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Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-products as influenced by harvest year and cultivar
Animal Feed Science and Technology 133 (2007) 320–334
Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-products as influenced by harvest year and cultivar T. Steiner a , R. Mosenthin a,∗ , B. Zimmermann b , R. Greiner c , S. Roth a a
Institute of Animal Nutrition, University of Hohenheim, 70599 Stuttgart, Germany b 64291 Darmstadt, Germany c Centre for Molecular Biology, Federal Research Centre for Nutrition and Food, 76131 Karlsruhe, Germany
Received 7 June 2005; received in revised form 27 March 2006; accepted 24 April 2006
Abstract Samples of legume seeds, cereals and cereal by-products (n = 113) grown in south-western Germany and originating from different cultivars and harvest years were analyzed for phytase activity, total phosphorus (P) and phytate P. Phytase activities determined by means of a direct incubation method were lowest in legume seeds and oats (262–496 U/kg dry matter), intermediate in cereals (except oats) (2323–6016 U/kg DM) and highest in cereal by-products (9241–9945 U/kg DM). However, the application of an extraction procedure for the determination of phytase activities in legume seeds resulted in values below the detection limit of 50 U/kg. On average, about 0.67 of total P in legume seeds, cereals and their by-products is bound to phytate. There was a significant influence (P<0.001) of harvest year (1998–2000) on phytate P contents in wheat. Furthermore, total P and phytate P concentrations differed (P<0.05) between different cultivars of wheat. Moreover, phytase activities differed (P=0.023) between different cultivars of barley. Total P and phytate P concentrations were highly correlated in legume seeds (r = 0.95) and cereal by-products (r = 0.96) and, to a smaller extent, in cereals (r = 0.66). Milling of cereal grains to bran and flour revealed that phytase
∗
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0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.04.007
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activity, total P and phytate P are highly concentrated (P<0.001) in the outer grain layers of cereals. The influence of preservation of intact legume seeds with propionic acid over a period of 4, 8 or 12 weeks resulted only in a marginal decrease in phytase activity. Due to high native phytase activities in cereals (except oats) and their by-products these feedstuffs may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals, whereas the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P seems to be rather limited. © 2006 Elsevier B.V. All rights reserved. Keywords: Phytase; Phosphorus; Phytate; Legume seeds; Cereals; By-products
1. Introduction Phytate, the salt of myo-inositol-1,2,3,4,5,6-hexaphosphoric acid, is the major storage form of phosphorus (P) in plants. About 0.50–0.80 of total P in plant feedstuffs is bound as phytate P. In this form, P is poorly available for non-ruminant animals (Pointillart et al., 1984), because they lack sufficient endogenous phytase, which releases orthophosphate from the phytate molecule in the gastrointestinal tract (Pointillart et al., 1984). Thus, environmental pollution results from excessive output of P in the manure. Furthermore, phytate chelates positively charged cations such as Ca2+ , Mg2+ , Zn2+ and Fe2+ under physiological conditions in the gut (Erdman, 1979), thereby reducing the availability of these minerals for pigs and poultry. However, phytase originating from microbes and plant tissues improves the availability of plant P and chelated minerals. High native phytase activities are present in cereals and cereal by-products, whereas lower activities have been reported for legume seeds (Eeckhout and De Paepe, 1994; Viveros et al., 2000; Zimmermann et al., 2002a). However, there is large variation in phytase activities among feedstuffs, depending on genetic and environmental factors such as cultivars (Barrier-Guillot et al., 1996; Cossa et al., 2000), cultivation or harvest years (Cossa et al., 2000). Moreover, the analytical methods used for phytase determination may also contribute to this variation (Zimmermann et al., 2002a; Greiner and Egli, 2003). For example, reports on phytase activities in legume seeds reveal extremely inconsistent values ranging between 0 U/kg (Eeckhout and De Paepe, 1994) and 258 U/kg (Viveros et al., 2000). The inclusion of homegrown legume seeds in diets for non-ruminant animals as alternative to soyabean products has been receiving growing attention in various European countries. Preservation procedures such as treatment with heat and organic acids are widely used to avoid microbial deterioration of legume seeds after harvest. However, no studies have been carried out so far in which the effect of application of organic acids on native phytase activity in legume seeds have been reported. The current study was conducted to assess phytase activities and the contents of total P and phytate P in samples of different legume seeds, cereals and cereal by-products as influenced by harvest years, cultivar and the analytical method used for the determination of phytase activities. In addition, the effect of propionic acid supplementation on native phytase activities in legume seeds was studied as influenced by storage time.
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Table 1 Cultivars and harvest years of feedstuffs, and methods applied for the determination of phytase activity Feedstuff
na
Number of cultivars
Harvest year
Determination of phytase activityb
Legume seedsc Field beans Peas Lupins
11 18 14
11 18 14
2000–2002 1999–2002 2000–2002
DI, EX DI, EX DI, EX
Cereals Oats Wheat Barleyd Triticalee Ryef
6 18 15 12 13
2 6 6 5 6
1998–2000 1998–2000 1998–2000 1998–2000 1998–2000
DI DI DI DI DI
3 3
3 3
1999 1999
DI DI
Cereal by-products Wheat bran Rye bran a b c d e f
Number of observations. DI: direct incubation (Greiner and Egli, 2003); EX: extraction procedure (Naumann and Bassler, 1976). In legume seeds, each cultivar originated either from 1999, 2000, 2001 or 2002. For barley, three cultivars originated from 1998 and 1999 only. For triticale, three cultivars originated from 1999 and 2000 only. For rye, five cultivars originated from 1999 and 2000 only.
2. Materials and methods 2.1. Experiment 1 In total 113 samples of legume seeds (field beans, peas and lupins, n = 43), cereals (oats, wheat, barley, triticale and rye, n = 64) and cereal by-products (wheat bran and rye bran, n = 6) were collected at breeding companies, feed mills, farms and the State Plant Breeding Institute (University of Hohenheim, 70599 Stuttgart, Germany). The cereal samples included different cultivars harvested in 1998, 1999 or 2000 (Table 1). All samples were ground to pass a 1-mm mesh sieve using a rotor mill (Retsch® ZM 1, Retsch GmbH & Co. KG, 42781 Haan, Germany). Native phytase activity was measured in triplicate analyses using the direct incubation method as outlined by Greiner and Egli (2003). One unit of phytase activity is equivalent to the amount of enzyme that liberates 1 mol of orthophosphate from 100 mol of sodium phytate at 37 ◦ C and pH 5.0 per minute. Additionally, phytase activities in legume seeds were assayed by means of the extraction procedure as outlined by the Association of German Agricultural Experimental and Research Stations (Naumann and Bassler, 1976). According to this method, one unit of phytase activity is equivalent to the amount of enzyme that liberates 1 mol of orthophosphate from 5.1 mmol of sodium phytate at 37 ◦ C and pH 5.5 per minute. With the method by Greiner and Egli (2003) the total phytase activity is determined, whereas the extraction procedure by Naumann and Bassler (1976) determines the activity of the soluble phytases only. The contents of total and phytate P were analyzed by the vanadate-molybdate method (Naumann and Bassler, 1976) and the anion-exchange method as described by Harland and Oberleas (1986), respectively.
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2.2. Experiment 2 Three cultivars of wheat or rye harvested in 1999 were selected to investigate the effect of milling on the distribution of phytase activity, total P and phytate P in these grains. Fine flour type 405 and bran were produced from the grains using a precision laboratory roller mill (Brabender® Quadrumat® Junior, Brabender OHG, 47055 Duisburg, Germany). The samples were analyzed for total and phytate P by means of the procedures as described in Experiment 1 and the phytase activities were determined according to Greiner and Egli (2003). 2.3. Experiment 3 One representative cultivar out of each of the legume seeds was selected to study the effect of preservation with propionic acid on native phytase activities determined 0, 4, 8 and 12 weeks after organic acid treatment. The original samples (20 kg) were soaked for approximately 45 min in aqua bidest. to lower the original dry matter (DM) content (895 ± 13 g/kg) to values between 850 and 870 g/kg. At this DM level legume seeds are susceptible to microbial deterioration (UFOP, 2004). Propionic acid (Luprosil® , BASF AG, 67056 Ludwigshafen, Germany) was used as preservative, and sprayed on the intact samples according to manufacturer’s recommendations (6 g/kg, wt./wt.). After thoroughly mixing, three subsamples of 350 g each were taken from each legume seed and stored in PVC-containers at 20 ◦ C room temperature. Samples of approximately 100 g were taken after 4, 8 and 12 weeks of storage and rinsed with aqua bidest. to remove any remaining propionic acid. Subsequently, the legume seeds were freeze-dried, ground to pass a 1-mm mesh sieve and analyzed for phytase activity according to Greiner and Egli (2003). 2.4. Statistical analyses The experimental data were subjected to analyses of variance using the GLM procedure of SAS (1999). The model includes in Experiment 1 the main effects of harvest year and cultivar; in Experiment 2 the effects of different milling treatments (seed, flour, bran); and in Experiment 3 the effect of storage time (0, 4, 8, 12 weeks). Significant differences between means were determined using the PDIFF option with PROC GLM (SAS, 1999). Additionally, correlation coefficients and linear relationships between the components analyzed in Experiment 1 were calculated using the CORR and REG procedure of SAS (1999), respectively.
3. Results and discussion 3.1. Phytase activities in legume seeds, cereals and cereal by-products (Experiment 1) When analyzed according to the direct incubation method of Greiner and Egli (2003), the native phytase activities are lowest in legume seeds (262–324 U/kg DM) and oats
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Table 2 Phytase activity, total phosphorus and phytate phosphorus content in legume seeds, cereals and cereal by-productsa nb
Legume seeds Field beans Peas Lupins
11 18 14
Cereals Oats Wheat Barley Triticale Rye
6 18 15 12 13
Cereal by-products Wheat bran 3 Rye bran 3 a b
Phytase activity (U/kg)
290 ± 78 262 ± 73 324 ± 59 496 2886 2323 2799 6016
± ± ± ± ±
35 645 648 501 1578
9945 ± 0427 9241 ± 1452
Total P (g/kg)
5.7 ± 0.92 4.1 ± 0.46 5.7 ± 1.48 3.7 4.0 4.2 4.0 3.6
± ± ± ± ±
0.14 0.40 0.42 0.34 0.19
8.8 ± 0.71 5.8 ± 0.25
Phytate P g/kg
Proportion of total P
3.9 ± 0.75 2.4 ± 0.45 3.5 ± 0.92
0.70 ± 0.052 0.58 ± 0.060 0.63 ± 0.037
2.5 2.9 2.6 2.8 2.4
± ± ± ± ±
0.17 0.37 0.31 0.30 0.23
7.9 ± 0.05 4.9 ± 0.23
0.67 0.73 0.63 0.70 0.67
± ± ± ± ±
0.054 0.081 0.035 0.054 0.050
0.90 ± 0.073 0.85 ± 0.020
Means ± standard deviation (dry matter basis). Number of observations.
(496 U/kg DM), intermediate in wheat (2886 U/kg DM), barley (2323 U/kg DM), triticale (2799 U/kg DM) and rye (6016 U/kg DM) and highest in the by-products (9945 and 9241 U/kg DM in wheat and rye bran, respectively) (Table 2). In contrast to the findings of the present study, literature data show in general a larger variation in phytase activities. For example, for legume seeds values range between 0 U/kg (Eeckhout and De Paepe, 1994) and 258 U/kg (Viveros et al., 2000). It can be questioned if these data are representative due to the low number of observations in most of the experiments. In the study of Viveros et al. (2000), for example, only three samples of beans and six samples of peas and lupins, respectively, were analyzed. Moreover, the phytase activities reported by Eeckhout and De Paepe (1994) for field beans and lupins were based on one individual sample, whereas in the present study 11, 18 and 14 observations for field beans, peas and lupins originating from different cultivars, locations and harvest years were obtained (Table 1). The phytase activities reported herein for wheat, barley and rye agree with those presented by Greiner and Egli (2003), amounting to 2931, 2093 and 6752 U/kg, respectively. However, according to several reports considerably lower values were obtained. For example, as reported by Cossa et al. (2000) phytase activities in wheat were only 520–1390 U/kg. For wheat, barley and triticale published values vary between 88 U/kg (Liu et al., 1998) and 3120 U/kg (Singh and Sedeh, 1979). For oats, reported phytase activities range between 0 U/kg (Eeckhout and De Paepe, 1994) and 412 U/kg (Shen et al., 2005), which is lower as compared to the present investigation (496 U/kg DM). In agreement with the findings of the present study, activities in rye are in general higher than in other cereals. However, there is still a considerable variation between studies ranging from 3790 U/kg (Fretzdorff and Weipert, 1986) to 6840 U/kg (Zimmermann et al., 2002b). Since phytase is predominantly located in the aleurone layer and scutellum of cereal grains (Fretzdorff and Weipert, 1986),
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by-products such as wheat bran and rye bran generally show the highest activities among plant feedstuffs. However, literature values exhibit again a considerable variation between different studies ranging between 600 U/kg (Pointillart, 1988) and 7400 U/kg (Pointillart, 1993) for cereal brans. There is growing evidence that the analytical procedure used for the determination of phytase activity in plant feedstuffs may have a major impact on the values obtained. As previously shown by Zimmermann et al. (2002a) and Greiner and Egli (2003), phytase activities in cereals based on extraction methods (e.g. Engelen et al., 1994; Barrier-Guillot et al., 1996; Liu et al., 1998; Cossa et al., 2000; Kim et al., 2002; Zimmermann et al., 2002a; Selle et al., 2003) are considerably lower than those obtained by means of direct incubation methods. For example, in the study by Zimmermann et al. (2002a) phytase activities in wheat and rye determined according to an extraction procedure (Engelen et al., 1994; with minor modifications) were 70 and 48%, respectively, lower in comparison to the values obtained by means of a direct incubation method. These findings are confirmed by the results for phytase activities in legume seeds. In the present study, phytase activities for peas, field beans and lupins determined according to the direct incubation method (Greiner and Egli, 2003) averaged 262, 290 and 324 U/kg DM, respectively, whereas the corresponding values obtained by means of the extraction procedure of Naumann and Bassler (1976) were below the detection limit of 50 U/kg. According to Greiner and Egli (2003) the incomplete extraction of plant phytases might be attributed to proteolytic degradation and a partial association of the enzymes with membrane structures. Differences in the composition of the cell walls, interaction of phytases with morphological grain fractions and differences in the location of the enzymes within the grains are considered to be additional factors responsible for the incomplete and different extraction rates of plant phytases (Zimmermann et al., 2002a). Therefore, specification of the analysis method used for determination of plant phytase activities is mandatory in order to allow for a comparison of results between different studies. Due to high native phytase activities, cereals (except oats) and cereal by-products have the potential to contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals. Although the present study revealed considerably higher phytase activities in legume seeds than previous literature reports, the absolute values are rather low in comparison to cereals and their by-products. Therefore, the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P for pigs and poultry seems to be of minor importance. 3.2. Contents of total P and phytate P in legume seeds, cereals and cereal by-products (Experiments 1 and 2) Among legume seeds, field beans and lupins show higher concentrations of total P (5.7 g/kg DM) and phytate P (3.5–3.9 g/kg DM) as compared to peas (4.1 and 2.4 g/kg DM, respectively). In cereals, the contents of total P range between 3.6 g/kg DM (rye) and 4.2 g/kg DM (barley), whereas phytate P contents range between 2.4 g/kg DM (rye) and 2.9 g/kg DM (wheat). The highest concentrations of total P and phytate P are found in wheat bran (8.8 and 7.9 g/kg DM, respectively), whereas rye bran contains only 5.8 and 4.9 g/kg DM total P and phytate P, respectively. These values are in the range of previously reported
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Table 3 Phytase activity, total phosphorus and phytate phosphorus content in cereals grains, flour and bran originating from wheat and ryea Treatment
Seed Flour Bran S.E.M.c
nb
6 6 6
Phytase activity (U/kg)
Total P (g/kg)
3639 b 2446 c 9593 a 377
3.9 b 1.1 c 7.3 a 0.14
Phytate P g/kg
Proportion of total P
2.6 b 0.5 c 6.4 a 0.06
0.66 b 0.44 c 0.87 a 0.018
Within a column, means followed by different letters (a, b, c) differ at P<0.05. a Least squares means (dry matter basis). b Number of observations. c S.E.M.: standard error of least squares means.
literature data (e.g. Lolas et al., 1976; De Boever et al., 1994; Eeckhout and De Paepe, 1994; Viveros et al., 2000; Shen et al., 2005). In the present study, on average, about two thirds (0.67 ± 0.088) of total P in legume seeds, cereals and by-products (n = 113) are bound to phytate. However, in cereal by-products the proportion of phytate P in relation to total P is higher (0.85–0.90) than in legume seeds and cereals (0.58–0.73), thus indicating an enrichment of phytate in brans. This is confirmed by the results obtained in Experiment 2, in which grains of wheat and rye were milled to flour and bran in order to determine phytase activities and concentrations of total P and phytate P in these fractions. Compared to the intact grains, the proportion of phytate P in relation to total P was higher (P<0.05) in wheat and rye bran (0.87 versus 0.66) (Table 3). On the other hand, total P and phytate P contents along with phytase activities were significantly (P<0.05) lower in the flours originating from wheat and rye (1.1 g/kg DM, 0.5 g/kg DM and 2446 U/kg DM, respectively) in comparison to both bran sources and intact grains. In agreement with previous studies by O’Dell et al. (1972), Singh and Reddy (1977), Fretzdorff and Weipert (1986) and Okot-Kotber et al. (2003), the results of the present study clearly confirm that phytase as well as total P and phytate P are predominantly associated with the outer layers of cereal grains. Extremely low phytate P concentrations, such as determined by Viveros et al. (2000) in field beans (0.8 g/kg DM) or by Eeckhout and De Paepe (1994) in lupins (0.5 g/kg DM), can be attributed to different methods used for the determination of phytate P. According to Selle et al. (2003) the application of the analysis method of Haug and Lantzsch (1983), which is based on the precipitation of phytate with an acidic iron (III) solution may have caused an underestimation of the actual phytate P concentrations in field beans and lupins in the aforementioned studies of Viveros et al. (2000) and Eeckhout and De Paepe (1994). Obviously, quantitative extraction of phytate in these feedstuffs was limited due to the strong binding of phytate to protein fractions. Selle et al. (2003) concluded, based on studies with field beans and lupins, that the anion-exchange column chromatography (Harland and Oberleas, 1986), which has been used in the present study, is more suitable for quantitative determination of phytate P.
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3.3. Influence of harvest year on phytase activities and contents of total P and phytate P in wheat and barley in Experiment 1 Other factors than the use of different analytical procedures for the determination of phytase activities and concentrations of total P and phytate P might be responsible for different results between studies. These factors include differences in cultivar (Miller et al., 1980a; Barrier-Guillot et al., 1996; Cossa et al., 2000), harvest year (Ockenden et al., 1997; Cossa et al., 2000), climatic conditions (Bassiri and Nahapetian, 1977; Kim et al., 2002), P content of the soil (Miller et al., 1980b; Saastamoinen, 1987) and growing location (Miller et al., 1980a; Cossa et al., 2000). Wheat and barley as major feed ingredients in diets for non-ruminant animals were selected as cereal grains to determine whether phytase activities and contents of total P and phytate P differed between harvest years and cultivars. In the present study, there was no significant influence (P>0.05) of harvest year (1998–2000) on phytase activities and total P concentrations in wheat and barley (Table 4), which is in agreement with reports by Ockenden et al. (1997) and Kim et al. (2002), whereas Cossa et al. (2000) obtained a significant influence of different harvest years (1995 versus 1996) on phytase activities in wheat samples. In the present study, however, there was a significant influence (P<0.001) of harvest year on total phytate P concentrations in wheat, which was confirmed as trend (P=0.059) in barley. For example, in wheat the highest phytate P contents were found in 2000 (3.2 g/kg DM), whereas in 1998 and 1999 phytate P concentrations amounted to 2.7 and 2.8 g/kg DM, respectively. Additionally, there was a tendency towards an influence of harvest year on phytate P concentrations, expressed as percentage of total P in wheat (P=0.057). Moreover, there was a tendency towards an effect of harvest year on total P contents in barley (P=0.074). The findings of the present study are confirmed by Cossa et al. (2000), who observed a significant influence of different harvest years (1995 versus 1996) on phytate P concentrations in wheat as well. Numerical, but not significant (P=0.08) effects of different harvest years (1999 versus 2000) on phytate P contents in wheat were reported by Kim et al. (2002). Furthermore, Ockenden et al. (1997) reported in barley samples a significant effect of different harvest years (1984–1994) on phytate P concentrations expressed as percentage of total P. 3.4. Influence of different cultivars of wheat and barley on phytase activities and contents of total P and phytate P in Experiment 1 The samples used in the present experiment represent six and three different cultivars of wheat and barley, respectively. In barley, phytase activity was significantly (P=0.023) affected by cultivar (Table 4). Among the barley cultivars, phytase activities range between 1665 U/kg DM (cv. Hanna) and 2857 U/kg DM (cv. Henni). Previously, the effect of cultivar on phytase activities in barley has not yet been studied. In the present study, there was no significant (P=0.107) influence of cultivar on phytase activities in wheat, which is in contrast to Barrier-Guillot et al. (1996), Cossa et al. (2000) and Kim et al. (2002). The total P and phytate P contents differ significantly (P<0.05) among the six different wheat cultivars (Table 4). Values for total P in wheat range between 3.5 g/kg DM (cv.
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Table 4 Influence of harvest year and cultivar on phytase activity, total phosphorus and phytate phosphorus content in wheat and barleya nb
Wheat Harvest year 1998 1999 2000 S.E.M.c P value Cultivar Bussard Estica Flair Toronto Transit Triso S.E.M.c P value Barley Harvest year 1998 1999 2000 S.E.M.c P value Cultivar Barke Hanna Henni S.E.M.c P value
Phytase activity (U/kg)
Total P (g/kg)
Phytate P g/kg
Proportion of total P
6 6 6
2854 2748 3057 225 0.628
3.8 4.1 4.0 0.11 0.137
2.7 b 2.8 b 3.2 a 0.05 <0.001
0.71 0.69 0.79 0.027 0.057
3 3 3 3 3 3
1956 3186 3106 3371 2881 2818 319 0.107
4.0 abc 3.5 c 3.8 bc 4.3 ab 4.0 ab 4.4 a 0.16 0.033
3.1 b 2.6 c 2.5 c 2.9 b 3.0 b 3.4 a 0.07 <0.001
0.78 0.75 0.66 0.68 0.74 0.77 0.038 0.250
3 3 3
2139 2128 2391 180 0.556
3.7 4.4 4.0 0.17 0.074
2.2 2.8 2.6 0.13 0.059
0.60 0.64 0.65 0.013 0.124
3 3 3
2136 b 1665 b 2857 a 180 0.023
4.1 3.8 4.2 0.17 0.460
2.6 2.4 2.7 0.13 0.303
0.64 0.61 0.64 0.013 0.333
For harvest years and cultivars, means within a column followed by different letters (a, b, c) differ at P<0.05. a Least squares means (dry matter basis). b Number of observations. c S.E.M.: standard error of least squares means.
Estica) and 4.4 g/kg DM (cv. Triso), and for phytate P between 2.5 g/kg DM (cv. Flair) and 3.4 g/kg DM (cv. Triso). In contrast, there was no effect (P>0.05) of cultivar on total P or phytate P concentrations in barley samples. Reports pertaining to the effect of cultivar on total P or phytate P contents in cereal grains are equivocal. In wheat samples, total P and phytate P levels were not significantly different between cultivars (Barrier-Guillot et al., 1996; Kim et al., 2002). However, Singh and Reddy (1977) and Feil and Fossati (1997) reported significant differences in the phytate content in different cultivars of triticale. Explanations for the conflicting findings obtained in the above-mentioned studies remain rather speculative. It may be hypothesized that in the different studies possible effects
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of harvest year and cultivar on phytase activity, total P and phytate P might have been confounded by various other factors such as analytical methods, growing location, degree of fertilization, climatic conditions or interactions between these factors. In the studies by Barrier-Guillot et al. (1996), Cossa et al. (2000) and Kim et al. (2002), for example, phytase activities were determined using an extraction procedure instead of a direct incubation method. Furthermore, Kim et al. (2002) assumed that environmental conditions have in general a higher impact on the chemical composition of grains than genetic influences. Likewise, Ockenden et al. (1997) indicated, for example, that the accumulation of total P and phytate P in barley may be influenced by temperature and precipitation level. It is well known that such environmental factors may vary considerably between different harvest years. It has to be emphasized that the cereal grains used in the present study were grown in the same geographic area (south-western Germany); however, possible effects of environmental factors such as growing location, degree of fertilization and climatic conditions on the results reported herein cannot be excluded. 3.5. Relationship between components analyzed in Experiment 1 Phytase activity was not significantly (P>0.05) correlated with total P or phytate P concentrations in any of the feedstuffs under investigation. In agreement with the conclusions drawn by Eeckhout and De Paepe (1994), Barrier-Guillot et al. (1996) and Viveros et al. (2000), native phytase activities in plant feedstuffs cannot be predicted from their total P or phytate P contents. It is assumed that measurements of in vitro phytase activities may not necessarily reflect the potential for phytate hydrolysis under in vivo conditions since intestinal and dietary factors such as pH and transit time may affect the degree of phytate degradation. A positive correlation and a significant linear relationship between phytate P and total P contents could be established for legume seeds (r = 0.95, P<0.001, Fig. 1), cereals (r = 0.66, P<0.001, Fig. 2) and cereal by-products (r = 0.96, P=0.002) as well. The regression equations calculated for legume seeds, cereals and cereal by-products are listed in Table 5. Significant correlations between total P and phytate P have been reported previously. In agreement with the present study, Eeckhout and De Paepe (1994) and Viveros et al. (2000) determined higher correlations between phytate P and total P in legume seeds as compared to cereals. In field beans (r = 0.92, P<0.001), peas (r = 0.92, P<0.001) and lupins
Fig. 1. Linear relationship between phytate P and total P content in legume seeds (n = 43).
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Fig. 2. Linear relationship between phytate P and total P in cereals (n = 64).
(r = 0.97, P<0.001), phytate P and total P contents are highly correlated, indicating that total P values can be used to predict the phytate P contents in legume seeds. In contrast, among cereals the relationships are comparatively low for wheat (r = 0.63, P=0.005), triticale (r = 0.63, P=0.028) and rye (r = 0.62, P=0.024), whereas for barley the correlation coefficient amounts to 0.86 (P<0.001). For oats, wheat bran and rye bran the correlation is not significant (P>0.05), presumably, because of the small number of samples analyzed (n = 6 for oats, n = 3 for wheat or rye bran, respectively). The correlation coefficients determined in the present study for wheat (r = 0.63) and barley (r = 0.86) show good agreement with literature values reported by Barrier-Guillot et al. (1996) and Ockenden et al. (1997) (r = 0.56 and 0.89, respectively). However, in contrast to these reports and the present study, Lolas et al. (1976) and Selle et al. (2003) found higher correlation coefficients (0.91–0.96) between phytate P and total P concentrations for samples of oats and wheat. Obviously, there is a closer relationship between phytate P and total P in feedstuffs with higher total P concentrations (cereal by-products, field beans, peas, lupins, barley) as compared to those with lower total P contents (wheat, triticale, rye) in the present study. Table 5 Linear relationship between phytate P (y) and total P (x) contents in legume seeds, cereals and their by-products Feedstuff
na
Regression equation
R2
P value
R.S.D.b
All feedstuffs
113
y = 0.81 ± 0.034x − 0.63 ± 0.161
0.83
<0.001
0.449
Legume seeds Field beans Peas Lupins
43 11 18 14
y = 0.73 ± 0.039x − 0.50 ± 0.203 y = 0.75 ± 0.108x − 0.31 ± 0.618 y = 0.88 ± 0.096x − 1.23 ± 0.399 y = 0.60 ± 0.040x + 0.09 ± 0.235
0.89 0.84 0.84 0.95
<0.001 <0.001 <0.001 <0.001
0.318 0.315 0.184 0.214
Cereals Oats Wheat Barley Triticale Rye
64 6 18 15 12 13
y = 0.58 ± 0.084x + 0.38 ± 0.333 y = −0.09 ± 0.612x + 2.80 ± 2.270 y = 0.58 ± 0.180x + 0.57 ± 0.723 y = 0.63 ± 0.104x − 0.01 ± 0.040 y = 0.56 ± 0.216x + 0.57 ± 0.864 y = 0.76 ± 0.291x − 0.33 ± 1.046
0.44 0.01 0.40 0.74 0.40 0.38
<0.001 0.888 0.005 <0.001 0.028 0.024
0.263 0.190 0.295 0.163 0.242 0.187
By-products
6
y = 0.91 ± 0.130x − 0.27 ± 0.967
0.93
0.002
0.503
a b
Number of observations. R.S.D.: residual standard deviation.
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Fig. 3. Effect of propionic acid on phytase activities in intact field beans, peas and lupins as influenced by different storage times. Data are least squares means and standard error of three observations per treatment. For each legume seed, means with different letters (a, b, c) differ at P<0.05.
The reasons for the inconsistent correlation coefficients reported in different studies have not yet been fully elucidated. However, the results of the present study indicate that the relationship between phytate P and total P contents is stronger in legume seeds (Fig. 1) and cereal by-products than in cereals (Fig. 2). As indicated by Raboy (1997), phytate P accumulation during the seed development functions to maintain a constant level of nonphytate P. However, until now the mechanisms involved in the homeostatic regulation of phytic acid synthesis have not been discovered sufficiently. 3.6. Influence of preservation with propionic acid on native phytase activities in legume seeds (Experiment 3) In order to avoid microbial degradation of legume seeds through bacteria, yeasts and fungi during storage, it is mandatory to preserve these feedstuffs if the moisture content of the grains exceeds 120 g/kg (UFOP, 2004). In practice, the preservation of legume seeds by means of organic acids receives growing attention. The antimicrobial effect of organic acid supplementation in feedstuffs can be attributed to a reduction in pH. The influence of preservation with propionic acid on phytase activity in intact legume seeds during the storage over a period of 12 weeks is presented in Fig. 3. The initial phytase activities in the cultivars of field beans (cv. Music), peas (cv. Lido) and lupins (cv. Borsaja) amounted to 455, 356 and 350 U/kg DM, respectively, at the beginning of the experiment. These values were close to or above the average values obtained for each of the legume seeds (Table 2). Albeit of small magnitude, the decrease in phytase activity over a period of 12 weeks is significant (P<0.05) due to an extremely low variation within each treatment under the standardized laboratory conditions as described herein. After 12 weeks of storage, the decline in phytase activities amounted to 4.1, 6.1 and 25.5% in field beans, peas and lupins, respectively. As shown in in vitro studies, the activity of purified plant phytase is highly influenced by pH conditions (Phillippy, 1999; Greiner et al., 2001; Greiner, 2002). It was therefore investigated if Luprosil® , which shows a pH value of 2.3 only, has a
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detrimental impact on phytase activities in legume seeds. However, the results of the present study show that propionic acid has no major effect on native phytase activity in material which has not been ground before treatment with organic acids. Presumably, phytases are protected against irreversible denaturizing by acid molecules in the seed matrix. This is supported by Greiner and Konietzny (1998) who concluded from studies with Phaseolus beans that the seed matrix might have a stabilizing effect on phytase. Because none of the treatments distinctly decreased the native phytase activity, conservation with propionic acid represents a suitable procedure to preserve moist legume seeds after harvest for at least 12 weeks. In conclusion, cereals (except oats) and cereal by-products may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals due to high native phytase activities, whereas the contribution of legume seeds in terms of improving the availability of plant P seems to be almost negligible. In wheat, there is a significant influence of harvest year on the phytate P concentrations. Additionally, total P and phytate P contents differ between different cultivars of wheat. Moreover, phytase activities differ between different cultivars of barley. Finally, preservation with propionic acid results in a marginal decrease in phytase activities in legume seeds only.
Acknowledgements This work was financially supported by UFOP (Union zur F¨orderung von Oel- und Proteinpflanzen e.V., Berlin, Germany). The authors would like to thank Edith Haller, Jasmin Sarikol (Federal Research Centre for Nutrition and Food, Karlsruhe, Germany) and BASF AG (Ludwigshafen, Germany) for analysis of phytase activity and Helga Ott and Melanie Berger (University of Hohenheim, Stuttgart, Germany) for analysis of total P contents.
References Barrier-Guillot, B., Casado, P., Maupetit, P., Jondreville, C., Gatel, F., Larbier, M., 1996. Wheat phosphorus availability: 1-In vitro study; factors affecting endogenous phytasic activity and phytic phosphorus content. J. Sci. Food Agric. 70, 62–68. Bassiri, A., Nahapetian, A., 1977. Differences in concentrations and interrelationships of phytate, phosphorus, magnesium, calcium, zinc, and iron in wheat varieties grown under dryland and irrigated conditions. J. Agric. Food Chem. 25, 1118–1122. Cossa, J., Oloffs, K., Kluge, H., Drauschke, W., Jeroch, H., 2000. Variabilities of total and phytate phosphorus contents as well as phytase activity in wheat. Tropenlandwirt 101, 119–126. De Boever, J.L., Eeckhout, W., Boucque, C.V., 1994. The possibilities of near infrared reflection spectroscopy to predict total-phosphorus, phytate-phosphorus and phytase activity in vegetable feedstuffs. Neth. J. Agric. Sci. 42, 357–369. 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., 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. Erdman Jr., J.W., 1979. Oilseed phytates: nutritional implications. J. Am. Oil Chem. Soc. 56, 736–741. Feil, B., Fossati, D., 1997. Phytic acid in triticale grains as affected by cultivar and environment. Crop Sci. 37, 916–921.
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Fretzdorff, B., Weipert, D., 1986. Phytins¨aure in Getreide und Getreideerzeugnissen. Mitteilung I: Phytins¨aure und Phytase in Roggen und Roggenprodukten. Z. Lebensm. Unters. Forsch. 182, 287–293. Greiner, R., 2002. Purification and characterization of three phytases from germinated lupine seeds (Lupinus albus var. Amiga). J. Agric. Food Chem. 50, 6858–6864. Greiner, R., Egli, I., 2003. Determination of the activity of acidic phytate-degrading enzymes in cereal seeds. J. Agric. Food Chem. 51, 847–850. Greiner, R., Konietzny, U., 1998. Endogenous phytate-degrading enzymes are responsible for phytate reduction while preparing beans (Phaseolus vulgaris). J. Food. Process. Preserv. 29, 321–331. Greiner, R., Muzquiz, M., Burbano, C., Cuadrado, C., Pedrosa, M.M., Goyoaga, C., 2001. Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba var. Alameda). J. Agric. Food Chem. 49, 2234–2240. Harland, B.F., Oberleas, D., 1986. Anion-exchange method for determination of phytate in foods: collaborative study. J. Assoc. Off. Anal. Chem. 69, 667–670. 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. Kim, J.C., Mullan, B.P., Selle, P.H., Pluske, J.R., 2002. Levels of total phosphorus, phytate-phosphorus, and phytase activity in three varieties of Western Australian wheats in response to growing region, growing season, and storage. Aust. J. Agric. Res. 53, 1361–1366. Liu, J., Ledoux, D.R., Veum, T.L., 1998. In vitro prediction of phosphorus availability in feed ingredients for swine. J. Agric. Food Chem. 46, 2678–2681. Lolas, G.M., Palamidis, N., Markakis, P., 1976. The phytic acid-total phosphorus relationship in barley, oats, soybeans, and wheat. Cereal Chem. 53, 867–871. Miller, G.A., Youngs, V.L., Oplinger, E.S., 1980a. Environmental and cultivar effects on oat phytic acid concentration. Cereal Chem. 57, 189–191. Miller, G.A., Youngs, V.L., Oplinger, E.S., 1980b. Effect of available soil phosphorus and environment on the phytic acid concentration in oats. Cereal Chem. 57, 192–194. Naumann, K., Bassler, R., 1997. Verband deutscher Landwirtschaftlicher Untersuchungs- und Forschungsanstalten. Methodenbuch Band III. Die chemische Untersuchung von Futtermitteln. Mit Erg¨anzungslieferungen 1983, 1993. VDLUFA-Verlag, Darmstadt, Germany. Ockenden, I., Falk, D.E., Lott, J.N.A., 1997. Stability of phytate in barley and beans during storage. J. Agric. Food Chem. 34, 1673–1677. O’Dell, B.L., de Boland, A.R., Koirtyohann, S.R., 1972. Distribution of phytate and nutritionally important elements among the morphological components of cereal grains. J. Agric. Food Chem. 20, 718–721. Okot-Kotber, M., Yong, K.-J., Bagorogoza, K., Liavoga, A., 2003. Phytase activity in extracts of flour and bran from wheat cultivars: enhanced extractability with -glucanase and endo-xylanase. J. Cereal Sci. 38, 307– 315. Phillippy, B.Q., 1999. Susceptibility of wheat and Aspergillus niger phytases to inactivation by gastrointestinal enzymes. J. Agric. Food Chem. 47, 1385–1388. Pointillart, A., 1988. Phytate phosphorus utilization in growing pigs. In: Proceedings of the 4th International Seminar on Digestive Physiology in Pigs. Polish Academy of Sciences, Jablonna, Poland, pp. 319–326. Pointillart, A., 1993. Importance of phytates and cereal phytases in the feeding of pigs. In: Proceedings of the 1st Symposium on Enzymes in Animal Nutrition. Kartause Ittingen, Switzerland, pp. 192–198. 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. 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 Publishers, Dordrecht, The Netherlands, pp. 441–477. Saastamoinen, M., 1987. Effect of nitrogen and phosphorus fertilization on the phytic acid content of oats. Cereal Res. Commun. 15, 57–64. SAS, 1999. SAS Online Doc® , Version 8. SAS Institute Inc., Cary, NC, USA. Selle, P.H., Walker, A.R., Bryden, W.L., 2003. Total and phytate-phosphorus contents and phytase activity of Australian-sourced feed ingredients for pigs and poultry. Aust. J. Exp. Agr. 43, 475–479. Shen, Y., Yin, Y., Chavez, E.R., Fan, M.Z., 2005. Methodological aspects of measuring phytase activity and phytate phosphorus content in selected cereal grains and digesta and feces of pigs. J. Agric. Food Chem. 53, 853–859.
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Singh, B., Reddy, N.R., 1977. Phytic acid and mineral compositions of triticales. J. Food Sci. 42, 1077–1083. Singh, B., Sedeh, H.G., 1979. Characteristics of phytase and its relationship to acid phosphatase and certain minerals in triticale. Cereal Chem. 56, 267–272. UFOP (Union zur F¨orderung von Oel und Proteinpflanzen e.V.), 2004. UFOP Praxisinformation. Inhaltsstoffe, Futterwert und Einsatz von Ackerbohnen in der Nutztierf¨utterung. Aktualisierte Auflage. UFOP, Bonn, Germany. 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. Zimmermann, B., Lantzsch, H.-J., Langbein, U., Drochner, W., 2002a. Determination of phytase activity in cereal grains by direct incubation. J. Anim. Physiol. a. Anim. Nutr. 86, 347–352. Zimmermann, B., Lantzsch, H.-J., Mosenthin, R., Sch¨oner, F.-J., Biesalski, H.K., Drochner, W., 2002b. Comparative evaluation of the efficacy of cereal and microbial phytases in growing pigs fed diets with marginal phosphorus supply. J. Sci. Food Agric. 82, 1298–1304.
Distribution of phytase activity, total phosphorus and phytate phosphorus in legume seeds, cereals and cereal by-products as influenced by harvest year and cultivar T. Steiner a , R. Mosenthin a,∗ , B. Zimmermann b , R. Greiner c , S. Roth a a
Institute of Animal Nutrition, University of Hohenheim, 70599 Stuttgart, Germany b 64291 Darmstadt, Germany c Centre for Molecular Biology, Federal Research Centre for Nutrition and Food, 76131 Karlsruhe, Germany
Received 7 June 2005; received in revised form 27 March 2006; accepted 24 April 2006
Abstract Samples of legume seeds, cereals and cereal by-products (n = 113) grown in south-western Germany and originating from different cultivars and harvest years were analyzed for phytase activity, total phosphorus (P) and phytate P. Phytase activities determined by means of a direct incubation method were lowest in legume seeds and oats (262–496 U/kg dry matter), intermediate in cereals (except oats) (2323–6016 U/kg DM) and highest in cereal by-products (9241–9945 U/kg DM). However, the application of an extraction procedure for the determination of phytase activities in legume seeds resulted in values below the detection limit of 50 U/kg. On average, about 0.67 of total P in legume seeds, cereals and their by-products is bound to phytate. There was a significant influence (P<0.001) of harvest year (1998–2000) on phytate P contents in wheat. Furthermore, total P and phytate P concentrations differed (P<0.05) between different cultivars of wheat. Moreover, phytase activities differed (P=0.023) between different cultivars of barley. Total P and phytate P concentrations were highly correlated in legume seeds (r = 0.95) and cereal by-products (r = 0.96) and, to a smaller extent, in cereals (r = 0.66). Milling of cereal grains to bran and flour revealed that phytase
∗
Corresponding author. Tel.: +49 711 459 3938; fax: +49 711 459 2421. E-mail address: [email protected] (R. Mosenthin).
0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2006.04.007
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activity, total P and phytate P are highly concentrated (P<0.001) in the outer grain layers of cereals. The influence of preservation of intact legume seeds with propionic acid over a period of 4, 8 or 12 weeks resulted only in a marginal decrease in phytase activity. Due to high native phytase activities in cereals (except oats) and their by-products these feedstuffs may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals, whereas the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P seems to be rather limited. © 2006 Elsevier B.V. All rights reserved. Keywords: Phytase; Phosphorus; Phytate; Legume seeds; Cereals; By-products
1. Introduction Phytate, the salt of myo-inositol-1,2,3,4,5,6-hexaphosphoric acid, is the major storage form of phosphorus (P) in plants. About 0.50–0.80 of total P in plant feedstuffs is bound as phytate P. In this form, P is poorly available for non-ruminant animals (Pointillart et al., 1984), because they lack sufficient endogenous phytase, which releases orthophosphate from the phytate molecule in the gastrointestinal tract (Pointillart et al., 1984). Thus, environmental pollution results from excessive output of P in the manure. Furthermore, phytate chelates positively charged cations such as Ca2+ , Mg2+ , Zn2+ and Fe2+ under physiological conditions in the gut (Erdman, 1979), thereby reducing the availability of these minerals for pigs and poultry. However, phytase originating from microbes and plant tissues improves the availability of plant P and chelated minerals. High native phytase activities are present in cereals and cereal by-products, whereas lower activities have been reported for legume seeds (Eeckhout and De Paepe, 1994; Viveros et al., 2000; Zimmermann et al., 2002a). However, there is large variation in phytase activities among feedstuffs, depending on genetic and environmental factors such as cultivars (Barrier-Guillot et al., 1996; Cossa et al., 2000), cultivation or harvest years (Cossa et al., 2000). Moreover, the analytical methods used for phytase determination may also contribute to this variation (Zimmermann et al., 2002a; Greiner and Egli, 2003). For example, reports on phytase activities in legume seeds reveal extremely inconsistent values ranging between 0 U/kg (Eeckhout and De Paepe, 1994) and 258 U/kg (Viveros et al., 2000). The inclusion of homegrown legume seeds in diets for non-ruminant animals as alternative to soyabean products has been receiving growing attention in various European countries. Preservation procedures such as treatment with heat and organic acids are widely used to avoid microbial deterioration of legume seeds after harvest. However, no studies have been carried out so far in which the effect of application of organic acids on native phytase activity in legume seeds have been reported. The current study was conducted to assess phytase activities and the contents of total P and phytate P in samples of different legume seeds, cereals and cereal by-products as influenced by harvest years, cultivar and the analytical method used for the determination of phytase activities. In addition, the effect of propionic acid supplementation on native phytase activities in legume seeds was studied as influenced by storage time.
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Table 1 Cultivars and harvest years of feedstuffs, and methods applied for the determination of phytase activity Feedstuff
na
Number of cultivars
Harvest year
Determination of phytase activityb
Legume seedsc Field beans Peas Lupins
11 18 14
11 18 14
2000–2002 1999–2002 2000–2002
DI, EX DI, EX DI, EX
Cereals Oats Wheat Barleyd Triticalee Ryef
6 18 15 12 13
2 6 6 5 6
1998–2000 1998–2000 1998–2000 1998–2000 1998–2000
DI DI DI DI DI
3 3
3 3
1999 1999
DI DI
Cereal by-products Wheat bran Rye bran a b c d e f
Number of observations. DI: direct incubation (Greiner and Egli, 2003); EX: extraction procedure (Naumann and Bassler, 1976). In legume seeds, each cultivar originated either from 1999, 2000, 2001 or 2002. For barley, three cultivars originated from 1998 and 1999 only. For triticale, three cultivars originated from 1999 and 2000 only. For rye, five cultivars originated from 1999 and 2000 only.
2. Materials and methods 2.1. Experiment 1 In total 113 samples of legume seeds (field beans, peas and lupins, n = 43), cereals (oats, wheat, barley, triticale and rye, n = 64) and cereal by-products (wheat bran and rye bran, n = 6) were collected at breeding companies, feed mills, farms and the State Plant Breeding Institute (University of Hohenheim, 70599 Stuttgart, Germany). The cereal samples included different cultivars harvested in 1998, 1999 or 2000 (Table 1). All samples were ground to pass a 1-mm mesh sieve using a rotor mill (Retsch® ZM 1, Retsch GmbH & Co. KG, 42781 Haan, Germany). Native phytase activity was measured in triplicate analyses using the direct incubation method as outlined by Greiner and Egli (2003). One unit of phytase activity is equivalent to the amount of enzyme that liberates 1 mol of orthophosphate from 100 mol of sodium phytate at 37 ◦ C and pH 5.0 per minute. Additionally, phytase activities in legume seeds were assayed by means of the extraction procedure as outlined by the Association of German Agricultural Experimental and Research Stations (Naumann and Bassler, 1976). According to this method, one unit of phytase activity is equivalent to the amount of enzyme that liberates 1 mol of orthophosphate from 5.1 mmol of sodium phytate at 37 ◦ C and pH 5.5 per minute. With the method by Greiner and Egli (2003) the total phytase activity is determined, whereas the extraction procedure by Naumann and Bassler (1976) determines the activity of the soluble phytases only. The contents of total and phytate P were analyzed by the vanadate-molybdate method (Naumann and Bassler, 1976) and the anion-exchange method as described by Harland and Oberleas (1986), respectively.
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2.2. Experiment 2 Three cultivars of wheat or rye harvested in 1999 were selected to investigate the effect of milling on the distribution of phytase activity, total P and phytate P in these grains. Fine flour type 405 and bran were produced from the grains using a precision laboratory roller mill (Brabender® Quadrumat® Junior, Brabender OHG, 47055 Duisburg, Germany). The samples were analyzed for total and phytate P by means of the procedures as described in Experiment 1 and the phytase activities were determined according to Greiner and Egli (2003). 2.3. Experiment 3 One representative cultivar out of each of the legume seeds was selected to study the effect of preservation with propionic acid on native phytase activities determined 0, 4, 8 and 12 weeks after organic acid treatment. The original samples (20 kg) were soaked for approximately 45 min in aqua bidest. to lower the original dry matter (DM) content (895 ± 13 g/kg) to values between 850 and 870 g/kg. At this DM level legume seeds are susceptible to microbial deterioration (UFOP, 2004). Propionic acid (Luprosil® , BASF AG, 67056 Ludwigshafen, Germany) was used as preservative, and sprayed on the intact samples according to manufacturer’s recommendations (6 g/kg, wt./wt.). After thoroughly mixing, three subsamples of 350 g each were taken from each legume seed and stored in PVC-containers at 20 ◦ C room temperature. Samples of approximately 100 g were taken after 4, 8 and 12 weeks of storage and rinsed with aqua bidest. to remove any remaining propionic acid. Subsequently, the legume seeds were freeze-dried, ground to pass a 1-mm mesh sieve and analyzed for phytase activity according to Greiner and Egli (2003). 2.4. Statistical analyses The experimental data were subjected to analyses of variance using the GLM procedure of SAS (1999). The model includes in Experiment 1 the main effects of harvest year and cultivar; in Experiment 2 the effects of different milling treatments (seed, flour, bran); and in Experiment 3 the effect of storage time (0, 4, 8, 12 weeks). Significant differences between means were determined using the PDIFF option with PROC GLM (SAS, 1999). Additionally, correlation coefficients and linear relationships between the components analyzed in Experiment 1 were calculated using the CORR and REG procedure of SAS (1999), respectively.
3. Results and discussion 3.1. Phytase activities in legume seeds, cereals and cereal by-products (Experiment 1) When analyzed according to the direct incubation method of Greiner and Egli (2003), the native phytase activities are lowest in legume seeds (262–324 U/kg DM) and oats
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Table 2 Phytase activity, total phosphorus and phytate phosphorus content in legume seeds, cereals and cereal by-productsa nb
Legume seeds Field beans Peas Lupins
11 18 14
Cereals Oats Wheat Barley Triticale Rye
6 18 15 12 13
Cereal by-products Wheat bran 3 Rye bran 3 a b
Phytase activity (U/kg)
290 ± 78 262 ± 73 324 ± 59 496 2886 2323 2799 6016
± ± ± ± ±
35 645 648 501 1578
9945 ± 0427 9241 ± 1452
Total P (g/kg)
5.7 ± 0.92 4.1 ± 0.46 5.7 ± 1.48 3.7 4.0 4.2 4.0 3.6
± ± ± ± ±
0.14 0.40 0.42 0.34 0.19
8.8 ± 0.71 5.8 ± 0.25
Phytate P g/kg
Proportion of total P
3.9 ± 0.75 2.4 ± 0.45 3.5 ± 0.92
0.70 ± 0.052 0.58 ± 0.060 0.63 ± 0.037
2.5 2.9 2.6 2.8 2.4
± ± ± ± ±
0.17 0.37 0.31 0.30 0.23
7.9 ± 0.05 4.9 ± 0.23
0.67 0.73 0.63 0.70 0.67
± ± ± ± ±
0.054 0.081 0.035 0.054 0.050
0.90 ± 0.073 0.85 ± 0.020
Means ± standard deviation (dry matter basis). Number of observations.
(496 U/kg DM), intermediate in wheat (2886 U/kg DM), barley (2323 U/kg DM), triticale (2799 U/kg DM) and rye (6016 U/kg DM) and highest in the by-products (9945 and 9241 U/kg DM in wheat and rye bran, respectively) (Table 2). In contrast to the findings of the present study, literature data show in general a larger variation in phytase activities. For example, for legume seeds values range between 0 U/kg (Eeckhout and De Paepe, 1994) and 258 U/kg (Viveros et al., 2000). It can be questioned if these data are representative due to the low number of observations in most of the experiments. In the study of Viveros et al. (2000), for example, only three samples of beans and six samples of peas and lupins, respectively, were analyzed. Moreover, the phytase activities reported by Eeckhout and De Paepe (1994) for field beans and lupins were based on one individual sample, whereas in the present study 11, 18 and 14 observations for field beans, peas and lupins originating from different cultivars, locations and harvest years were obtained (Table 1). The phytase activities reported herein for wheat, barley and rye agree with those presented by Greiner and Egli (2003), amounting to 2931, 2093 and 6752 U/kg, respectively. However, according to several reports considerably lower values were obtained. For example, as reported by Cossa et al. (2000) phytase activities in wheat were only 520–1390 U/kg. For wheat, barley and triticale published values vary between 88 U/kg (Liu et al., 1998) and 3120 U/kg (Singh and Sedeh, 1979). For oats, reported phytase activities range between 0 U/kg (Eeckhout and De Paepe, 1994) and 412 U/kg (Shen et al., 2005), which is lower as compared to the present investigation (496 U/kg DM). In agreement with the findings of the present study, activities in rye are in general higher than in other cereals. However, there is still a considerable variation between studies ranging from 3790 U/kg (Fretzdorff and Weipert, 1986) to 6840 U/kg (Zimmermann et al., 2002b). Since phytase is predominantly located in the aleurone layer and scutellum of cereal grains (Fretzdorff and Weipert, 1986),
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by-products such as wheat bran and rye bran generally show the highest activities among plant feedstuffs. However, literature values exhibit again a considerable variation between different studies ranging between 600 U/kg (Pointillart, 1988) and 7400 U/kg (Pointillart, 1993) for cereal brans. There is growing evidence that the analytical procedure used for the determination of phytase activity in plant feedstuffs may have a major impact on the values obtained. As previously shown by Zimmermann et al. (2002a) and Greiner and Egli (2003), phytase activities in cereals based on extraction methods (e.g. Engelen et al., 1994; Barrier-Guillot et al., 1996; Liu et al., 1998; Cossa et al., 2000; Kim et al., 2002; Zimmermann et al., 2002a; Selle et al., 2003) are considerably lower than those obtained by means of direct incubation methods. For example, in the study by Zimmermann et al. (2002a) phytase activities in wheat and rye determined according to an extraction procedure (Engelen et al., 1994; with minor modifications) were 70 and 48%, respectively, lower in comparison to the values obtained by means of a direct incubation method. These findings are confirmed by the results for phytase activities in legume seeds. In the present study, phytase activities for peas, field beans and lupins determined according to the direct incubation method (Greiner and Egli, 2003) averaged 262, 290 and 324 U/kg DM, respectively, whereas the corresponding values obtained by means of the extraction procedure of Naumann and Bassler (1976) were below the detection limit of 50 U/kg. According to Greiner and Egli (2003) the incomplete extraction of plant phytases might be attributed to proteolytic degradation and a partial association of the enzymes with membrane structures. Differences in the composition of the cell walls, interaction of phytases with morphological grain fractions and differences in the location of the enzymes within the grains are considered to be additional factors responsible for the incomplete and different extraction rates of plant phytases (Zimmermann et al., 2002a). Therefore, specification of the analysis method used for determination of plant phytase activities is mandatory in order to allow for a comparison of results between different studies. Due to high native phytase activities, cereals (except oats) and cereal by-products have the potential to contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals. Although the present study revealed considerably higher phytase activities in legume seeds than previous literature reports, the absolute values are rather low in comparison to cereals and their by-products. Therefore, the contribution of native phytases originating from legume seeds in terms of improving the availability of plant P for pigs and poultry seems to be of minor importance. 3.2. Contents of total P and phytate P in legume seeds, cereals and cereal by-products (Experiments 1 and 2) Among legume seeds, field beans and lupins show higher concentrations of total P (5.7 g/kg DM) and phytate P (3.5–3.9 g/kg DM) as compared to peas (4.1 and 2.4 g/kg DM, respectively). In cereals, the contents of total P range between 3.6 g/kg DM (rye) and 4.2 g/kg DM (barley), whereas phytate P contents range between 2.4 g/kg DM (rye) and 2.9 g/kg DM (wheat). The highest concentrations of total P and phytate P are found in wheat bran (8.8 and 7.9 g/kg DM, respectively), whereas rye bran contains only 5.8 and 4.9 g/kg DM total P and phytate P, respectively. These values are in the range of previously reported
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Table 3 Phytase activity, total phosphorus and phytate phosphorus content in cereals grains, flour and bran originating from wheat and ryea Treatment
Seed Flour Bran S.E.M.c
nb
6 6 6
Phytase activity (U/kg)
Total P (g/kg)
3639 b 2446 c 9593 a 377
3.9 b 1.1 c 7.3 a 0.14
Phytate P g/kg
Proportion of total P
2.6 b 0.5 c 6.4 a 0.06
0.66 b 0.44 c 0.87 a 0.018
Within a column, means followed by different letters (a, b, c) differ at P<0.05. a Least squares means (dry matter basis). b Number of observations. c S.E.M.: standard error of least squares means.
literature data (e.g. Lolas et al., 1976; De Boever et al., 1994; Eeckhout and De Paepe, 1994; Viveros et al., 2000; Shen et al., 2005). In the present study, on average, about two thirds (0.67 ± 0.088) of total P in legume seeds, cereals and by-products (n = 113) are bound to phytate. However, in cereal by-products the proportion of phytate P in relation to total P is higher (0.85–0.90) than in legume seeds and cereals (0.58–0.73), thus indicating an enrichment of phytate in brans. This is confirmed by the results obtained in Experiment 2, in which grains of wheat and rye were milled to flour and bran in order to determine phytase activities and concentrations of total P and phytate P in these fractions. Compared to the intact grains, the proportion of phytate P in relation to total P was higher (P<0.05) in wheat and rye bran (0.87 versus 0.66) (Table 3). On the other hand, total P and phytate P contents along with phytase activities were significantly (P<0.05) lower in the flours originating from wheat and rye (1.1 g/kg DM, 0.5 g/kg DM and 2446 U/kg DM, respectively) in comparison to both bran sources and intact grains. In agreement with previous studies by O’Dell et al. (1972), Singh and Reddy (1977), Fretzdorff and Weipert (1986) and Okot-Kotber et al. (2003), the results of the present study clearly confirm that phytase as well as total P and phytate P are predominantly associated with the outer layers of cereal grains. Extremely low phytate P concentrations, such as determined by Viveros et al. (2000) in field beans (0.8 g/kg DM) or by Eeckhout and De Paepe (1994) in lupins (0.5 g/kg DM), can be attributed to different methods used for the determination of phytate P. According to Selle et al. (2003) the application of the analysis method of Haug and Lantzsch (1983), which is based on the precipitation of phytate with an acidic iron (III) solution may have caused an underestimation of the actual phytate P concentrations in field beans and lupins in the aforementioned studies of Viveros et al. (2000) and Eeckhout and De Paepe (1994). Obviously, quantitative extraction of phytate in these feedstuffs was limited due to the strong binding of phytate to protein fractions. Selle et al. (2003) concluded, based on studies with field beans and lupins, that the anion-exchange column chromatography (Harland and Oberleas, 1986), which has been used in the present study, is more suitable for quantitative determination of phytate P.
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3.3. Influence of harvest year on phytase activities and contents of total P and phytate P in wheat and barley in Experiment 1 Other factors than the use of different analytical procedures for the determination of phytase activities and concentrations of total P and phytate P might be responsible for different results between studies. These factors include differences in cultivar (Miller et al., 1980a; Barrier-Guillot et al., 1996; Cossa et al., 2000), harvest year (Ockenden et al., 1997; Cossa et al., 2000), climatic conditions (Bassiri and Nahapetian, 1977; Kim et al., 2002), P content of the soil (Miller et al., 1980b; Saastamoinen, 1987) and growing location (Miller et al., 1980a; Cossa et al., 2000). Wheat and barley as major feed ingredients in diets for non-ruminant animals were selected as cereal grains to determine whether phytase activities and contents of total P and phytate P differed between harvest years and cultivars. In the present study, there was no significant influence (P>0.05) of harvest year (1998–2000) on phytase activities and total P concentrations in wheat and barley (Table 4), which is in agreement with reports by Ockenden et al. (1997) and Kim et al. (2002), whereas Cossa et al. (2000) obtained a significant influence of different harvest years (1995 versus 1996) on phytase activities in wheat samples. In the present study, however, there was a significant influence (P<0.001) of harvest year on total phytate P concentrations in wheat, which was confirmed as trend (P=0.059) in barley. For example, in wheat the highest phytate P contents were found in 2000 (3.2 g/kg DM), whereas in 1998 and 1999 phytate P concentrations amounted to 2.7 and 2.8 g/kg DM, respectively. Additionally, there was a tendency towards an influence of harvest year on phytate P concentrations, expressed as percentage of total P in wheat (P=0.057). Moreover, there was a tendency towards an effect of harvest year on total P contents in barley (P=0.074). The findings of the present study are confirmed by Cossa et al. (2000), who observed a significant influence of different harvest years (1995 versus 1996) on phytate P concentrations in wheat as well. Numerical, but not significant (P=0.08) effects of different harvest years (1999 versus 2000) on phytate P contents in wheat were reported by Kim et al. (2002). Furthermore, Ockenden et al. (1997) reported in barley samples a significant effect of different harvest years (1984–1994) on phytate P concentrations expressed as percentage of total P. 3.4. Influence of different cultivars of wheat and barley on phytase activities and contents of total P and phytate P in Experiment 1 The samples used in the present experiment represent six and three different cultivars of wheat and barley, respectively. In barley, phytase activity was significantly (P=0.023) affected by cultivar (Table 4). Among the barley cultivars, phytase activities range between 1665 U/kg DM (cv. Hanna) and 2857 U/kg DM (cv. Henni). Previously, the effect of cultivar on phytase activities in barley has not yet been studied. In the present study, there was no significant (P=0.107) influence of cultivar on phytase activities in wheat, which is in contrast to Barrier-Guillot et al. (1996), Cossa et al. (2000) and Kim et al. (2002). The total P and phytate P contents differ significantly (P<0.05) among the six different wheat cultivars (Table 4). Values for total P in wheat range between 3.5 g/kg DM (cv.
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Table 4 Influence of harvest year and cultivar on phytase activity, total phosphorus and phytate phosphorus content in wheat and barleya nb
Wheat Harvest year 1998 1999 2000 S.E.M.c P value Cultivar Bussard Estica Flair Toronto Transit Triso S.E.M.c P value Barley Harvest year 1998 1999 2000 S.E.M.c P value Cultivar Barke Hanna Henni S.E.M.c P value
Phytase activity (U/kg)
Total P (g/kg)
Phytate P g/kg
Proportion of total P
6 6 6
2854 2748 3057 225 0.628
3.8 4.1 4.0 0.11 0.137
2.7 b 2.8 b 3.2 a 0.05 <0.001
0.71 0.69 0.79 0.027 0.057
3 3 3 3 3 3
1956 3186 3106 3371 2881 2818 319 0.107
4.0 abc 3.5 c 3.8 bc 4.3 ab 4.0 ab 4.4 a 0.16 0.033
3.1 b 2.6 c 2.5 c 2.9 b 3.0 b 3.4 a 0.07 <0.001
0.78 0.75 0.66 0.68 0.74 0.77 0.038 0.250
3 3 3
2139 2128 2391 180 0.556
3.7 4.4 4.0 0.17 0.074
2.2 2.8 2.6 0.13 0.059
0.60 0.64 0.65 0.013 0.124
3 3 3
2136 b 1665 b 2857 a 180 0.023
4.1 3.8 4.2 0.17 0.460
2.6 2.4 2.7 0.13 0.303
0.64 0.61 0.64 0.013 0.333
For harvest years and cultivars, means within a column followed by different letters (a, b, c) differ at P<0.05. a Least squares means (dry matter basis). b Number of observations. c S.E.M.: standard error of least squares means.
Estica) and 4.4 g/kg DM (cv. Triso), and for phytate P between 2.5 g/kg DM (cv. Flair) and 3.4 g/kg DM (cv. Triso). In contrast, there was no effect (P>0.05) of cultivar on total P or phytate P concentrations in barley samples. Reports pertaining to the effect of cultivar on total P or phytate P contents in cereal grains are equivocal. In wheat samples, total P and phytate P levels were not significantly different between cultivars (Barrier-Guillot et al., 1996; Kim et al., 2002). However, Singh and Reddy (1977) and Feil and Fossati (1997) reported significant differences in the phytate content in different cultivars of triticale. Explanations for the conflicting findings obtained in the above-mentioned studies remain rather speculative. It may be hypothesized that in the different studies possible effects
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of harvest year and cultivar on phytase activity, total P and phytate P might have been confounded by various other factors such as analytical methods, growing location, degree of fertilization, climatic conditions or interactions between these factors. In the studies by Barrier-Guillot et al. (1996), Cossa et al. (2000) and Kim et al. (2002), for example, phytase activities were determined using an extraction procedure instead of a direct incubation method. Furthermore, Kim et al. (2002) assumed that environmental conditions have in general a higher impact on the chemical composition of grains than genetic influences. Likewise, Ockenden et al. (1997) indicated, for example, that the accumulation of total P and phytate P in barley may be influenced by temperature and precipitation level. It is well known that such environmental factors may vary considerably between different harvest years. It has to be emphasized that the cereal grains used in the present study were grown in the same geographic area (south-western Germany); however, possible effects of environmental factors such as growing location, degree of fertilization and climatic conditions on the results reported herein cannot be excluded. 3.5. Relationship between components analyzed in Experiment 1 Phytase activity was not significantly (P>0.05) correlated with total P or phytate P concentrations in any of the feedstuffs under investigation. In agreement with the conclusions drawn by Eeckhout and De Paepe (1994), Barrier-Guillot et al. (1996) and Viveros et al. (2000), native phytase activities in plant feedstuffs cannot be predicted from their total P or phytate P contents. It is assumed that measurements of in vitro phytase activities may not necessarily reflect the potential for phytate hydrolysis under in vivo conditions since intestinal and dietary factors such as pH and transit time may affect the degree of phytate degradation. A positive correlation and a significant linear relationship between phytate P and total P contents could be established for legume seeds (r = 0.95, P<0.001, Fig. 1), cereals (r = 0.66, P<0.001, Fig. 2) and cereal by-products (r = 0.96, P=0.002) as well. The regression equations calculated for legume seeds, cereals and cereal by-products are listed in Table 5. Significant correlations between total P and phytate P have been reported previously. In agreement with the present study, Eeckhout and De Paepe (1994) and Viveros et al. (2000) determined higher correlations between phytate P and total P in legume seeds as compared to cereals. In field beans (r = 0.92, P<0.001), peas (r = 0.92, P<0.001) and lupins
Fig. 1. Linear relationship between phytate P and total P content in legume seeds (n = 43).
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Fig. 2. Linear relationship between phytate P and total P in cereals (n = 64).
(r = 0.97, P<0.001), phytate P and total P contents are highly correlated, indicating that total P values can be used to predict the phytate P contents in legume seeds. In contrast, among cereals the relationships are comparatively low for wheat (r = 0.63, P=0.005), triticale (r = 0.63, P=0.028) and rye (r = 0.62, P=0.024), whereas for barley the correlation coefficient amounts to 0.86 (P<0.001). For oats, wheat bran and rye bran the correlation is not significant (P>0.05), presumably, because of the small number of samples analyzed (n = 6 for oats, n = 3 for wheat or rye bran, respectively). The correlation coefficients determined in the present study for wheat (r = 0.63) and barley (r = 0.86) show good agreement with literature values reported by Barrier-Guillot et al. (1996) and Ockenden et al. (1997) (r = 0.56 and 0.89, respectively). However, in contrast to these reports and the present study, Lolas et al. (1976) and Selle et al. (2003) found higher correlation coefficients (0.91–0.96) between phytate P and total P concentrations for samples of oats and wheat. Obviously, there is a closer relationship between phytate P and total P in feedstuffs with higher total P concentrations (cereal by-products, field beans, peas, lupins, barley) as compared to those with lower total P contents (wheat, triticale, rye) in the present study. Table 5 Linear relationship between phytate P (y) and total P (x) contents in legume seeds, cereals and their by-products Feedstuff
na
Regression equation
R2
P value
R.S.D.b
All feedstuffs
113
y = 0.81 ± 0.034x − 0.63 ± 0.161
0.83
<0.001
0.449
Legume seeds Field beans Peas Lupins
43 11 18 14
y = 0.73 ± 0.039x − 0.50 ± 0.203 y = 0.75 ± 0.108x − 0.31 ± 0.618 y = 0.88 ± 0.096x − 1.23 ± 0.399 y = 0.60 ± 0.040x + 0.09 ± 0.235
0.89 0.84 0.84 0.95
<0.001 <0.001 <0.001 <0.001
0.318 0.315 0.184 0.214
Cereals Oats Wheat Barley Triticale Rye
64 6 18 15 12 13
y = 0.58 ± 0.084x + 0.38 ± 0.333 y = −0.09 ± 0.612x + 2.80 ± 2.270 y = 0.58 ± 0.180x + 0.57 ± 0.723 y = 0.63 ± 0.104x − 0.01 ± 0.040 y = 0.56 ± 0.216x + 0.57 ± 0.864 y = 0.76 ± 0.291x − 0.33 ± 1.046
0.44 0.01 0.40 0.74 0.40 0.38
<0.001 0.888 0.005 <0.001 0.028 0.024
0.263 0.190 0.295 0.163 0.242 0.187
By-products
6
y = 0.91 ± 0.130x − 0.27 ± 0.967
0.93
0.002
0.503
a b
Number of observations. R.S.D.: residual standard deviation.
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Fig. 3. Effect of propionic acid on phytase activities in intact field beans, peas and lupins as influenced by different storage times. Data are least squares means and standard error of three observations per treatment. For each legume seed, means with different letters (a, b, c) differ at P<0.05.
The reasons for the inconsistent correlation coefficients reported in different studies have not yet been fully elucidated. However, the results of the present study indicate that the relationship between phytate P and total P contents is stronger in legume seeds (Fig. 1) and cereal by-products than in cereals (Fig. 2). As indicated by Raboy (1997), phytate P accumulation during the seed development functions to maintain a constant level of nonphytate P. However, until now the mechanisms involved in the homeostatic regulation of phytic acid synthesis have not been discovered sufficiently. 3.6. Influence of preservation with propionic acid on native phytase activities in legume seeds (Experiment 3) In order to avoid microbial degradation of legume seeds through bacteria, yeasts and fungi during storage, it is mandatory to preserve these feedstuffs if the moisture content of the grains exceeds 120 g/kg (UFOP, 2004). In practice, the preservation of legume seeds by means of organic acids receives growing attention. The antimicrobial effect of organic acid supplementation in feedstuffs can be attributed to a reduction in pH. The influence of preservation with propionic acid on phytase activity in intact legume seeds during the storage over a period of 12 weeks is presented in Fig. 3. The initial phytase activities in the cultivars of field beans (cv. Music), peas (cv. Lido) and lupins (cv. Borsaja) amounted to 455, 356 and 350 U/kg DM, respectively, at the beginning of the experiment. These values were close to or above the average values obtained for each of the legume seeds (Table 2). Albeit of small magnitude, the decrease in phytase activity over a period of 12 weeks is significant (P<0.05) due to an extremely low variation within each treatment under the standardized laboratory conditions as described herein. After 12 weeks of storage, the decline in phytase activities amounted to 4.1, 6.1 and 25.5% in field beans, peas and lupins, respectively. As shown in in vitro studies, the activity of purified plant phytase is highly influenced by pH conditions (Phillippy, 1999; Greiner et al., 2001; Greiner, 2002). It was therefore investigated if Luprosil® , which shows a pH value of 2.3 only, has a
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detrimental impact on phytase activities in legume seeds. However, the results of the present study show that propionic acid has no major effect on native phytase activity in material which has not been ground before treatment with organic acids. Presumably, phytases are protected against irreversible denaturizing by acid molecules in the seed matrix. This is supported by Greiner and Konietzny (1998) who concluded from studies with Phaseolus beans that the seed matrix might have a stabilizing effect on phytase. Because none of the treatments distinctly decreased the native phytase activity, conservation with propionic acid represents a suitable procedure to preserve moist legume seeds after harvest for at least 12 weeks. In conclusion, cereals (except oats) and cereal by-products may contribute substantially to the gastrointestinal hydrolysis of phytate in non-ruminant animals due to high native phytase activities, whereas the contribution of legume seeds in terms of improving the availability of plant P seems to be almost negligible. In wheat, there is a significant influence of harvest year on the phytate P concentrations. Additionally, total P and phytate P contents differ between different cultivars of wheat. Moreover, phytase activities differ between different cultivars of barley. Finally, preservation with propionic acid results in a marginal decrease in phytase activities in legume seeds only.
Acknowledgements This work was financially supported by UFOP (Union zur F¨orderung von Oel- und Proteinpflanzen e.V., Berlin, Germany). The authors would like to thank Edith Haller, Jasmin Sarikol (Federal Research Centre for Nutrition and Food, Karlsruhe, Germany) and BASF AG (Ludwigshafen, Germany) for analysis of phytase activity and Helga Ott and Melanie Berger (University of Hohenheim, Stuttgart, Germany) for analysis of total P contents.
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