Synergic effect of different phytase preparations in feed

143 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-65 Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 143-152 This paper is available online at SYNERGIC EFFECT OF DIFFERENT PHYTASE PREPARATIONS IN FEED Tran Thi Thuy and Dao Thi Nu Faculty of Biology, Hanoi National University of Education Abstract: Phytase is an important feed enzyme which is used to remove phytate, the main phosphate storage and also an anti nutrient factor (ANF) of grains in feedstuff. Commercial phytases in the feed market now are the histidine acid phytases which perform well their activity at low pH in stomach of monogastric animals. This study showed a potential of combining a histidine acid phytase (OptiPhos®) and an alkaline phytase (BacP) which performs activity at neutral and slightly alkaline condition of small intestine. Synergic activity of these two phytases (combination of 1:1 between OptiPhos® and BacP phytase) showed wider pH optimum (pH 4.5 - 7.5) of phytase activity. In comparison to both single phytases, this combination has improved the hydrolytic catalysis of phytate in rice bran, corn and soybean powder; resulted in higher amount of inorganic phosphate released in two phases – in vitro digestion. Further studies of this combination on monogastric animals shall be carried on to confirm the synergic effect of these two phytases. Keywords: alkaline phytase, histidine acid phytase, feedstuff, in vitro digestion, synergic effect. 1. Introduction Phytase is an important feed enzyme which catalyzes the hydrolysis of phytate, a salt form of phytic acid present in plant material, to less phosphorylated myo-inositol derivatives with concomitant release of inorganic phosphate [1]. Phytate (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate; IP6) is the main phosphate storage in legume and many kinds of cereals which contain 1-5% (w/w) dry weight of phytate. It has been considered as an anti-nutrient factor in feed due to the high affinity to essential minerals, proteins and polysaccharides in gastro intestinal tract of monogatric animals [2]. Supplementation of phytase to the feed will increase the bioavailability of not only inorganic phosphate but also minerals, proteins and some polysaccharides for feed during the digestion time [3]. It also reduces or eliminates the supplementation of inorganic phosphate to the feed contributing to lower the pollution of phosphate in surface and ground water due to the Received October 31, 2017. Revised December 7, 2017. Accepted December 14, 2017. Contact: Tran Thi Thuy, e-mail address: thuy_tt@hnue.edu.vn Synergic effect of different phytase preparations in feed 144 high concentration of manure at the intensive breeding areas [2, 4]. Available phytase preparations in the feed market now are the histidine acid phytases (HAP) derived from Aspegillus niger or Escherichia coli. These phytases are active at low pH from 3.5 to 6.0 therefore they work at acidic condition as in stomach of monogatric animals [1]. Phytases working at neutral and alkaline condition are from Bacillus sp. and pollen of Thypha lattifolia and Lilium longiflorum [5, 6]. These phytases are active at pH 6.0 - 8.0 as in the small intestine of monogastric animals; they have also been well studied on structure and characterization. Combination of these two enzyme groups in feed may lead to increase the active time of phytase in gastrointestinal tract of livestock. Elkhalil et al., 2007, has reported the in vivo effect of phytases derived from Aspergillus, Bacillus, E. coli and Klebsiella in the utilisation of phosphorus of broiler chickens and reported that they were equally effective in improving P utilisation regardless of their proposed intestinal site of activity. They also proposed that the combination of phytases acting in the gizzard with phytases acting in the intestine may be a promising way to further improving in vivo efficacy of phytases in poultry [7]. In this study, in vitro effect of two phytases preparation, an E. coli-derived phytases (OptiPhos® from JBS United, Inc.) and a Bacilli phytase (BacP, our in-house preparation) were evaluated separately and in combination in order to from a suitable proportion to combine them in vivo. 2. Content 2.1. Materials and methods * Materials - OptiPhos® 2500G (granulated), an E. coli-derived phytase [8], was purchased from JBS United, Inc. USA. - BacP, a Bacilli phytase, was prepared in our laboratory from culture broth of recombinant E. coli BL21 carrying phytase gene from Bacillus subtilis [9]. - Chemicals used in all experiments are analytical grade from Merck, Prolabo and Schalau. * Methods Phytase activity The phytase activity was assayed according to the method described by Shimizu [10]. Phytase preparation (BacP) was diluted in 0.1 M Tris-HCl buffer, pH 7 containing 5 mM CaCl2 or 0.1 M acetate buffer pH 5.5 containing 5 mM CaCl2 (for OptiPhos®), and mix with 1.5 mM sodium phytate in the same buffer. Mixture was incubated for 20 minutes at 50C for OptiPhos® phytase or at 70C for BacP phytase. An equal volume of 15% trichloroacetic acid (TCA) solution was added to stop the reaction. Clear supernatant (100 µL) after centrifugation was transferred to a 96 well plate. The same amount of ammonium molybdate solution (containing 1:4 mixtures of 2.7% FeSO4 and 1.5% ammonium molybdate in 5.5% H2SO4) was added to each well and incubated for 5 min at room temperature. After that, absorbance at 700 nm was read and used to calculate the Tran Thi Thuy and Dao Thi Nu 145 concentration of Pi from a standard curve which was made by sodium dihydrogen phosphate solution in the concentration range of 0 - 600 nmol/mL Pi. The amount of enzyme that releases 1 µmole of Pi per minute under the assay conditions was defined as one unit of phytase activity. All the samples were assayed in triplicates. Protein electrophoresis The purity of the phytase preparations (OptiPhos® and BacP) was checked on 12.5 % (w/v) polyacrylamide gel by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Two phases - in vitro digestion Rice bran, corn powder, soybean powder and mixture of these three substrates were used as feedstuff in two phases - in vitro digestion [11]. These feedstuffs were dry heated at 90C for two hours in order to denature all enzymes from grains and mimic the heating temperature during feed pelleting. In each test, 0.1 g of feedstuff was used, except sodium phytate (using 0.3 mL of 1.67 mM solution). Triplicates were done for all tests. At the stomach phase, feedstuff (0.1 g) or 0.3 mL of 1.67 mM sodium phytate was added to each test tube and then 3 mL of 0.1M HCl containing 2000 IU/mL of pepsin was added. Phytase preparation (OptiPhos® or BacP or combination of 1 OptiPhos®: 1 BacP based on phytase activity) was added to each test tube to reach 0.5 U/mL. All the mixtures were incubated at 40 C for 45 minutes, periodically mixed to imitate the movement of stomach. Samples (0.3 mL) were taken before adding phytase preparation and at the end of stomach phase for Pi analysis. At the intestine phase, one milliliter of 1 M NaHCO3 containing pancreatin (2 mg/mL) was added to each of above test tube. They then were incubated at 40C for two more hours, periodically mixed to imitate the movement of intestinal tract. At the end of intestine phase (after 165 minutes of the reaction), a sample of 0.3 mL were taken for Pi analysis. 2.2. Result and discussion 2.2.1. Biophysical and Biochemical properties of OptiPhos® and BacP phytase Feed enzymes are usually crude extracts from fermentation broth of producing microorganisms or preliminary purified preparations. The purity of two feed enzyme preparations (OptiPhos® and BacP) has been checked on SDS-PAGE (Figure 1); their molecular weight, temperature and pH for optimal activity were reported in Table 1. Result from SDS-PAGE showed the purity of BacP was higher than OptiPhos® (Figure 1) correspond to higher specific activity of BacP compared to OptiPhos®. Therefore both of these two phytases are preliminary purified preparation. Molecular weight of OptiPhos® is 53 kDa and BacP is 47.5 kDa (Table 1) which are similar to other phytase enzymes from bacteria except the one from Klebsiella aerogenes [11]. BacP is a thermostable phytase; therefore, its thermo optimal activity was higher (70 - 75ºC) than OptiPhos®’s (50 - 55ºC). This result is similar to previous report on alkaline phytases from Bacillus spp [1, 9]. Optimum pH of BacP is at neutral environment (pH 7.0) when OptiPhos® prefers to work at acidic condition (pH 5.5) as the Synergic effect of different phytase preparations in feed 146 recommendation from the manufacturer [8]. Most of histidine acid phytases show their optimal activity at pH 4.5 - 6.0 that is the pH of gizzard or upper part of stomach [1, 13]. Apart from that, alkaline phytases were reported to work well at the lower part of intestine. Table 1. Some biophysical and biochemical properties of phytase preparations Figure 2 shows the effect of different pH values (from 2.0 to 9.0) to the activity and stability of OptiPhos® (A) and BacP (B). Base on that, OptiPhos® showed good activity (more than 50% of its highest activity) at pH 4.0 - 8.0; this phytase enzyme acts well (showed more than 90% of its highest activity) at pH 5.0 - 6.0; out of that pH range, phytase activity was reduced significantly (Figure 2A). OptiPhos® phytase was quite stable at acidic environment, it could remain more than 80% activity after 2 hours treatment at pH 4.0 - 7.0; however, it was rapidly losing activity at pH above 7.0 (Figure 2A). Figure 2. Activity and stability of phytase preparations at different pH (A): OptiPhos®; (B): BacP Properties OptiPhos® BacP Specific activity (U/mg) 3.5 6.4 Optimum temperature (ºC) 50 - 55 70 - 75 Optimum pH 5.5 7 Molecular weight (kDa) 53 47.5 Figure 1. Molecular weight of phytase preparations (Lane 1: Biorad- all blue protein ladder; lane 2: BacP; and lane 3: OptiPhos®) Tran Thi Thuy and Dao Thi Nu 147 BacP phytase worked well (showed more than 50% its highest activity) at pH 4.0 - 8.5; highest activity (more than 90% of the highest activity) was seen at pH 6.0 - 7.5; at pH lower than 4.0 and higher than 8.5, its phytase activity reduced rapidly (Figure 2B). Figure 2B also shows a good stability of BacP phytase remaining more than 80% of its residual activity at neutral and alkaline environment (pH 5.5 - 9.0); at acidic environment (pH lower than 5.5), residual activity reduce gradually and remain less than 50% when pH was lower than 3.0. 2.2.2. Activity of OptiPhos® and BacP phytase in two phases - in vitro digestion In order to estimate the activity of two tested phytase preparations in monogastric animal, two phases - in vitro digestion of sodium phytate has been performed with these two phytase preparations (Table 2). The pH value of stomach phase was between 4.0 and 4.5, and pH of intestine phase was about 7.0 - 8.0. Results showed that inorganic phosphate released by OptiPhos® phytase (2.02 ± 0.11 µmol/mL) was higher than that of BacP phytase (1.65 ± 0.06 µmol/mL) at stomach phase. At intestine phase, BacP phytase was more active than OptiPhos®; it catalysed the hydrolysis of sodium phytate and released 2.4 ± 0.27 µmol/mL of inorganic phosphate, while OptiPhos® phytase catalysed a releasing of 2.14 ± 0.19 µmol/mL. These correlates well to the above studies on activity and stability of these two phytases at different pH values. Some activity of these two phytases might be lost due to the hydrolytic catalysis of pepsin in the stomach phase or pancreatin in the intestine phase. Simon and Igbasan (2002) have reported that alkaline phytases from B. subtilis were more susceptible to pepsin than histidine phytases from E. coli were [14]. Table 2. Activity of OptiPhos® and BacP phytase in two phases - in vitro digestion of sodium phytate Enzyme Total inorganic phosphate, Pi, released (µmol/mL) Pi released from each phase Starting (a) Stomach phase (b) Intestine phase (c) (b) - (a) (c) - (b) (c) - (a) OptiPhos® 0.08 ± 0.01 2.02 ± 0.21 4.16 ± 0.33 2.02 ± 0.11 2.14 ± 0.27 4.16 ± 0.19 BacP 0.09 ± 0.01 1.65 ± 0.11 4.05 ± 0.27 1.65 ± 0.06 2.4 ± 0.19 4.05 ± 0.13 Note: Total activity of each test was the same and equal to 0.5 U/mL Three common cereal-base ingredients of feedstuff (rice bran, corn powder and soybean powder) were used as the substrate for OptiPhos® and BacP phytase in two phases - in vitro digestion (Table 3). Comparing to sodium phytate, these starting materials already contain some phosphate and during the two phases - in vitro digestion, some endogenous enzymes from these grains did catalyse the hydrolysis of phytate from these materials to release inorganic phosphates; however, these enzyme activities were very low, the highest one is from rice bran (Table 3). Supplement of OptiPhos® and BacP phytase has made significant improvement in the concentration of phosphate released. Synergic effect of different phytase preparations in feed 148 Table 3. Activity of OptiPhos® and BacP phytase in two phases - in vitro digestion of some common feedstuffs Substrate + enzyme Total inorganic phosphate, Pi, released (µmol/mL) Pi released from each phase Starting (a) Stomach phase (b) Intestine phase (c) (b) – (a) (c) – (b) (c) – (a) Rice bran 3.51 ± 0.31 4.1 ± 0.39 4.53 ± 0.23 0.56 ± 0.35 0.47 ± 0.31 1.02 ± 0.33 Corn powder 1.78 ± 0.14 2.23 ± 0.19 2.47 ± 0.27 0.46 ± 0.17 0.24 ± 0.23 0.7 ± 0.20 Soybean powder 2.17 ± 0.11 2.46 ± 0.16 2.89 ± 0.31 0.29 ± 0.14 0.435 ± 0.24 0.72 ± 0.19 Rice bran + OptiPhos® 3.51 ± 0.21 9.66 ± 0.47 13.64 ± 0.51 6.15 ± 0.34 3.98 ± 0.49 10.13 ± 0.42 Rice bran + BacP 3.51 ± 0.21 6.76 ± 0.30 11.92 ± 0.71 3.25 ± 0.26 5.16 ± 0.51 8.41 ± 0.39 Corn powder + OptiPhos® 1.77 ± 0.21 6.18 ± 0.41 9.33 ± 0.37 4.41 ± 0.31 3.15 ± 0.39 7.56 ± 0.35 Corn powder + BacP 1.77 ± 0.21 4.94 ± 0.41 9.86 ± 0.42 3.17 ± 0.31 4.92 ± 0.42 8.09 ± 0.37 Soybean powder + OptiPhos® 2.19 ± 0.23 6.5 ± 0.39 10.03 ± 0.51 4.31 ± 0.31 3.53 ± 0.45 7.84 ± 0.38 Soybean powder + BacP 2.19 ± 0.23 4.77 ± 0.31 9.66 ± 0.39 2.58 ± 0.26 4.89 ± 0.35 7.47 ± 0.31 Note: Total activity of each test was the same and equal to 0.5 U/mL In the condition of low pH, pepsin available at stomach phase and pancreatin available at intestine phase, OptiPhos® and BacP phytase could still show their activity in different feedstuff ingredients. The high content of phosphate did not affect much to phytase activity of these preparations; therefore, both of them worked well with three tested feedstuffs (Table 3). However, they worked better with rice bran (released from 8.41 ± 0.39 to 10.13 ± 0.42 µmol/mL of inorganic phosphate). Table 3 also showed higher activity of OptiPhos® at stomach phase compared to BacP phytase, while BacP worked better at intestine phase: the highest Pi release by the action of OptiPhos® phytase was in rice bran after 45 minutes of stomach phase (6.15 ± 0.34 µmol/mL), and by BacP phytase was also in rice bran after two hours of intestine phase (5.16 ± 0.51 µmol/mL). These results showed the possibility of combining these two phytase preparations which could improve the total effect of phytase in feedstuff. 2.2.3. Synergic activity of OptiPhos® and BacP phytase Phytases have a huge application in feedstuff; therefore, a phytase which is active at wide range of pH and can perform their catalysis activity along digestive tract from the upper part of stomach to the lower part of intestinal tract would be a preference [7]. However, there is no nature phytase can work at such wide pH range (from very low pH in stomach to slightly high pH in small intestine). The combination of various phytases Tran Thi Thuy and Dao Thi Nu 149 which are active at different pH optimum cold be a possible choice. In this experiment, OptiPhos® (optimum pH is 5.5) and BacP phytase (optimum pH is 7.0) was combined in different proportions based on their activities in order to screen for a best combination for feed additive. Table 4. Activity of the combinations between OptiPhos® and BacP phytase in two phases - in vitro digestion of sodium phytate Proportions of combination (OptiPhos®:BacP) Total inorganic phosphate, Pi, released (µmol/mL) Pi released from each phase Starting (a) Stomach phase (b) Intestine phase (c) (b) - (a) (c) - (b) (c) - (a) 1:1 0.08 ± 0.01 1.89 ± 0.11 4.7 ± 0.25 1.89 ± 0.06 2.81 ± 0.18 4.70 ± 0.12 9:1 0.09 ± 0.01 2.35 ± 0.17 4.62 ± 0.21 2.35 ± 0.09 2.27 ± 0.19 4.62 ± 0.14 1:9 0.09 ± 0.01 1.93 ± 0.11 4.31 ± 0.31 1.93 ± 0.06 2.38 ± 0.22 4.31 ± 0.14 7:3 0.08 ± 0.01 2.18 ± 0.12 4.61 ± 0.29 2.18 ± 0.07 2.43 ± 0.21 4.61 ± 0.14 3:7 0.07 ± 0.01 1.82 ± 0.07 4.36 ± 0.33 1.82 ± 0.04 2.54 ± 0.20 4.36 ± 0.12 Note: Total activity of each combination was the same and equal to 0.5 U/mL At the end of two phases - in vitro digestion, the amount of phosphate released from sodium phytate by the action of OptiPhos® phytase was 4.16 ± 0.19 µmol/mL, and by the action of BacP phytase was 4.05 ± 0.13 µmol/mL (Table 2). Results from Table 4 proved the combination of these two phytase has improved significantly the total phosphate released due to the synergic activity of OptiPhos® and BacP phytase in two phases - in vitro digestion of sodium phytate. The highest amount of phosphate released (4.61 - 4.70 µmol/mL) was from the combinations of 1:1, 9:1 and 7:3 between OptiPhos® and BacP phytase (Table 4). These combinations have also been checked in some common feedstuffs (Table 5). Using the same amount of phytase unit (0.5 U/mL), the combinations of 1:1, 1:9, 9:1, 3:7 and 7:3 between OptiPhos® and BacP phytase did not showed significant differences (P ≤ 0.1) in rice bran (Table 5) although all of these combinations worked better in rice bran than they did in soybean and corn powder. The combinations of 1:1 and 9:1 between OptiPhos® and BacP phytase showed better activity in soybean and corn powder (Table 5). These combinations could catalyse and release 5.59 - 5.76 µmol/mL of inorganic phosphate after two phases - in vitro digestion of corn powder, and 6.68 - 6.94 µmol/mL of inorganic phosphate after two phases - in vitro digestion of soybean powder (Table 5). Synergic effect of different phytase preparations in feed 150 Table 5. Activity of the combinations between OptiPhos® and BacP phytase in two phases - in vitro digestion of some common feedstuffs Substrate Phytase combinations of OptiPhos® and BacP Total inorganic phosphate, Pi, released (µmol/mL) Pi released from each phase Starting (a) Stomach phase (b) Intestine phase (c) (b) – (a) (c) – (b) (c) – (a) Rice bran 1:1 3.51 ± 0.61 8.98 ± 0.55 14.79 ± 0.73 5.47 ± 0.58 5.81 ± 0.64 11.28 ± 0.61 9:1 3.51 ± 0.61 8.86 ± 0.44 14.51 ± 0.81 5.35 ± 0.53 5.65 ± 0.63 11.00 ± 0.58 1:9 3.51 ± 0.61 6.87 ± 0.54 13.03 ± 0.45 3.36 ± 0.58 6.16 ± 0.50 9.52 ± 0.54 7:3 3.51 ± 0.61 8.04 ± 0.67 13.59 ± 0.76 4.53 ± 0.64 5.55 ± 0.72 10.08 ± 0.68 3:7 3.51 ± 0.61 7.27 ± 0.37 13.52 ± 0.32 3.76 ± 0.49 6.25 ± 0.35 10.01 ± 0.42 Corn powder 1:1 1.77 ± 0.11 4.21 ± 0.25 7.53 ± 0.39 2.44 ± 0.18 3.32 ± 0.32 5.76 ± 0.25 9:1 1.77 ± 0.11 4.63 ± 0.33 7.36 ± 0.23 2.86 ± 0.22 2.73 ± 0.28 5.59 ± 0.25 1: 9 1.77 ± 0.11 4.2 ± 0.19 6.78 ± 0.31 2.43 ± 0.15 2.58 ± 0.25 5.11 ± 0.20 7: 3 1.77 ± 0.11 4.46 ± 0.27 6.99 ± 0.35 2.68 ± 0.19 2.53 ± 0.31 5.21 ± 0.25 3: 7 1.77 ± 0.11 4.36 ± 0.32 6.66 ± 0.35 2.58 ± 0.22 2.3 ± 0.34 4.88 ± 0.28 Soybean powder 1: 1 2.17 ± 0.15 5.12 ± 0.43 9.11 ± 0.43 2.95 ± 0.29 3.99 ± 0.43 6.94 ± 0.36 9: 1 2.17 ± 0.15 5.76 ± 0.27 8.84 ± 0.39 3.6 ± 0.21 3.08 ± 0.33 6.68 ± 0.27 1: 9 2.17 ± 0.15 5.48 ± 0.23 7.87 ± 0.42 3.31 ± 0.19 2.39 ± 0.33 5.7 ± 0.26 7: 3 2.17 ± 0.15 5.98 ± 0.22 8.01 ± 0.55 3.81 ± 0.12 2.03 ± 0.39 5.84 ± 0.26 3: 7 2.17 ± 0.15 5.69 ± 0.31 8.16 ± 0.61 3.52 ± 0.23 2.47 ± 0.46 5.99 ± 0.35 Note: Total activity of each test was the same and equal to 0.5 U/mL Base on these above results we decided to choose the combination of 1:1 between OptiPhos® and BacP phytase for further studies. Optimal activity of this phytase combination (1 OptiPhos® : 1 BacP) was in the range of pH 4.5 - 7.5 (Figure 3) which was wider than the optimal pH for activity of both OptiPhos® (pH 5.0 - 6.0) and BacP (pH 6.0 - 7.5). This wider pH optimum explained the better activity of phytase combination in two phases – in vitro digestion of sodium phytate (Table 4) and different types of feedstuff (Table 5). Tran Thi Thuy and Dao Thi Nu 151 Figure 3. Activity of phytase combination (OptiPhos® and BacP phytase 1:1) at different pH values 3. Conclusion Using phytase enzyme as a feed additive has been an important strategy in feed processing to remove phytate, the main phosphate storage and also an anti nutrient factor (ANF) of grains in feedstuff. This study showed a potential of combining OptiPhos® and BacP in one phytase preparation to add in feedstuff. 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