In vitro digestion effect on mineral bioaccessibility and antioxidant bioactive compounds of plant-based beverages.

Consumption of plant-based beverages (PBB) is a growing trend; and have been used as viable substitutes for dairy based products. To date, no study has comparatively analyzed mineral composition and effect of in vitro digestion on the bioaccessibility of different PBB. The aim of this research was to investigate the content of essential minerals (calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn)) and to estimate the effect of in vitro digestion in plant-based beverages, and their antioxidant bioactive compounds (phenolic compounds and antioxidant capacity). Moreover, the presence of antinutritional factors, such as myo-inositol phosphates fractions, were evaluated. Samples of PBB (rice, cashew nut, almond, peanut, coconut, oat, soy, blended or not with another ingredients, fortified with minerals or naturally present) and milk for comparison were evaluated. TPC ranged from 0.2 mg GAEq/L for coconut to 12.4 mg GAEq/L for rice and, the antioxidant capacity (DPPH) ranged from 3.1 to 306.5 µmol TE/L for samples containing peanut and oat, respectively. Only a few samples presented myo-inositol phosphates fractions in their composition, mostly IP5 and IP6, especially cashew nut beverages. Mineral content showed a wide range for Ca, ranging from 10 to 1697.33 mg/L for rice and coconut, respectively. The Mg content ranged from 6.29 to 251.23-268.43 mg/L for rice and cashew nut beverages, respectively. Fe content ranged from 0.76 mg/L to 12.89 mg/L for the samples of rice. Zinc content ranged from 0.57 mg/L to 8.13 mg/L for samples of oat and soy, respectively. Significant variation was observed for Ca (8.2-306.6 mg/L) and Mg (1.9-107.4 mg/L) dialyzed between the beverages, with lower concentrations of Fe (1.0 mg/L) and Zn (0.5 mg/L) in dialyzed fractions. This study provides at least 975 analytically determined laboratory results, providing important information for characterization and comparison of different plant-based beverages.

Effects of Moringa oleifera leaf extract on ruminal methane and carbon dioxide production and fermentation kinetics in a steer model.

Ruminal fermentation produces greenhouse gases involved in global warming. Therefore, the effect of nutrient combinations on methane, carbon dioxide, and biogas production as well as ruminal fermentation kinetics was evaluated in in vitro studies. In total mixed rations, dietary corn grain was partially replaced by two levels of soybean hulls (a highly reusable residue), and a Moringa oleifera extract (a natural extract) at three concentration levels was added. Higher levels of both soybean hulls and M. oleifera extract delayed the initiation of methane production and resulted in a lower methane and carbon dioxide production. Thus, total biogas production was also lower. Replacement of corn grain by soybean hulls tended to lower methane production rates and asymptotic carbon dioxide production, and a delay in biogas and methane formation was observed. Asymptotic biogas and carbon dioxide production, however, were increased. The presence of M. oleifera extract tended to delay methane formation and to decrease methane production rate as well as asymptotic methane production. Higher M. oleifera extract levels decreased asymptotic biogas production with the control and the highest soybean hull levels. In the presence of M. oleifera extract, asymptotic carbon dioxide production was shown to be quadratically increased with the control and lowest soybean hull levels, but quadratically decreased with the highest soybean hull level. With the exception of fermentation pH, the interaction of substrate type and M. oleifera extract level was shown to have an effect on all fermentation parameters. Most fermentation parameters were shown to be higher when replacing corn grain by soybean hulls, including fermentation pH. Thus, the conclusion could be drawn that corn grain replacement by soybean hulls (an agricultural residue) in the presence of M. oleifera extract (a sparing leaf product) could ameliorate greenhouse gas emissions and improve digestion.

Influence of phytase enzyme on ruminal biogas production and fermentative digestion towards reducing environmental contamination.

Environmental impact of livestock production has received a considerable public scrutiny because of the adverse effects of nutrient run-offs, primarily N and P, from agricultural land harboring intensive energy livestock operations. Hence, this study was designed to determine the efficacy of dietary phytase supplementation on fermentation of a sorghum grain-based total mixed ration (TMR) using a ruminal in vitro digestion approach. Phytase was supplemented at three doses: 0 (control), 540 (P540), and 720 (P720) g/t dry matter, equivalent to 0, 2.7 × 106, and 3.6 × 106 CFU/t DM, respectively. Compared to P720 and the control, gas production was higher for P540 after 12 h (P = 0.02) and 24 h (P = 0.03) of fermentation suggesting a higher microbial activity in response to phytase supplementation at lower phytase levels. Correspondingly, dry matter degradability was found to have improved in P540 and P720 compared to the control by 13 and 11% after 24 h of incubation (P = 0.05). For ammonia nitrogen (NH3-N), a tendency towards lower values was only observed for P540 at 24 h of fermentation (P = 0.07), while minimal treatment effects were observed at other fermentation times. The concentrations of total volatile fatty acids (VFA) were higher (P < 0.05) after 48 h of fermentation for P540 and P720 compared to the control (P = 0.03) by 10% and 14%, respectively. Ruminal acetate tended towards higher values in the presence of phytase after 12 h of fermentation (P = 0.10), but towards lower values after 24 h of fermentation (P = 0.02), irrespective of the phytase dose applied. A trend towards lower ruminal propionate levels was observed in the presence of phytase after 6 h (P = 0.10) and 12 h (P = 0.06) of fermentation, while no effects were found at other fermentation times. In conclusion, phytase supplementation has the potential to improve metabolic energy activity of rumen microorganisms and the use of feed constituents. Thus, phytase supplementation could help to reduce environmental contamination in areas of ruminant production.

Effect of enzymatic treatment on phytate content and mineral bioacessability in soy drink.

The objective of this study was to evaluate in vitro bioaccessibility of calcium (Ca), iron (Fe) and zinc (Zn) in soy drink after phytase treatment and correlate it with the content of myo-inositol phosphates. Samples of commercial soy drink products and one sample produced in the laboratory by maceration were evaluated. Phytase was applied using 300 U per liter in 60 min considering the phosphate release. The content of myo-inositol tris-, tetrakis-, pentakis and hexakisphosphate was not observed after phytase treatment. The solubility assay showed an increase from 2.0% to 20.8% for Ca, 2.2% to 37.1% for Fe and 38.8% to 67.4% for Zn after phytase treatment with significant differences (p ≤ 0.05) for most samples. Dialysis assay demonstrated 1.0% to 9.5% for Ca after phytase treatment (p ≤ 0.05) except for one commercial sample. The phytase treatment is a valuable alternative process for improving mineral natural availability in soy drink and decreased the use of salts in the fortification.

The effect of exogenous phytase supplementation on nutrient digestibility, ruminal fermentation and phosphorous bioavailability in Rambouillet sheep.

BACKGROUND: The effect of phytase supplementation with respect to a high sorghum grain diet on sheep voluntary feed intake, apparent nutrient digestibility, ruminal fermentation, phosphorus (P) excretion and blood serum P concentration was evaluated. RESULTS: Phytase supplementation significantly decreased fecal P excretion (P = 0.003), resulting in a 26% decrease in relation to the phytase free diet. Dry matter intake, nutrient digestibility, ruminal butyrate and serum P were not (P > 0.10) affected by the phytase level. Neutral detergent digestibility showed a tendency to increase linearly (P = 0.10) with increasing phytase levels. Ruminal pH was lower for phytase supplemented sheep, with a significant decrease (P = 0.007) at 9 h post feeding, whereas ruminal ammonia-N at 3 h post feeding was lower (P = 0.004) for the phytase treatment groups, resulting in a decreasing linear response (P = 0.001) with an increasing phytase dose. Duodenal pH was significantly reduced at 6 h post feeding. Propionate tended (P = 0.051) to be increased linearly as the phytase supplementation level increased. CONCLUSION: Exogenous phytase supplementation of high sorghum grain diets significantly decreased fecal P excretion in Rambouillet rams. Phytase supplementation appears to affect neutral detergent fiber digestibility, duodenal and ruminal pH, ammonia and propionate. © 2018 Society of Chemical Industry.

Effects of natural blends of garlic and eucalypt essential oils on biogas production of four fibrous feeds at short-term of incubation in the ruminal anaerobic biosystem.

BACKGROUND: The present study explored the effect of garlic and/or eucalypt oils on biogas production during in vitro ruminal fermentation of four agro industry byproducts. For this, 0-180 mg oil L-1 incubation medium was added and gas volumes were recorded from 2 to 48 h of incubation. Dry matter substrate degradability and neutral as well as acid detergent fibre were determined after 72 h. RESULTS: Gas production and nutrient degradability was oil type dependent. The oils enhanced (P < 0.05) biogas and asymptotic biogas production for corn stalks and oat straw, although no effect was observed on asymptotic biogas production for sorghum straw and sugarcane bagasse. Addition of both oils decreased (P < 0.05) fermentation pH for corn stalks, sorghum straw and oat straw and also increased (P < 0.05) dry matter degradability for all four byproducts. Neutral detergent fibre degradability for all byproducts was higher (P < 0.05) with garlic oil. Eucalypt oil, however, decreased (P < 0.05) neutral detergent fibre degradability for sugarcane bagasse and corn stalks, although only weak effects were observed for sorghum and oat straws. CONCLUSION: With respect to ruminal biogas production, the addition of garlic oil showed better environmental effects than the addition of eucalypt oil and increasing oil concentrations resulted in enhanced fermentation characteristics. © 2018 Society of Chemical Industry.

Screening and Characterization of Phytases from Bacteria Isolated from Chilean Hydrothermal Environments.

Phytases are enzymes involved in organic phosphorus cycling in nature and widely used as feed additives in animal diets. Thermal tolerance is a desired property of phytases. The objectives of this study were to screen and characterize bacterial phytases from Chilean hydrothermal environments. In this study, 60% (30 of 63) of screened thermophilic (60 °C) isolates showed phytase activity in crude protein extracts. The characterization of phytase from two selected isolates (9B and 15C) revealed that both isolates produce phytases with a pH optimum at 5.0. The temperature optimum for phytate dephosphorylation was determined to be 60 and 50 °C for the phytases from the isolates 9B and 15C, respectively. Interestingly, the phytase from the isolate 15C showed a residual activity of 46% after incubation at 90 °C for 20 min. The stepwise dephosphorylation of phytate by protein extracts of the isolates 9B and 15C was verified by HLPC analysis. Finally, the isolates 9B and 15C were identified by partial sequencing of the 16S rRNA gene as members of the genera Bacillus and Geobacillus, respectively.

Bioaccessibility of calcium, iron and magnesium in residues of citrus and characterization of macronutrients.

The aim of this study was to estimate bioaccessibility of Ca, Fe and Mg in residues of orange, lime, and their mixture, in order to evaluate the effects of cooking in water on mineral bioaccessibility and also to determine the composition of macronutrients and myo-inositol phosphate content. The citrus samples contained on average 9.53g/100 g moisture, 6.09g/100 g protein, 3.23g/100g ash, 3.15g/100g lipids, 34.26g/100g insoluble fiber, 27.88g/100g soluble fiber and 25.64g/100g carbohydrates. The percentage of soluble and dialyzable minerals ranged from 19.36 to 77.33% and from 5.59 to 69.06% for Fe, from 33.34 to 60.84% and 14.71 to -26.13% for Ca, and from 29.95 to 94.20% and 34.42 to 62.51%, for Mg, respectively. It was verified that cooking influenced the minerals bioaccessibility and increased the dialyzable fraction of Fe and Mg, but decreased the fraction of Ca dialysate, except to orange. No myoinositol phosphate esters were detected. The Principal Component Analysis allowed the separation of different types of citrus residues, but did not discriminate the raw and cooked samples. This study pointed the potential of citrus residue to be used for human consumption and contribute to the necessary dietary minerals and macronutrients, with high content of soluble and insoluble fibers.

Effect of different iron compounds on rheological and technological parameters as well as bioaccessibility of minerals in whole wheat bread.

This study aimed at investigating the effect of iron compounds used in whole wheat flour (WWF) fortification, both on rheological properties of the dough and on bread technological quality. Furthermore, bioaccessibility of iron (Fe), zinc (Zn) and calcium (Ca) in the final breads was determined. Rheological properties (mainly dough development time, stability, mixing tolerance index, resistance to extension and ratio number) of the dough and the technological quality of bread (mainly oven spring and cut opening) were altered. However, producing roll breads fortified with different iron compounds was still possible. NaFeEDTA (ferric sodium ethylene diamine tetra acetic acid) proved to be the most effective iron compound in the fortification of WWF, since it presented the highest levels of solubility (44.80%) and dialysability (46.14%), followed by microencapsulated ferrous fumarate (FFm). On the other hand, the microencapsulated ferrous sulfate (FSm) and reduced iron presented the lowest solubility (5.40 and 18.30%, respectively) and dialysability (33.12 and 31.79%, respectively). Zn dialysis was positively influenced by NaFeEDTA, FSm, and ferrous fumarate. As for Ca, dialysis was positively influenced by FSm and negatively influenced by FFm. The data indicated that there is a competitive interaction for the absorption of these minerals in whole wheat roll breads, but all studied minerals can be considered bioaccessible.

Phytase-Producing Bacteria from Extreme Regions in Indonesia

ABSTRACTIn this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genusBacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity toB. cereus, while isolate MC8 exhibited 99% sequence identity toB. aryabhatti and D6 99% sequence identity toB. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

Studies towards the stabilisation of a mushroom phytase produced by submerged cultivation.

A novel phytase from Ganoderma australe G24 was produced by submerged cultivation and recovery. Liquid and solid forms of phytase were developed; both types of product were formulated using different additives. Ganoderma australe G24 phytase was very stable in liquid form with NaCl and sodium acetate buffer. Solid form products were obtained by spray-drying using different polymers to encapsulate the phytase and the capsules obtained were analyzed by electron microscopy. Micrographs confirmed micro and nanoparticles formed with maltodextrin (300 nm to 7-8 µm) without the presence of agglomerates. The use of maltodextrin for solid formulation of G. australe G24 phytase is recommended, and resulted in good stability after the drying process and during storage (shelf life). Kinetic models of phytase inactivation in the microencapsulated powders over time were proposed for the different stabilizing additives. Inactivation rate constants, half-lives and D values (decimal reduction time) were obtained. Phytase encapsulated with maltodextrin remained stable after 90 days, with k 0.0019 day(-1) and a half-life (t1/2) of 367.91 days(-1).

Chitosan nanoparticles loaded the herbicide paraquat: the influence of the aquatic humic substances on the colloidal stability and toxicity.

Polymeric nanoparticles have been developed for several applications, among them as carrier system of pesticides. However, few studies have investigated the fate of these materials in the environment in relation to colloidal stability and toxicity. In nature, humic substances are the main agents responsible for complexation with metals and organic compounds, as well as responsible for the dynamics of these nanoparticles in aquatic and terrestrial environments. In this context, the evaluation of the influence of aquatic humic substances (AHS) on the colloidal stability and toxicity of polymeric nanoparticles of chitosan/tripolyphosphate with or without paraquat was performed. In this study, the nanoparticles were prepared by the ionic gelation method and characterized by size distribution measurements (DLS and NTA), zeta potential, infrared and fluorescence spectroscopy. Allium cepa genotoxicity studies and ecotoxicity assays with the alga Pseudokirchneriella subcapitata were used to investigate the effect of aquatic humic substances (AHS) on the toxicity of this delivery system. No changes were observed in the physical-chemical stability of the nanoparticles due to the presence of AHS using DLS and NTA techniques. However some evidence of interaction between the nanoparticles and AHS was observed by infrared and fluorescence spectroscopies. The ecotoxicity and genotoxicity assays showed that humic substances can decrease the toxic effects of nanoparticles containing paraquat. These results are interesting because they are important for understanding the interaction of these nanostructured carrier systems with species present in aquatic ecosystems such as humic substances, and in this way, opening new perspectives for studies on the dynamics of these carrier systems in the ecosystem.

Chitosan/tripolyphosphate nanoparticles loaded with paraquat herbicide: an environmentally safer alternative for weed control.

Paraquat is a fast acting nonselective contact herbicide that is extensively used worldwide. However, the aqueous solubility and soil sorption of this compound can cause problems of toxicity in nontarget organisms. This work investigates the preparation and characterization of nanoparticles composed of chitosan and sodium tripolyphosphate (TPP) to produce an efficient herbicidal formulation that was less toxic and could be used for safer control of weeds in agriculture. The toxicities of the formulations were evaluated using cell culture viability assays and the Allium cepa chromosome aberration test. The herbicidal activity was investigated in cultivations of maize (Zea mays) and mustard (Brassica sp.), and soil sorption of the nanoencapsulated herbicide was measured. The efficiency association of paraquat with the nanoparticles was 62.6 ± 0.7%. Encapsulation of the herbicide resulted in changes in its diffusion and release as well as its sorption by soil. Cytotoxicity and genotoxicity assays showed that the nanoencapsulated herbicide was less toxic than the pure compound, indicating its potential to control weeds while at the same time reducing environmental impacts. Measurements of herbicidal activity showed that the effectiveness of paraquat was preserved after encapsulation. It was concluded that the encapsulation of paraquat in nanoparticles can provide a useful means of reducing adverse impacts on human health and the environment, and that the formulation therefore has potential for use in agriculture.

Identification of ß-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils.

Phytate is one of the most abundant sources of organic phosphorus (P) in soils, but must be mineralized by phytase-producing bacteria to release P for plant uptake. Microbial inoculants based on Bacillus spp. have been developed commercially, but few studies have evaluated the ecology of these bacteria in the rhizosphere or the types of enzymes that they produce. Here, we studied the diversity of aerobic endospore-forming bacteria (EFB) with the ability to mineralize phytate in the rhizosphere of pasture plants grown in volcanic soils of southern Chile. PCR methods were used to detect candidate phytase-encoding genes and to identify EFB bacteria that carry these genes. This study revealed that the phytate-degrading EFB populations of pasture plants included species of Paenibacillus and Bacillus, which carried genes encoding ß-propeller phytase (BPP). Assays of enzymatic activity confirmed the ability of these rhizosphere isolates to degrade phytate. The phytase-encoding genes described here may prove valuable as molecular markers to evaluate the role of EFB in organic P mobilization in the rhizosphere.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3 percent referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55ºC. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37ºC. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Current and projected applications of nanotechnology in the food sector: [review] / Aplicações atuais e futuras da nanotecnologia no setor alimentício: [revisão]

The prospects for applications of nanotechnology to the food sector have become more apparent over the last few years. Nanotechnology applications are expected to bring changes to the food sector, including improved production and processing techniques, improved food contact materials, modification of taste, texture and sensation, monitoring food quality and freshness, reduced fat content, enhanced nutrient absorption, and improved traceability and security of food products. A variety of food ingredients, additives, encapsulation systems and food contact materials is already available in some countries and the market for nanotechnology-derived food products and food contact materials is expected to grow worldwide. However, no clear information about the actual use of nanotechnology in the food industry is available and data on the benefits, improvements and risks of nanotechnology applications in the food sector as well as their economical competitiveness are still almost lacking.

Las posibilidades de aplicación de la nanotecnología al sector de alimentos se tornaron más evidentes en los últimos años. Se espera que su empleo introduzca cambios en el sector de alimentos, incluyendo aumentos de productividad; perfeccionamiento de los procesos; mejora de los materiales de contacto con alimentos; modificaciones de sabor, textura y sensación; vigilancia de la calidad y el frescor; reducción del contenido de grasa; aumento de la absorción de nutrientes y perfeccionamiento de la trazabilidad y seguridad de los productos alimenticios. Una variedad de ingredientes de alimentos, aditivos, sistemas de encapsulación y materiales de contacto con los alimentos ya están disponibles en algunos países y se espera que el mercado de productos alimenticios con origen en la nanotecnología aumente en todo el mundo. Sin embargo, no existe en el momento información sobre como la nanotecnología esta siendo usada en la industria de alimentos, ni datos sobre los beneficios, progresos y riesgos de su aplicación al sector de alimentos o de la competitividad económica de esta tecnología.

As perspectivas de aplicações da nanotecnologia ao setor alimentício se tornaram mais aparentes nos últimos anos. Espera-se que as aplicações da nanotecnologia trarão mudanças ao setor, incluindo técnicas aperfeiçoadas de produção e processamento, materiais que têm contato com o alimento melhorados, modificação de sabor, textura e sensação, monitoria da qualidade e frescor dos alimentos, reduzido teor de gordura, absorção de nutrientes aumentada, e melhor rastreabilidade e segurança dos produtos alimentícios. Uma variedade de ingredientes, aditivos, sistemas de encapsulação e materiais que entram em contato com o alimento já estão disponíveis em alguns países e espera-se que o mercado para produtos alimentícios derivados da nanotecnologia e materiais que entram em contato com os alimentos cresça mundialmente. Entretanto, não há claras informações disponíveis sobre o real uso da nanotecnologia na indústria de alimentos e dados sobre os benefícios, melhorias e riscos da aplicação da nanotecnologia na indústria de alimentos, bem como sua competitividade econômica ainda são quase ausentes.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9.

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be KM = 54 µmol l(-1) and kcat = 190 sec(-1) at pH 5.0 and 37°C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37°C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Production of D-myo-inositol(1,2,4,5,6)pentakisphosphate using alginate-entrapped recombinant Pantoea agglomerans glucose-1-phosphatase

The glucose-1-phosphatase encoding gene (agp) of Pantoea agglomerans was sequenced and heterologously expressed in Escherichia coli. The enzyme showed very high homology to periplasmatic glucose-1-phosphatases of other members of the Enterobacteriaceae family. It was isolated from transformed Escherichia coli cells in a single step in high yields (32.3 ± 1.2 mg per litre of culture) by Ni-NT agarose affinity chromatography to >95 percent purity as calculated from specific activity determinations. The purified glucose-1-phosphatase was entrapped in alginate beads with an entrapment efficiency of >80 percent. Temperature stability was enhanced as a consequence of entrapment, whereas pH dependence of enzyme activity was not affected. Maximum catalytic activity of entrapped glucose-1-phosphatase was found at 70°C, whereas the free enzyme exhibited maximal activity at 60°C. A single pH optimum at pH 4.5 was determined for the free and the entrapped enzyme. Kinetic parameters for the hydrolysis of sodium phytate were found to be affected by entrapment. They were determined to be K M = 0.84 mmol l-1 and k cat = 8 s-1 at pH 4.5 and 37°C for the entrapped glucose-1-phosphatase and K M = 0.35 mmol l-1 and k cat = 20.5 s-1 for the free enzyme. Complete conversion of phytate into one single myo-inositol pentakisphosphate isomer, identified as D-myo-inositol(1,2,4,5,6)pentakis-phosphate, was shown to be feasible by using the enzyme-loaded alginate beads in batch operations. The entrapped enzyme showed a high operational stability by retaining almost full activity even after ten uses.

myo-inositol phosphate isomers generated by the action of a phytase from a malaysian waste-water bacterium.

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P(5), D-I(2,3,4,5)P(4), D-I(2,3,4)P(3), D-I(2,3)P(2) to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P(5). Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P(5), D/L-I(1,2,4,5)P(4), D/L-I(1,2,4)P(3), D/L-I(2,4)P(2) to finally I(2)P.