic pens of 2.0 3 1.5 m, in a mechanically ventilated room at an ambient temperature of 188C. A 2 3 2 3 2 factorial arrangement plus an additional positive control treatment (4.6 g kg 21 monocalcium phosphate monohy-

drate [MCP; Aliphos, Tessenderlo Chemie, Tessenderlo, Belgium] to provide 1.0 g P kg 21 diet) was carried out according to a balanced six rows (periods) by six columns (pigs) design, as given by Shah (1977), which allowed for four replicates per treatment. Experimental variables were Aspergillus niger phytase (Natuphos  , Gist-brocades, Delft, The Netherlands) at doses of 0 and 900 FTU kg 21 , lactic acid in liquid form (85% [wt wt 21 ], purity 98%; Sigma, St. Louis, MO) at doses of 0 and 30 g kg 21 diet and Na phytate (C 6 H 6 O 24 P6 Na 12 , dodecasodium 21 salt from maize, M 5 923.8 g mol , Sigma, St. Louis, MO) at doses of 0 and 7.4 g kg 21 (to provide 1.5 g phytic acid P kg 21 diet).

2.2. Experimental diets and feeding Experimental diets were formulated to be isoproteinous (Table 1) and isoenergetic (14.2 MJ ME kg 21 ). The major components were maize and extracted soybean meal, in which intrinsic phytase activity is low ( , 100 FTU kg 21 ), and phytate content relatively high. Sodium carbonate was substituted for Na phytate to maintain constant Na levels. Sodium phytate, Na carbonate, MCP and(or) microbial phytase premix were exchanged for maize starch. The marker Cr 2 O 3 and the microbial phytase preparation were added to the diets in the form of premixes in order to obtain a homogenous distribution. Chromic oxyde was mixed with maize starch (1:3 wt wt 21 ), and subsequently ground in a hammer mill through a sieve with a pore size of 0.5 mm (Jongbloed et al., 1991). The Natuphos  preparation and maize starch were mixed thoroughly to a concentration of 900 FTU g 21 . These premixes were mixed with another premix containing L-lysine ? HCl, L-threonine, L-tryptophan, limestone, choline chloride, maize starch, MCP, Na phytate and Na carbonate, and a mix of trace elements and vitamins. The other ingredients were mixed into one batch, serving as stock, to prepare the experimental diets. Finally, the premixes and other ingredients were mixed together to obtain the experimental diets. Daily rations were given to pigs twice (at 06:00 and 18:00 h) in a wet, mash form (water:feed ratio 5 2.5 vol wt 21 ). Drinking water and lactic acid were mixed with the feed directly before feeding. Feeding level was 2.3 times maintenance requirement ( 5 418

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

109

Table 1 Diet formulation and analyzed chemical composition (g kg 21 ) Diet

1 6a

2 7a

3 8a

4 9a

5

1 – –

– – –

– 1 1

– 1 –

– – 1

Maize Soybean meal extr. Trace mineral-vitamin premix b Cr 2 0 3 -starch premix L-lysine ? HCl L-threonine L-tryptophan Choline chloride (50%) Limestone Maize starch Monocalcium phosphate 1H 2 O Phytase premix (900 FTU g 21 ) Na phytate (C 6 H 6 O 24 P6 Na 12 ) Na carbonate (Na 2 CO 3 )

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 12.7 0.7 4.6 – – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 3.3 – – – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 – – 1.0 7.4 –

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 2.3 – 1.0 – 5.1

858.0 112.6 1.4 2.0 2.2 0.2 0.2 0.3 14.7 1.0 – – 7.4 –

DM OM CP (Nx6.25) Total P Phytic acid Phytic acid P Na K Phytase activity (FTU kg 21 ) Essential amino acids Arg Asp Cys His Ile Leu Lys Met Phe Thr Try Val Nonessential amino acids Ala Glu Gly Pro Ser Tyr Dietary pH c

863 796 129 4.0 9.7 2.7 2.1 5.1 75

862 828 128 3.0 9.4 2.6 2.0 5.0 71

862 826 130 4.2 14.0 4.0 1.9 5.1 957

862 827 131 3.0 9.3 2.6 2.0 5.0 1072

863 827 129 4.2 14.3 4.0 1.8 5.3 61

7.4 11.1 2.4 3.3 5.5 13.8 6.8 2.2 6.7 5.4 1.3 6.9

7.3 10.8 2.6 3.7 5.4 13.7 6.8 2.3 6.5 5.2 1.3 6.7

7.4 11.2 2.4 3.3 5.6 13.8 6.8 2.1 6.8 5.4 1.3 6.9

7.5 11.2 2.5 3.7 5.6 14.0 7.0 2.3 6.6 5.5 1.3 6.9

7.6 11.2 2.4 3.4 5.7 14.0 7.0 2.2 6.7 5.4 1.3 6.9

8.3 24.2 5.0 9.8 6.5 5.4 6.8

8.1 24.0 4.9 9.7 6.4 5.1 7.3

8.3 24.4 5.0 9.8 6.6 5.5 6.5

8.3 24.6 5.1 9.9 6.7 5.3 7.3

8.3 24.6 5.1 10.0 6.7 5.5 6.8

Monocalcium phosphate Microbial phytase Na phytate

Diets 6–9 were identical to diets 2–5, respectively, except that they were acidified with 30 g of lactic acid kg 21 . The mix of trace elements and vitamins supplied (mg kg 21 diet): 40 CuSO 4 ? 5H 2 O (10 Cu); 430 FeSO 4 ? 7H 2 O (86 Fe); 50 MnO (39 Mn); 155 ZnSO 4 ? 7H 2 O (35 Zn); 2 KI (1.5 I); 0.7 Na 2 SeO 3 (0.32 Se); 2.4 Vitamin A (8,000 IU); 0.04 Vitamin D 3 (1,600 IU); 16 Vitamin E (16.0 IU); 0.02 Vitamin B 12 ; 4.0 riboflavin; 20 niacin; 7.6 Ca pantothenate; 125 antioxidant (4–5% BHA, 4–5% ethoxyquin, 4–5% citric acid, 2–3% o-phosphoric acid, 2–3% E471 fatty acid and SiO 2 as a carrier); 547 maize starch as a carrier. c The diets 6, 7, 8, and 9 had pH values of 5.4, 5.1, 5.4, and 5.1, respectively. a

b

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P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

kJ ME BW 20.75 ), and pigs had no access to drinking water between meals. The levels of ileal digestible essential amino acids were estimated to be 80% of the allowances for a 60 kg growing pig (CVB, 1990). The diets were formulated to contain 6.0 g Ca kg 21 .

2.3. Collection procedures Each experimental period lasted 15 days, including 3 days of transition, 9 days of adaptation and, on days 13 and 15, ileal digesta collection. The quantitative collection of ileal digesta was carried out for 12 h on each day. Digesta were collected in polyethylene bags and frozen immediately after collection. Fresh ileal digesta were homogenized and two subsamples were taken for determination of N. Subsequently, ileal digesta were freeze dried, weighed, ground to pass a 1 mm sieve and analysed for DM, N, Cr, and amino acid contents. Samples of the diets were taken immediately after production and during the collection periods. Each diet was analysed sixfold to determine DM, N, Cr, pH, amino acids, phytic acid and phytase activity.

2.4. Analytical procedures Dry matter and Kjeldahl N were determined according to AOAC (1980). Amino acids (except methionine, cystine and tryptophan) were assayed by ion-exchange column chromatography after hydrolysis for 23 h in HCl (6 mol L 21 ). Threonine, serine, valine and isoleucine contents were corrected according to Lenis et al. (1990), the correction factors being 1.05, 1.10, 1.07 and 1.08, respectively. Cystine and methionine were determined as cysteic acid and methionine sulfone after oxidation with performic acid before hydrolysis (Schram et al., 1954). Tryptophan was determined according to Sato et al. (1984). Content of Cr was analysed by atomic absorption spectrophotometry after ashing the samples at 550EC for four hours. The Cr digestion was performed in phosphoric-manganese sulphate solution / potassium bromate solution (Williams et al., 1962). Phytic acid was assayed according to Bos et al. (1991) and phytic acid content was calculated using molar ratios. Phytase activity was assayed

according to Engelen et al. (1994). The AID was calculated, using Cr as an indigestible marker.

2.5. Statistical analysis Each individual animal was an experimental unit. The experimental data were subjected to analysis of variance for factorial effects and their interactions and the factors animal and period. The effect of the positive control was compared to all other treatments. The data were statistically analysed, using Genstat 5 (Payne et al., 1993). Treatment means were compared by the Student t-test. Significance levels of P , 0.05 and 0.05 $ P , 0.10 were defined as statistically significant and as tendency for statistical significance, respectively. When interactions were statistically significant over a wide range of response parameters, they were presented in the table of the Results section. When they were not statistically significant over this wide range, only the main effects were presented.

3. Results

3.1. Chemical composition of the experimental diets The assayed chemical composition of the experimental diets (Table 1) was close to the calculated composition. In the positive control diet, MCP was supplemented as planned. When Na phytate (7.4 g kg 21 ) was added to Diets 3 and 5, the total P content increased by 1.2 gkg 21 , whereas phytic acid P content was increased by 1.4 g kg 21 . This discrepancy was within the analytical error of phytic acid analysis. Calcium content of the diets was slightly lower than expected (5.3 to 5.9 g kg 21 , Kemme et al., 1999). Diets 3 and 4, which were supplemented with microbial phytase, had a slightly higher phytase activity than anticipated.

3.2. Animal health Despite the cannulation required to collect ileal digesta, the pigs grew normally (514 g d 21 ) at the feeding level of 2.3 times the amount required for maintenance. One pig was replaced because it de-

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

veloped a small, local irritation of the abdominal wall exposed to a permanent contact with the flange of the silicone cannula. The test of homogeneity of variance (Bartlett’s test, Cochran and Snedecor, 1989) showed that both pigs had similar variations of the parameters measured, and it was thus concluded that this replacement did not interfere with the outcome of the statistical analysis.

3.3. Apparent ileal digestibility of N and amino acids The AID of the essential amino acids lysine, methionine, threonine, isoleucine, and arginine were improved when lactic acid was added to the diet, as well as the AID of the nonessential amino acids valine, aspartic acid, glutamine, and alanine (P , 0.05; Table 2). The AID of leucine, serine, and glycine tended to be improved by lactic acid addition (P , 0.10). The AID of N, cystine, tryptophan, histidine, phenylalanine, proline, and tyrosine remained unaffected. Moreover, a significant interaction between lactic acid and Na phytate was found for the AID of methionine (P 5 0.026) and cystine (P 5 0.014; data not shown), and for isoleucine and valine as a tendency (P , 0.10). In general, the AID of these amino acids was the lowest in the diet containing Na phytate but lacking lactic acid, and the highest in the diet containing both. The AID in the other two diets fell in between. The AID of most amino acids was significantly affected by the interaction between Na phytate and microbial phytase. When Na phytate was added to the diet, the AID of essential amino acids (except for methionine, leucine and histidine) and nonessential amino acids (except for glutamine and proline) were improved (P , 0.05) compared to the diet without any supplementation. However, it did not affect the AID of cystine. It improved the AID of N as main effect (P 5 0.026; data not shown). When microbial phytase alone was added to the diet, the AID of N tended to be improved (P 5 0.056). The AID of the essential amino acids lysine, tryptophan, threonine and isoleucine were improved (P , 0.05) and the AID of arginine and phenylalanine tended to be stimulated by microbial phytase (P , 0.10). The AID of methionine and histidine were unaffected. Among the nonessential amino acids, aspartic acid, glycine,

111

alanine and tyrosine had improved AID (P , 0.05). The AID of serine and glutamine tended to be enhanced (P , 0.10), while proline AID was not affected when microbial phytase alone was added to the diet. The AID of cystine tended to decrease (P 5 0.076) by microbial phytase addition, as main effect. Adding both Na phytate and microbial phytase to the diet slightly decreased the AID of most amino acids in comparison to adding only microbial phytase. Only the AID of histidine was decreased (P , 0.05) and the AID of methionine tended to decrease (P , 0.10), when Na phytate was added to the diet with microbial phytase. No significant differences could be shown in AID of amino acids when the latter two diets were compared to the diet with only added Na phytate, except for the AID of histidine, which decreased (P , 0.05) when phytase was added to diet with Na phytate. Adding MCP to the diet did not change the AID of N and amino acids compared to the average of all other treatments. No significant interaction between lactic acid and microbial phytase on the amino acid AID was observed.

4. Discussion The objective of this experiment was to determine the apparent ileal digestibility (AID) of dietary protein and amino acids in growing–finishing pigs, which were fed a maize-soybean meal diet with or without added microbial phytase, Na phytate, and lactic acid, so that we were able to test the hypothesis that the effect of microbial phytase on the AID of amino acids would become greater when lactic acid was added to the diet. However, we did not observe any interaction between acidifying the diet and adding phytase, which indicates that there was no synergistic effect of the two supplements. In essence, acidifying diets may induce three processes. First, it may reduce the rate of stomach emptying (Mayer, 1994), allowing more time for protein to digest in the stomach, and thus to stimulate amino acid AID. Second, it also may stimulate phytase to hydrolyse dietary phytate by providing a more optimal pH of gastric digesta during the time just after feeding (Jongbloed et al., 1992; Kemme et al.,

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

112

Table 2 Apparent ileal digestibility (AID) of N and amino acids as influenced by added monocalcium phosphate (MCP), lactic acid, and the interaction Na phytatexmicrobial phytase (FTU)a Interaction phytatexphytase MCP, g kg AID, %

0 (2–9)

N

Arg

Asp

Cys

His

Ile

Leu

Lys

Met

Phe

Thr

Try

Val

Ala

Glu

75.8

85.0

77.2

72.9

81.2

79.6

84.7

79.4

81.0

81.5

70.7

71.7

77.9

79.9

85.1

4.6

21

SED b

Lactic acid, g kg P-value

(1)

76.2

84.1

76.5

72.7

79.9

78.4

84.5

78.0

79.7

81.0

70.2

72.6

77.1

79.5

84.6

0 (2–5

0.94

0.76

0.70

1.02

0.78

1.01

0.90

0.90

0.88

0.95

1.33

1.63

0.97

0.89

0.85

0.753

0.229

0.470

0.168

0.115

0.280

0.871

0.164

0.168

0.609

0.699

0.597

0.440

0.673

0.638

75.3

84.4

76.5

72.8

80.9

78.8

84.1

78.4

80.3

80.9

69.8

71.2

77.1

79.1

84.4

30

SED

21

FTU kg P-value

21

Na phytate, g kg 21

0

0 (2, 6)

7.4 (5, 9)

900

(3, 7)

(4, 8)

0

74.2

77.1

900 0

75.8 83.8

76.1 86.1

900 0

85.3 75.2

84.9 78.5

900 0

77.7 73.4

77.5 73.7

900 0

73.0 81.4

71.4 81.5

900 0

82.1 77.9

79.8 80.9

900 0

80.0 83.8

79.5 85.5

900 0

85.1 77.5

84.2 80.4

900 0

79.9 80.6

79.7 81.4

900 0

81.7 80.0

80.3 82.7

900 0

81.7 68.3

81.5 73.0

900 0

71.2 68.3

70.4 73.2

900 0

72.7 76.1

72.7 79.3

900 0

78.1 78.3

78.1 81.1

900 0

80.2 84.1

80.0 85.7

900

85.6

84.9

SED

P-value

0.89

0.056

0.71

0.014

0.91

0.015

0.96

0.181

0.74

0.037

0.95

0.021

0.85

0.052

0.85

0.016

0.83

0.071

0.90

0.037

1.25

0.006

1.53

0.037

0.91

0.023

0.84

0.020

0.80

0.068

(6–9)

76.3

85.7

78.0

72.9

81.5

80.3

85.2

80.3

81.7

82.0

71.7

72.3

78.6

80.7

85.7

0.63

0.50

0.65

0.68

0.52

0.67

0.60

0.60

0.58

0.63

0.89

1.08

0.65

0.59

0.56

0.154

0.019

0.033

0.877

0.239

0.037

0.090

0.006

0.033

0.113

0.043

0.307

0.033

0.016

0.030

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

113

Table 2. Continued Interaction phytatexphytase MCP, g kg AID, %

0 (2–9)

Gly

Pro

Ser

Tyr a b

66.5

82.4

79.6

81.8

4.6

21

SED b

Lactic acid, g kg P-value

(1)

67.4

83.6

79.0

81.9

0 (2–5

1.73

0.98

0.90

0.96

0.620

0.236

0.573

0.943

65.4

82.4

79.0

81.3

30

SED

21

FTU kg P-value

21

Na phytate, g kg 21

0

0 (2, 6)

7.4 (5, 9)

900

(3, 7)

(4, 8)

0

63.7

68.7

900 0

67.2 81.6

66.3 83.6

900 0

82.6 78.1

81.9 81.2

900 0

79.8 79.7

79.2 83.4

900

81.8

82.4

SED

P-value

1.63

0.021

0.93

0.047

0.85

0.007

0.90

0.027

(6–9)

67.5

82.5

80.1

82.3

1.15

0.66

0.60

0.64

0.088

0.873

0.081

0.121

The treatment(s) that yielded the means are shown between parentheses. SED 5 standard error of difference of means.

1998). Because of its effect on gastric retention time, it also allows more time for phytase action in the stomach. These two processes combined may result in the synergistic effect. However, also a third process is induced by acidifying the diet. Okubo et al. (1976) found that in the pH range from 2.5 to 4.9 complexes of protein and phytate were formed, the extent of binding increasing with decreasing pH. This process will result in lower amino acid AID. Since we were not able to detect a synergistic effect of acidification with lactic acid and supplementation of microbial phytase on the AID of amino acids in our experiment, the stimulatory effects of acidification on protein digestibility and phytase action as such may be concealed by the detrimental effect of the formation of protein-phytate complexes. However, we cannot exclude the possibility that different doses of organic acid might produce a synergistic effect of the acid and microbial phytase on the AID of amino acids. In practice, diets for weanling pigs are often acidified with organic acids to avoid a post-weaning lag phase (Easter, 1988), while the effect of feed acidification decreases with increasing live weight (Giesting et al., 1991). Diet acidification would stimulate protein digestion in pigs by reducing the rate of gastric emptying, which allows more time for

protein to be digested in the stomach (Mayer, 1994), and(or) by stimulating pancreatic exocrine secretion of proteolytic enzymes (Harada et al., 1986; Sano et al., 1995), and stimulating the epithelial cell proliferation in the gastrointestinal mucosa (Sakata et al., 1995). The experiment was not designed to determine to what extent these factors apart contribute to the observed rise in amino acid AID. In general, lactic acid stimulated the AID of amino acids in this trial, although the increases measured were not statistically significant for all of them. Organic acids seem to have a slight positive effect on the total tract digestibility of N (e.g., Eckel et al., 1992; Eidelsburger et al., 1992a), although others reported no increase (e.g., Giesting and Easter, 1991; Eidelsburger et al., 1992b). Little is known about the effect of organic acids on the AID of N and amino acids. Giesting and Easter (1991) showed that the AID of N increased when they added fumaric acid to the diet, whereas Bolduan et al. (1988) found that formic acid did not affect the AID of N. Mosenthin et al. (1992) showed that adding propionic acid to the diet improved AID of several essential amino acids. Neither formic (Gabert et al., 1995) nor fumaric acid (Gabert and Sauer, 1995) supplementation improved the AID of amino acids. It is not known to what extent acidifying the diet of pigs has

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an effect on the hydrolysis of phytate or on the digestibility of phytate–protein complexes. Phytate present in plant feedstuffs may form complexes not only with several cations, but also with protein (Cheryan, 1980; Cosgrove, 1980; Maga, 1982; Pallauf et al., 1994). According to Hartman (1979), 2 to 3% of protein in soy protein isolate are strongly complexed to phytate. Factors that affect complex formation of phytate with protein are the type of protein (O’Dell and De Boland, 1976; Honig and Wolf, 1991; Hussain and Bushuk, 1992), pH (Hartman, 1979), dietary contents of Ca and Mg (Appurao and Narasinga Rao, 1975; Prattley et al., 1982; Caldwell, 1992), solubility rate of protein (Gifford and Clydesdale, 1990), and the interaction between proteolytic enzymes, phytate and protein (Knuckles et al., 1985; 1989; Caldwell, 1992). The complex formation may lead to a decreased protein digestibility in farm animals (Atwal et al., 1980; Thompson and Serraino, 1986). However, to predict the extent to which phytate–protein complexes exist and how they affect protein digestibility is very complicated. This may also explain why results of the stimulatory effect of microbial phytase on N and amino acid digestibility in animal trials are sometimes contradictory. Results showed that adding Aspergillus niger phytase to the diet increased the AID of N and most amino acids significantly or as a tendency. These results agree with those of our earlier study (Mroz et al., 1994), in which the AID of N increased by 2.5%-units, as a tendency, and the AID of most amino acids was enhanced to about the same magnitude. In the current trial, however, the increase in AID of more amino acids reached statistical significance because of a more efficient statistical design. Yi (1995), in a study using turkey poults, also reported that adding microbial phytase to their diet increased the AID of N and amino acids, and the increases were similar to those we measured. However, other studies report increases of a higher magnitude. Officer and Batterham (1992) found that adding microbial phytase to a diet with Linola TM meal (linseed meal after oil separation) for growing pigs greatly increased the AID of N (by 12 percentage units) and all amino acids (by 4 to 13%units). However, only the increases of lysine and histidine AID were significant. Khan and Cole

(1993) also reported such an increase in ileal N digestibility (by 13 percentage units) in pigs fed a barley-based diet. However, their data can be questioned, since they reported large experimental errors and exceptionally large amounts of total P (40%) disappeared from the large intestine. Khan and Cole (1993) and Wenk et al. (1993) found that adding microbial phytase to pig diets increased faecal digestibility of N. However, in contrast, ¨ and Helander (1994), and Ketaren et al. (1993), Nasi Pallauf et al. (1994) found no improvement. Increasing phytic acid P level in the diet by 1.4 g kg 21 surprisingly improved the AID of most amino acids. This finding cannot be explained by differences in Na content of the diets as such, because Na content was balanced in the diets. However, it may be related to differences in Na solubility or absorbability between Na carbonate and Na phytate. In the animal, Na seems to be the primary driving force for intestinal peptide transport, because it is involved in the Na 1 –H 1 exchange in the brush border membrane coupled with the Na 1 –K 1 -ATPase in the laterobasal membrane (Ganapathy and Leibach, 1985). A higher availability of Na will, therefore, facilitate a more efficient peptide transport. In the in vitro studies by Knuckles and co-workers (Knuckles et al., 1985, 1989), supplemental Na phytate decreased the digestion with pepsin of casein and, to a lesser extent, of bovine serum albumin. This finding may be consistent with ours, because in in vitro studies, added Na cannot influence amino acid absorption. Using normal diets and diets with reduced plant phytate content, Thompson and Serraino (1986) found that phytate from rapeseed had no effect on protein and amino acid digestibilities in rats. However, Atwal et al. (1980) observed that when rats were fed diets containing various amounts of phytic acid (varying from 0.01 to 1.24%), their performance was inversely correlated with the amount of dietary phytic acid. As the amount of phytic acid was increased, their weight gain, diet consumption and efficiency of protein utilization were worsened. Overall, it is possible that Na phytate differs from intrinsic phytates and Na carbonate in its ability to dissolve in gastrointestinal contents and to bind nutrients. However, we cannot explain our finding that the AID of N and some amino acids was increased in the presence of Na phytate.

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The finding that the AID decreased marginally when Na phytate was added to the diet with microbial phytase suggests that microbial phytase preferentially hydrolyzes free Na phytate rather than complexed dietary phytate. Access to complexes of phytate and protein by microbial phytase may be lowered because they may be insoluble (Gifford and Clydesdale, 1990), or because the stereometric configuration of protein around the phytate molecules (Okubo et al., 1976) prevents phytase from hydrolyzing phytate.

5. Conclusion Adding Aspergillus niger phytase (Natuphos  ) at a dose of 900 FTU kg 21 to a maize-soybean meal based diet tended to increase the apparent ileal digestibility of crude protein by 1.6 percentage units. Phytase also stimulated the ileal digestibility of the essential amino acids lysine, tryptophan, threonine, and isoleucine, as well as that of most nonessential amino acids. The implication is that adding microbial phytase to the diet may result in a higher utilization of dietary protein, and therefore, reduce nitrogen excretion. Lactic acid increased the ileal digestibility of lysine, methionine, threonine, isoleucine, and arginine and some non-essential amino acids. However, lactic acid and microbial phytase had no synergistic effect on ileal amino acid digestibility.

Acknowledgements The authors acknowledge the Dutch Fund for Manure and Ammonia Research (FOMA) for partly financing this research, the members of the Feed Evaluation Working Group of the Dutch Product Board for Animal Feed for their valuable advice, and The Royal Gist-brocades BV, Delft, The Netherlands, for supplying the microbial phytase preparation.

References AOAC, 1980. Official methods of analysis. Association of Official Analytical Chemists, Inc. Arlington. Virginia.

115

Appurao, A.G., Narasinga Rao, M.S., 1975. Binding of Ca(II) by the 11S fraction of soybean proteins. Cereal Chem. 52, 21–33. Atwal, A.S., Eskin, N.A.M., McDonald, B.E., Vaisey-Genser, M., 1980. The effects of phytate on nitrogen utilization and zinc metabolism in young rats. Nutr. Rep. Int. 21, 257–267. Bolduan, G., Jung, H., Schneider, R., Block, J., Klenke, B., 1988. ¨ Die Wirkung von Propion- und Ameisensaure in der Ferkelaufzucht. J. Anim. Physiol. Anim. Nutr. 59, 72–78. Bos, K.D., Verbeek, C., van Eeden, C.H.P., Slump, P., Wolters, M.G.E., 1991. Improved determination of phytate by ionexchange chromatography. J. Agric. Food Chem. 39, 1770– 1772. ´ Camus, M.-C., Laporte, J.C., 1976. Inhibition de la proteolyse ˆ de l’acide phytique des issues. ´ Role pepsique in vitro par le ble. Ann. Biol. Anim. Bioch. Biophys. 16, 719–729. Caldwell, R.A., 1992. Effect of calcium and phytic acid on the activation of trypsinogen and the stability of trypsin. J. Agric. Food Chem. 40, 43–46. Cheryan, M., 1980. Phytic acid interactions in food systems. CRC Crit. Rev. Food Sci. 13, 297–335. Cochran, W.G., Snedecor, G.W., 1989. Statistical methods. 8th edition. Iowa state University Press, Ames, IA. Cosgrove, D.J., 1980. Inositol phosphates. Their chemistry, biochemistry and physiology. Elsevier scientific publishers, Amsterdam, The Netherlands. CVB, 1990. [Preliminary table of apparent ileal digestibilities of amino acids in feedstuffs for pigs.] CVB-reeks nr. 2. Lelystad, The Netherlands. Cromwell, G.L., 1992. The biological availability of phosphorus in feedstuffs for pigs. Pig News Info. 13, 75N. ¨ Dungelhoef, M., Rodehutscord, M., 1995. Wirkung von Phytasen ¨ auf die Verdaulichkeit des Phosphors beim Schwein. Ubers. ¨ Tierernahr. 23, 133–157. Easter, R.A., 1988. Acidification of diets for pigs. In: Haresign, W., Cole, D.J.A. (Eds.), Recent advances in animal nutrition. Butterworths, London, UK, pp. 61–71. Eckel, B., Kirchgeßner, M., Roth, F.X., 1992. Zum Einfluß von ¨ ¨ Ameisensaure auf tagliche Zunahmen, Futteraufnahme, Futterverwertung und Verdaulichkeit. 1. Mitteilung. Untersuchungen ¨ zur nutritiven Wirksamkeit von organischen Sauren in der Ferkelaufzucht. J. Anim. Physiol. Anim. Nutr. 67, 93–100. Eidelsburger, U., Kirchgeßner, M., Roth, F.X., 1992. Zum Einfluß ¨ von Ameisensaure, Calciumformiat und Natriumhydrogencar¨ bonat auf pH-Warte, Trockenmassegehalt, Konzentration an ¨ Carbonsauren und Ammoniak in verschiedenen Segmenten des Gastrointestinaltraktes. 8. Mitteilung. Untersuchungen zur nut¨ ritiven Wirksamkeit von organischen Sauren in der Ferkelaufzucht. J. Anim. Physiol. Anim. Nutr. 68, 20–32. Eidelsburger, U., Kirchgeßner, M., Roth, F.X., 1992. Zum Einfluß ¨ ¨ von Fumarsaure, Salzsaure, Natriumformiat, Tylosin und ¨ Toyocerin auf tagliche Zunahmen, Futteraufnahme, Futterverwertung und Verdaulichkeit. 11. Mitteilung. Untersuchungen ¨ zur nutritiven Wirksamkeit von organischen Sauren in der Ferkelaufzucht. J. Anim. Physiol. Anim. Nutr. 68, 82–92. Engelen, A.J., van der Heeft, F.C., Randsdorp, P.H.G., Smit, E.L.C., 1994. Simple and rapid determination of phytase activity. J. AOAC Int. 77, 760–764.

116

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117

Gabert, V.M., Sauer, W.C., 1995. The effect of fumaric acid and sodium formate supplementation to diets for weanling pigs on amino acid digestibility and volatile fatty acid concentrations in ileal digesta. Anim. Feed Sci. Technol. 53, 243–254. Gabert, V.M., Sauer, W.C., Schmidtz, M., Ahrens, F., Mosenthin, R., 1995. The effect of formic acid and buffering capacity on the ileal digestibilities of amino acids and bacterial populations and metabolites in the small intestine of weanling piglets fed semipurified fish meal diets. Can. J. Anim. Sci. 75, 615–623. Ganapathy, V., Leibach, F.H., 1985. Is intestinal peptide transport energized by a proton gradient?. Am. J. Physiol. 249, G153. Giesting, D.W., Easter, R.A., 1991. Effect of protein source and fumaric acid supplementation on apparent ileal digestibility of nutrients by young pigs. J. Anim. Sci. 69, 2497–2503. Giesting, D.W., Roos, M.A., Easter, R.A., 1991. Evaluation of the effect of fumaric acid and sodium bicarbonate addition on performance of starter pigs fed diets of different types. J. Anim. Sci. 69, 2489–2496. Gifford, S.R., Clydesdale, M., 1990. Interactions among calcium, zinc and phytate with three protein sources. J. Food Sci. 55, 1720–1724. Harada, E., Niiyama, M., Syuto, B., 1986. Comparison of pancreatic exocrine secretion via endogenous secretin by intestinal infusion of hydrochloric acid and monocarboxylic acid in anesthetized pigs. Japan. J. Physiol. 36, 843–856. Hartman, Jr. G.H., 1979. Removal of phytate from soy protein. J. Am. Oil Chem. Soc. 56, 731–735. Honig, D.H., Wolf, W.J., 1991. Phytate–mineral–protein composition of soybeans, gel filtration studies of soybean meal extracts. J. Agric. Food Chem. 39, 1037–1042. Hussain, A., Bushuk, W., 1992. Interference of phytic acid with extraction of proteins from grain legumes and wheat with acetic acid. J. Agric. Food Chem. 40, 1938–1942. Jongbloed, A.W., 1987. Phosphorus in the feeding of pigs. Effect of the diet on the absorption and retention of phosphorus by growing pigs. Ph.D. Thesis. Univ. of Wageningen, The Netherlands. Jongbloed, A.W., Bakker, J.G.M., Goedhart, P.W., Krol-Kramer, F., 1991. Evaluation of chromic oxide with lower concentration and of HCl-insoluble ash as markers for measuring overall apparent digestibility of some dietary nutrients for pigs. In: Verstegen, M.W.A., Huisman, J., den Hartog, L.A. (Eds.), Proc. Vth Int. Symp. Digestive Physiology in the Pig. EAAP Publication no. 54, 325–329. Jongbloed, A.W., Mroz, Z., Kemme, P.A., 1992. The effect of supplementary Aspergillus niger phytase in diets for pigs on concentration and apparent digestibility of dry matter, total phosphorus, and phytic acid in different sections of the alimentary tract. J. Anim. Sci. 70, 1159–1168. Kemme, P.A., Jongbloed, A.W., Mroz, Z., Beynen, A.C., 1998. Diurnal variation in degradation of phytic acid by plant phytase in the pig stomach. Livest. Prod. Sci. 54, 33–44. Kemme, P.A., Jongbloed, A.W., Mroz, Z., Beynen, A.C., 1999. Digestibility of nutrients in growing-finishing pigs is affected by Aspergillus niger phytase, phytate and lactic acid levels. 2. Apparent total tract digestibility of phosphorus, calcium and magnesium and ileal degradation of phytic acid. Livest. Prod. Sci. 58, 119–127.

Ketaren, P.P., Batterham, E.S., White, E., Farrell, D.J., Milthorpe, B.K., 1993. Phosphorus studies in pigs. Effect of phytase supplementation on the digestibility and availability of phosphorus in soya-bean meal for grower pigs. Br. J. Nutr. 70, 289–311. Khan, N., Cole, D.J.A., 1993. The effect of dietary inclusions of phytase and yeast on apparent phosphorus digestibility in pigs. Proc. Winter Meeting of the British Society of Animal Production. Scarborough, England. Knuckles, B.E., Kuzmicky, D.D., Betschart, A.A., 1985. Effect of phytate and partially hydrolysed phytate on in vitro protein digestibility. J. Food Sci. 50, 1080–1082. Knuckles, B.E., Kuzmicky, D.D., Gumbmann, M.R., Betschart, A.A., 1989. Effect of myo-inositol phosphate esters on in vitro and in vivo digestion of protein. J. Food Sci. 54, 1348–1350. Lenis, N.P., van Diepen, J.Th.M., Goedhart, P.W., 1990. Amino acid requirements for methionine and cystine, threonine and tryptophan of fast-growing boars and gilts, fed ad libitum. Neth. J. Agric. Sci. 38, 577–595. Maga, J.A., 1982. Phytate: its chemistry, occurrence, food interactions, nutritional significance, and methods of analysis. J. Agric. Food Chem. 30, 1–7. Mayer, E.A., 1994. The physiology of gastric storage and emptying. In: Johnson, L.R., Alpers, D.H., Christensen, J., Jacobson, E.D., Walsh, J.H. (Eds.), Physiology of the gastrointestinal tract. Volume 1. Raven Press, New York, NY, pp. 929–976. Mosenthin, R., Sauer, W.C., Ahrens, F., de Lange, C.F.M., Bornholdt, U., 1992. Effect of dietary supplements of propionic acid, siliceous earth or a combination of these on the energy, protein and amino acid digestibilities and concentrations of microbial metabolites in the digestive tract of growing pigs. Anim. Feed Sci. Technol. 37, 245–255. Mroz, Z., Bakker, G.C.M., Jongbloed, A.W., Dekker, R.A., Jongbloed, R., van Beers, A., 1996. Apparent digestibility of nutrients in diets with different energy density, as estimated by direct and marker methods for pigs with or without ileo-cecal cannulas. J. Anim. Sci. 74, 403–412. Mroz, Z., Jongbloed, A.W., Kemme, P.A., 1994. Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. J. Anim. Sci. 72, 126–132. ¨ Nasi, M., Helander, E., 1994. Effects of microbial phytase supplementation and soaking of barley-soybean meal on availability of plant phosphorus for growing pigs. Acta Agric. Scand. Sect. A, Animal Sci. 44, 79–86. O’Dell, B.L., De Boland, A., 1976. Complexation of phytate with proteins and cations in corn germ and oilseed meals. J. Agric. Food Chem. 24, 804–808. Officer, D.I., Batterham, E.S., 1992. Enzyme supplementation of Linola TM meal. Proc. of Wollongbar Pig Industry Seminar on Feed Enzymes, pp. 56–57. Okubo, K., Myers, D.V., Iacobucci, G.A., 1976. Binding of phytic acid to glycinin. Cereal Chem. 53, 513–524. Pallauf, J., Rimbach, G., Pippig, S., Schindler, B., Most, E., 1994. Effect of phytase supplementation to a phytase-rich diet based on wheat, barley and soya on the bioavailability of dietary phosphorus, calcium, magnesium, zinc and protein in piglets. Agribiol. Res. 47, 39–48.

P. A. Kemme et al. / Livestock Production Science 58 (1999) 107 – 117 Payne, R.W., Lane, P.W., Digby, P.G.N., Harding, S.A., Leech, P.K., Morgan, G.W., Todd, A.D., Thompson, R., Tunnicliffe Wilson, G., Welham, S.J., White, R.P., 1993. Genstat 5. Release 3. Reference manual. Clarendon Press, Oxford, UK. Prattley, C.A., Stanley, D.W., Van De Voort, F.R., 1982. Protein– phytate interactions in soybeans. II. Mechanism of protein– phytate binding as affected by calcium. J. Food Biochem. 6, 255–271. Sakata, T., Adachi, M., Hashida, M., Sato, N., Kojima, T., 1995. Effect of n-butyric acid on epithelial cell proliferation of pig ¨ colonic mucosa in short-term culture. Dtsch. Tierartztl. Wschr. 102, 163–164. Sano, H., Nakamaura, E., Takahashi, H., Terashima, Y., 1995. Plasma insulin and glucagon responses to acute challenges of acetate, propionate, n-butyrate and glucose in growing gilts (Sus scrofa). Comp. Biochem. Physiol. 110A, 375–378. Sato, H., Seino, T., Kobayashi, T., Murai, A., Yugari, Y., 1984. Determination of tryptophan content of feed and feedstuffs by ion exchange liquid chromatography. Agric. Biol. Chem. 48, 2961–2969.

117

Schram, E., Moore, S., Bigwood, E.W., 1954. Chromatographic determination of cystine as cystic acid. Biochem. J. 57, 35–37. Shah, K.R., 1977. Analysis of designs with two-way elimination of heterogenity. J. Statist. Planning Inference 1, 207–216. Singh, M., Krikorian, A.D., 1982. Inhibition of trypsin activity in vitro by phytate. J. Agric. Food Chem. 30, 799–800. Thompson, L.U., Serraino, M.R., 1986. Effect of phytic acid reduction on rapeseed protein digestibility and amino acid absorption. J. Agric. Food Chem. 34, 468–469. Wenk, C., Weiss, E., Bee, G., Messikommer, R., 1993. Phytases in rations for growing pigs: influence on the nutrient utilization. In: Wenk, C., Boessinger, M. (Eds.), Enzymes in animal nutrition. Proc. of the 1st Symposium, Kartause Ittingen, Switzerland, pp. 226–230. Williams, C.H., David, D.J., Iismaa, O., 1962. The determination of chromic oxide in faeces samples by atomic absorption spectrophotometry. J. Agric. Sci. 59, 381–385. Yi, Z., 1995. Supplementing microbial phytase to diets for swine and poultry. PhD Thesis, Virginia Polytechnic Institute and State University, Blacksburg, VA.