Browsing by Subject "Phytatabbau"

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Entwicklung und ernährungsphysiologische Bewertung mikrobieller Hybrid-Phytasen
(2023) Metten, Alexander; Rodehutscord, Markus
To degrade the organic phosphate storage in the best possible way, it is necessary to increase phytase efficiency in vivo. Both a better understanding of the influencing factors limiting phytate degradation in vivo and a continuous improvement of the biochemical properties of phytases to be best adapted to the conditions in the digestive tract of non-ruminants will help to achieve this. Therefore, the main objective of this work was the generation of a large number of sequentially unique hybrid phytases by directed recombination of known phytase genes with the goal to achieve improved biochemical properties compared to the wild-type phytases used. The focus of this work was the biochemical and nutritional evaluation of the newly generated hybrid phytases with respect to their suitability as feed supplements. All hybrid phytases examined showed more efficient InsP6 degradation at pH 3.0 than at pH 5.5, although the phytase activity supplemented was the same at both pH values. While InsP6 was dephosphorylated to InsP1-2 in many cases at pH 3.0, accumulation of the Ins(1,2,5,6)P4 isomer occurred at pH 5.5. In an in vitro model simulating the digestive tract of broilers, hybrid phytases with high sequential homology to the E. coli and C. braakii phytase showed high accumulation of InsP4 isomers. Interestingly, these phytases preferentially formed the Ins(1,2,5,6)P4 isomer. In contrast, other hybrid phytases were able to degrade all InsP4 isomers and in some cases high InsP2 concentrations were observed. Another in vitro experiment with a complex feed matrix consisting of soybean meal, rapeseed meal, and wheat with a high mineral content, illustrated the negative influence of certain feed-related factors on phytase efficiency. InsP6 present in the feed was significantly less degraded by all phytases used compared to a corn and soy-based feed matrix with a low mineral content. While a hybrid phytase was able to completely dephosphorylate the InsP6 of the corn and soy-based feed matrix down to the InsP3 isomer resulting in high InsP2 concentrations, the InsP6 were still detectable in the in vitro model with the more complex feed matrix and high mineral content, despite identical reaction conditions. In a final feeding trial with broilers, one of the hybrid phytases was supplemented at two doses each (500 and 1500 FTU/kg) to evaluate its suitability as a feed supplement. Also, a commercial phytase was included in the study design at the same doses setting the benchmark for phytase efficiency. A low phosphorus experimental feed based on corn and soybean meal was used. The supplementation of the used hybrid phytase resulted in a dose-dependent increase in broiler performance data such as daily weight gain, feed intake and significantly improved feed efficiency compared to the basal ration without enzyme supplementation. In addition, foot ash content was increased by 21.6% at a dose of 1500 FTU/kg phytase, indicating significantly improved bone mineralization due to the released InsP6 phosphate. By analyzing InsP6 concentration and its degradation products in different segments of the digestive tract, efficient InsP6 degradation was observed. In contrast to the in vitro experiments, no accumulation of InsP3-4 isomers could be detected in crop, gizzard or small intestine. In addition to a high exogenous phytase activity, this result also suggests a high endogenous phytase as well as phosphatase activity in the digestive tract of broilers. It can be assumed that the absence of monocalcium phosphate in the experimental rations may have induced the expression of endogenous phytases and phosphatases. This assumption is confirmed by the high precaecal InsP6 degradation, which was 63.5% in the basal ration without phytase supplementation. Nevertheless, the used hybrid phytase significantly increased the precaecal InsP6 degradation to 76.3%. The high phytase efficiency was also reflected in the measured precaecal phosphorus digestibility, which was increased by 6.8% compared to the basal ration. The commercial phytase used showed comparable improvement in broiler performance data to the non optimized hybrid phytase. This project demonstrated the development of a variety of sequentially unique hybrid phytases by recombination of known phytase genes, which exceeded the biochemical properties of the wild-type phytases in some relevant aspects. Some of the phytases showed very efficient phytate degradation when simulating the digestive tract of broilers in vitro. Also, the suitability of the tested hybrid phytase as feed additives was demonstrated by the increased performance data of broilers. The higher performance data of the broilers could be attributed to efficient phytate degradation. To achieve maximum InsP6 degradation in vivo, the feed-related and animal-related factors on phytase efficiency need to be better understood.
Phytate hydrolysis and formation of inositol phosphates in the digestive tract of broilers
(2015) Zeller, Ellen; Rodehutscord, Markus
Phytate (any salt of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) or InsP6) represents the major binding form of phosphorus (P) in plant seeds. In the digestive tract, availability of P from plant seeds and feedstuffs obtained thereof largely depends on the enzymatic hydrolysis of InsP6 and less phosphorylated inositol phosphate isomers (InsPs). High prices of mineral P supplements and environmental burden linked with excessive P excretion of animals as well as exhaustion of the global rock phosphate stores demand for maximization of phytate-P utilization in animal feeding. The major objective of this thesis was to understand better InsP6 hydrolysis and formation of lower InsPs in different segments of the digestive tract of broilers and how they can be influenced by different dietary factors. In the first study (Manuscript 1), broilers (n=10 pens per dietary treatment) were fed low-P (5.2 g/kg DM) corn-soybean meal-based diets without (basal diet) or with one of three different phytase supplements (an Aspergillus and two E. coli derived phytases) from days 16 to 25 of age. InsP6 hydrolysis until the lower ileum (74%) of birds fed the basal diet indicated a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP pattern in different gut segments suggested the involvement of phosphatases of mucosal or microbial origin. Supplemented phytases significantly increased InsP6 hydrolysis in the crop but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time in broilers. Presence of InsP4 and InsP5 isomers specifically formed by different supplemented phytases indicated activity of these enzymes still in the small intestine. InsP4 accumulation differed between the 6- and 3-phytases in the anterior segments of the gut. In the second study (Manuscript 2), effects of supplemental mineral P were studied using different basal diets. Semi-synthetic and corn-soybean meal-based basal diets (experiment 1), or corn-based and wheat-based basal diets were used (experiment 2). Anhydrous monosodium phosphate (MSPa) or monocalcium phosphate monohydrate (MCPh) was supplemented to increment the P concentration by 0.05, 0.10, and 0.15% or by 0.075 and 0.150% in experiment 1 and 2, respectively. In experiment 1, total excreta were collected from day 20 to 24 of age (7 replicated birds per diet). In experiment 2, digesta from the terminal ileum was collected when broilers were 22 days old (5 replicated pens per diet, 19 birds per pen). No differences were found in InsP6 hydrolysis between the maize- and wheat-based diets (experiment 2). Mineral P supplements significantly decreased InsP6 hydrolysis from the InsP-containing diets in both experiments. The choice of the basal diet did not affect the evaluation of the supplemented mineral P sources. This lead to the conclusion that calculated availability values for mineral P sources need to be adjusted for the decline in hydrolysis of InsP contained in the basal diet resulting from the P supplement. In the third study (Manuscript 3), broilers (20 birds per pen; n=8 pens per treatment) were fed two low-P corn-soybean meal-based diets without (BD-; 4.4 g P/kg DM) or with monocalcium phosphate (MCP) (BD+; 5.2 g P/kg DM) and without or with added phytase at 500 or 12,500 FTU/kg from days 15 to 24 of age. Digesta samples were taken from the duodenum/jejunum and lower ileum. Another 180 broilers (n=6 pens per treatment, 10 birds each) were fed the three BD+ diets from day 1 to 21 of age to assess the influence of supplemented phytase on tibia mineralization and strength. Interactions between MCP and phytase affected InsP6 hydrolysis and the concentrations of specific lower InsPs. Supplementation with 12,500 FTU/kg phytase resulted in 92% prececal InsP6 hydrolysis and strong degradation of InsP5. This resulted in higher P net absorption, affirmed by higher body weight gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg of phytase. MCP supplementation reduced InsP6 hydrolysis and the degradation of specific lower InsPs in birds fed diets without phytase or with 500 FTU/kg of phytase, but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Hence effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. In the fourth study (Manuscript 4), broilers (15 birds per pen, n=8 pens per treatment) were fed a wheat-soybean meal diet low in P (4.8 g/kg DM) and containing either microwave-treated (BDTW; 121 U/kg of phytase) or non-microwave treated (BDUTW; 623 U/kg of phytase) wheat meal from d 16 to 23 of age. Diets were used without or with supplementation of a phytase, alone or in combination with a xylanase. Interactions between microwave treatment and enzyme supplementation were found for InsP6 hydrolysis in the ileum and P net absorption in the duodenum/jejunum and ileum. In the ileum, P net absorption was similar, but InsP6 hydrolysis was significantly higher for BDTW (78%) than for BDUTW (69%) in the absence of supplemental phytase. Microwaving may have disrupted wheat aleurone structures in ways that increased the accessibility of the phytate and may have encouraged higher levels of activity among specific phytases of microbial or endogenous mucosal origin in the lower small intestine. In both segments, InsP6 hydrolysis and P net absorption were significantly increased by supplementation of phytase, but no further by additional supplementation of xylanase. In birds that were fed the phytase-supplemented diets, microwave treatment of wheat had no effect on InsP6 hydrolysis, but it significantly reduced P net absorption in both segments. The fifth study compromised two experiments (Manuscript 5) in which the influence of different dietary factors on InsP6 degradation in the crop was investigated. The experimental designs was as mentioned for Manuscript 3 (experiment 2) and 4 (experiment 1) since the samples were taken in the same trials. In experiment 1, InsP6 hydrolysis in the crop was significantly increased by supplementation of phytase, but not further by the additional supplementation of xylanase. Microwave treatment of wheat reduced InsP6 hydrolysis and degradation of InsP5, due to reduction in intrinsic enzyme activity. The effect of 500 FTU/kg of supplemental phytase on InsP6 hydrolysis was much higher in broilers fed the maize- compared to those fed the wheat-based diets (experiment 2 and 1). Thus, for supplemental phytase the accessibility of phytate in wheat seems to be lower than in maize, perhaps due to different storage sites. Supplementation of 12,500 FTU/kg of phytase caused high InsP6 hydrolysis (up to 80%) and stronger degradation of InsP3-5 than supplementation of 500 FTU/kg (experiment 2). In both experiments, degradation of Ins(1,2,5,6)P4 was a limiting step in the breakdown process of InsP6 by the supplemented phytase. However, upon phytase supplementation Ins(1,2,5,6)P4 accumulated in BDTW diets whereas InsP4 degradation proceeded in untreated wheat diets (experiment 1). Ins(1,2,5,6)P4 seemed to be degraded synergistically by intrinsic wheat phosphatases and the supplemented phytase. Taking all studies together, it can be concluded that broilers and their gut microbiota have a very high potential to hydrolyze InsP6 in the digestive tract when diets low in P and Ca are fed. Differences in the concentrations of lower InsPs showed that the initial step of InsP6 hydrolysis is not the only catabolic step influenced by different dietary factors. To optimize efficacy of phytases and achieve a maximal InsP degradation and minimal P excretions the separate and interactive effects of different dietary influencing factors on InsP hydrolysis need to be better understood and considered in future diet formulations.