Browsing by Subject "Hydrolyse"

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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.
Enzymatic hydrolysis of vegetable and insect proteins using technical enzyme preparations
(2021) Großmann, Kora Kassandra; Fischer, Lutz
The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing.
Studies on the extent of ruminal degradation of phytate from different feedstuffs
(2017) Haese, Eva; Rodehutscord, Markus
The predominant storage form of phosphorus (P) in plant seeds and grains is phytate (InsP6). To cleave the phosphate group and, thus, make the bound P available for absorption by the animal, the enzyme phytase is required. Rumen microorganisms show substantial phytase activity, however, recent studies have suggested that the extent of InsP6 hydrolysis in ruminants is variable leading to an incomplete hydrolysis of InsP6 in specific conditions followed by the excretion of P from undegraded InsP6. As P is an essential element for the metabolism in animals it is important to ensure that the animals’ requirements are met. Diets for ruminants are often supplemented with mineral P (Pi). However, the global phosphate resources are finite and the excretion of surplus P contributes to eutrophication of surface water when applied to the farmland with manure in excessive amounts. Thus, dietary P supply is of environmental concern. Better knowledge about ruminal InsP6 hydrolysis could help to optimise the utilisation of InsP6 and, thus, reduce the use of Pi as well as unnecessary excretion of P. Hence, the objectives of the present thesis were to examine the InsP6 hydrolysis from different feedstuffs in ruminants and to identify factors that might affect the extent of InsP6 hydrolysis. In the first study, the total digestive tract disappearance of InsP6 from diets differing in amount and source of P was determined in lactating dairy cows. The results confirmed the high potential of rumen microorganisms to hydrolyse InsP6, but the composition of the diet influenced the extent of hydrolysis in vivo. In the second study, two in vitro experiments were conducted in order to determine the InsP6 hydrolysis from maize grain and RSM. In experiment 1, two diets differing in P- and InsP6-P concentration were fed to the donor animals of rumen fluid. In experiment 2, a diet similar to the high P diet of experiment 1 was fed to the donor animals of rumen fluid and the rumen fluid was mixed with artificial saliva containing Pi (PI: 120 mg Pi/l) or no Pi. Maize and RSM were incubated for 3, 6, 12, and 24 h in both experiments and the InsP6 concentration was analysed in fermenter fluids and bag residues. InsP6 disappearance from maize proceeded faster than from RSM. The disappearance of InsP6 was higher when the diet with high P concentration was fed (experiment 1) and lower when the rumen fluid was mixed with Pi containing buffer (experiment 2). In the third study, the in situ disappearance of InsP6 from five different concentrates was examined. Maize, wheat, RSM, heat treated RSM (hRSM), and soybean meal were incubated in the rumen of fistulated dairy cows fed with three diets differing in P- and InsP6-P concentration. Concentrations of InsP6 and isomers of InsP5, InsP4, and InsP3 were determined in the bag residues after 2, 4, 8, 16, and 24 h of incubation. The disappearance of InsP6 from cereals proceeded faster than from oilseed meals, however, averaged over the diets, after 24 h of incubation 95% had disappeared from all concentrates except for hRSM (57%). Feeding the diet with high InsP6 concentrations increased InsP6 disappearance from oilseed meals but not from cereals, while feeding the high Pi diet did not influence ruminal InsP6 hydrolysis from any concentrate. The results derived from analysis of lower InsPs suggested that intrinsic plant phytase activity plays only a minor role in the rumen and that active phytases in the rumen react differently to changes in the ruminal environment. The results of the present thesis suggest that the composition of the diet fed to ruminants affects the extent of ruminal InsP6 hydrolysis. While high InsP6 concentrations have the potential to increase InsP6 hydrolysis, a decrease of InsP6 hydrolysis can occur after addition of Pi to the diet. Differences in the pace of InsP6 hydrolysis between concentrates occurred which could be of importance at high ruminal passage rates when the time available for ruminal hydrolysis decreases.