Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

Background: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. Aim: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. Methods: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). Results: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. Conclusions: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).

Strategies to Optimize Biosensors Based on Impedance Spectroscopy to Detect Phytic Acid Using Layer-by-Layer Films

Impedance spectroscopy has been proven a powerful tool for reaching high sensitivity in sensor arrays made with nanostructured films in the so-called electronic tongue systems, whose distinguishing ability may be enhanced with sensing units capable of molecular recognition. In this study we show that for optimized sensors and bio-sensors the dielectric relaxation processes involved in impedance measurements should also be considered, in addition to an adequate choice of sensing materials. We used sensing units made from layer-by-layer (LbL) films with alternating layers of the polyeletrolytes, poly(allylamine) hydrochloride (PAH) and poly(vinyl sulfonate) (PVS), or LbL films of PAH alternated with layers of the enzyme phytase, all adsorbed on gold interdigitate electrodes. Surprisingly, the detection of phytic acid was as effective in the PVS/PAH sensing system as with the PAH/phytase system, in spite of the specific interactions of the latter. This was attributed to the dependence of the relaxation processes on nonspecific interactions such as electrostatic cross-linking and possibly on the distinct film architecture as the phytase layers were found to grow as columns on the LbL film, in contrast to the molecularly thin PAH/PVS films. Using projection techniques, we were able to detect phytic acid at the micromolar level with either of the sensing units in a data analysis procedure that allows for further optimization.

Equilibrium, Thermoanalytical and Spectroscopic Studies to Characterize Phytic Acid Complexes with Mn(II) and Co(II)

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Fabrication of phytic acid sensor based on mixed phytase-lipid Langmuir-Blodgett films

This paper reports the surface activity of phytase at the air-water interface, its interaction with lipid monolayers, and the construction of a new phytic acid biosensor on the basis of the Langmuir-Blodgett (LB) technique. Phytase was inserted in the subphase solution of dipalmitoylphosphatidylglycerol (DPPG) Langmuir monolayers, and its incorporation to the air-water interface was monitored with surface pressure measurements. Phytase was able to incorporate into DPPG monolayers even at high surface pressures, ca. 30 mN/m, under controlled ionic strength, pH, and temperature. Mixed Langmuir monolayers of phytase and DPPG were characterized by surface pressure-area and surface potential-area isotherms, and the presence of the enzyme provided an expansion in the monolayers ( when compared to the pure lipid at the interface). The enzyme incorporation also led to significant changes in the equilibrium surface compressibility (in-plane elasticity), especially in liquid-expanded and liquid-condensed regions. The dynamic surface elasticity for phytase-containing interfaces was investigated using harmonic oscillation and axisymmetric drop shape analysis. The insertion of the enzyme at DPPG monolayers caused an increase in the dynamic surface elasticity at 30 mN m(-1), indicating a strong interaction between the enzyme and lipid molecules at a high-surface packing. Langmuir-Blodgett (LB) films containing 35 layers of mixed phytase-DPPG were characterized by ultraviolet-visible and fluorescence spectroscopy and crystal quartz microbalance nanogravimetry. The ability in detecting phytic acid was studied with voltammetric measurements.

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

Dietary Phytase: an ideal approach for a cost effective and low-polluting aquafeed

Global fishmeal production from wild-catch sources cannot continue to increase indefinitely; suitable alternatives have to be found for sustainable aquaculture. Plant-based aquafeed seems to be the ideal alternative to this, but has its own limitations. Plant ingredients are rich in phytic acid, which reduces the bioavailability of nutrients like minerals and protein to the fish, thereby causing aquaculture pollution. Dietary phytase treatment reduces the aquaculture pollution by improving the bioavailability of nutrients, and reduces the feed cost as evident from poultry and piggery. Phytase activity is highly dependent upon the pH of the gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In this article, the authors attempt to summarise various effects of phytase on nutrient utilization, growth of fish and aquatic pollution.

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P5 pool

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30-40% (p<0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P(5) levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p<0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to D/L-Ins(1,2,4,5,6)P5, levels of which increased 60% (p<0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.

Studies on the Bioactive Natural Antioxidants From Oilseeds

Antioxidants are substances that when present at low concentrations compared to that of an oxidisable substrate significantly delays or inhibits oxidation of that substrate in food products or in living systems. Antioxidants are either endogenous to the body or derived from the diet. Several types of synthetic antioxidants like BHT, BHA, TBHQ etc. are also used in the food industry. However, findings and subsequent publicity has fostered significant consumer resistance to the use of synthetic food additives as antioxidants, colourants etc. and therefore food industry is in search of potential natural antioxidants from edible sources.The major dietary sources of antioxidant phytochemicals are cereals, legumes, fruits, vegetables, oilseeds, beverages, spices and herbs. In the present study, we have focused on rice bran and its byproducts. Rice is one of the oldest of food crops and has been a staple food in India from very ancient times. It is also the staple food for about 60% of the world's population. Rice bran is a byproduct of the rice milling industry and is a potential commercial source of a healthy edible oil viz. rice bran oil and a variety of bio-active phytochemicals.Defatted rice bran (DRB), a byproduct of rice bran oil extraction, is also a good source of insoluble dietary fiber, protein, phytic acid, inosito I, vitamin B and a variety of other phytochemicals. Though the antioxidant potential of DRB has been demonstrated, it still remained a relatively unexplored source material, which demanded further investigation especially with regard to its detailed phytochemical profile leading to practical application. The focus of the present investigation therefore has been on DRB primarily to establish its phytochemical status and feasibility of using it as a source of bio-active phytochemicals and natural antioxidants leading to value addition of DRB otherwise used as cattle feed. To gain a better understanding of the value of rice bran as a source of phytochemicals, five popular rice varieties of the region viz. PTB 50, PTB 39, PTB 38, JA Y A, and MO 10 and a wild variety (oryza nivara) that is mainly used for medicinal applications in traditional ayurvedic system were characterized along with commercial samples of rice bran. The present study also explains the feasibility of a process for the extraction, enrichment, and isolation of antioxidant compounds from DRB. The antioxidant potential of the extracts were evaluated both in bulk oils and in food relevant model emulsions, using standard in vitro models. Radical scavenging effects, indicative of possible biological effects, were also evaluated.

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)2SO4 precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS–PAGE it was found to be a monomeric protein with molecular mass 72±2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 μM) and highest specificity constant (Vmax/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 μM and Ki=205 μM, respectively).

Quartz crystal microbalance monitoring of density changes in mesoporous TiO2 phytate films during redox and ion exchange processes

Nanofilm deposits of TiO2 nanoparticle phytates are formed on gold electrode surfaces by 'directed assembly' methods. Alternate exposure of a 3-mercapto-propionic acid modified gold surface to (i) a TiO2 sol and (ii) an aqueous phytic acid solution (pH 3) results in layer-by-layer formation of a mesoporous film. Ru(NH3)(6)(3+) is shown to strongly adsorb/accumulate into the mesoporous structure whilst remaining electrochemically active. Scanning the electrode potential into a sufficiently negative potential range allows the Ru(NH3)(6)(3+) complex to be reduced to Ru(NH3)(6)(2+) which undergoes immediate desorption. When applied to a gold coated quartz crystal microbalance (QCM) sensor, electrochemically driven adsorption and desorption processes in the mesoporous structure become directly detectable as a frequency response, which corresponds directly to a mass or density change in the membrane. The frequency response (at least for thin films) is proportional to the thickness of the mass-responsive film, which suggests good mechanical coupling between electrode and film. Based on this observation, a method for the amplified QCM detection of small mass/density changes is proposed by conducting measurements in rigid mesoporous structures. (C) 2003 Elsevier Science B.V. All rights reserved.

Effect of dietary phytate and microbial phytase on mineral and trace element bioavailability- a literature review

Phytic acid (PA) is the main phosphorus storage compound in cereals, legumes and oil seeds. In human populations where phytate-rich cereals such as wheat, maize and rice are a staple food, phytate may lead to mineral and trace element deficiency. Zinc appears to be the trace element whose bioavailability is most influenced by PA. Furthermore, several studies in humans as well as in monogastric animals clearly indicate an inhibition of non-haem iron absorption at marginal iron supply due to phytic acid. In fact PA seems to be, at least partly, responsible for the low absorption efficiency and high incidence of iron deficiency anaemia evident in most developing countries, where largely vegetarian diets are consumed Microbial phytases have provided a realistic means of improving mineral availability from traditionally high-phytate diets. In fact it has been consistently shown that Aspergillus phytases significantly enhance the absorption of calcium, magnesium and zinc in pigs and rats. Furthermore there are a few studies in humans indicating an improvement of iron bioavailability due to microbial phytase.

Phytase immobilization on modified electrodes for amperometric biosensing

Phytase (myo-inositol hexaphosphate phosphohydrolase) and phytic acid (myo-inositol hexaphosphate) play an important environmental role, in addition to being a health issue in food industry. Phytic acid is antinutritional due to its ability to chelate metal ions and may also react with proteins decreasing their bioavailability. In this work, we produced biosensors with phytase immobilized in Layer-by-Layer (LbL) films, which could detect phytic acid with a detection limit of 0.19 mmol L-1, which is sufficient to detect phytic acid in seeds of grains and vegetables. The biosensosrs consisted of LbL films containing up to eight bilayers of phytase alternated with poly(allylamine) hydrochloride (PAH) deposited onto an indium-tin oxide (ITO) substrate modified with Prussian Blue. Amperometric detection was conducted in an acetate buffer solution (at pH 5.5) at room temperature, with the biosensor response attributed to the formation of phosphate ions. In subsidiary experiments with the currents measured at 0.0 V (vs. SCE), we demonstrated the absence of effects from some interferents, pointing to a good selectivity of the biosensor. (c) 2007 Elsevier B.V. All rights reserved.

Phytase immobilization on modified electrodes for amperometric biosensing

Phytase (myo-inositol hexaphosphate phosphohydrolase) and phytic acid (myo-inositol hexaphosphate) play an important environmental role, in addition to being a health issue in food industry. Phytic acid is antinutritional due to its ability to chelate metal ions and may also react with proteins decreasing their bioavailability. In this work, we produced biosensors with phytase immobilized in Layer-by-Layer (LbL) films, which could detect phytic acid with a detection limit of 0.19 mmol L-1, which is sufficient to detect phytic acid in seeds of grains and vegetables. The biosensosrs consisted of LbL films containing up to eight bilayers of phytase alternated with poly(allylamine) hydrochloride (PAH) deposited onto an indium-tin oxide (ITO) substrate modified with Prussian Blue. Amperometric detection was conducted in an acetate buffer solution (at pH 5.5) at room temperature, with the biosensor response attributed to the formation of phosphate ions. In subsidiary experiments with the currents measured at 0.0 V (vs. SCE), we demonstrated the absence of effects from some interferents, pointing to a good selectivity of the biosensor. (c) 2007 Elsevier B.V. All rights reserved.

Enhancement of heterologous expression of alkaline phytase in Pichia pastors

Phytic acid is the major storage form of phosphorus and inositol in seeds and legumes. It forms insoluble phytate salts by chelating with positively charged mineral ions. Non-ruminant animals are not able to digest phytate due to the lack of phytases in their GI tracks, thus the undigested phytate is excreted leading to environmental contamination. Supplementation with phytases in animal feed has proven to be an effective strategy to alleviate nutritional and environmental issues. The unique catalytic and thermal stability properties of alkaline phytase from lily pollen (LlALP) suggest that it has the potential to be useful as a feed supplement. Our goal is to develop a method for the production of substantial amounts of rLlALP for animal feed and structural studies. rLlALP2 has been successfully expressed in the yeast, Pichia pastoris. However, expression yield was modest (8-10 mg/L). Gene copy number has been identified as an important parameter in enhancing protein yields. Multicopy clones were selected using Zeocin-resistance-based vectors and challenging transformants to high Zeocin levels under different conditions. Data indicate that increasing selection pressure led to the generation of clones with amplification of both rLlAlp2 and Zeor genes and the two genes were not equally amplified. Additionally, clones generated by step-wise methods led to clones with greater amplification. The effects of transgene copy number and gene sequence optimization on expression levels of rLlALP2 were examined. The data indicate that increasing the copy number of rLlAlp2 in transformed clones was detrimental to expression level. The use of a sequence-optimized rLlAlp2 (op-rLlAlp2) increased expression yield of the active enzyme by 25-50%, suggesting that transcription and translation efficiency are not major bottlenecks in the production of rLlALP2. Lowering induction temperature to 20 oC led to an increase in enzyme activity of 1.2 to 20-fold, suggesting that protein folding or post-translational processes may be limiting factors for rLlALP2 production. Cumulatively, optimization of copy number, gene sequence optimization and reduced temperature led to increase of rLlALP2 enzyme activity by three-fold (25-30 mg/L). In an effort to simplify the purification process of rLlALP2, extracellular expression of phytase was investigated. Extracellular expression is dependent on the presence of an appropriate secretion signal upstream of the transgene native signal peptide(s) present in the transgene may also influence secretion efficiency. The data suggest that deletion of both N- and C-terminal signal peptides of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion of LlALP2 by four-fold. The secretion signal peptide of chicken egg white lysozyme was ineffective in secretion rLlALP2 in P. pastoris. To enhance rLlALP2 secretion, effectiveness of the strong inducible promoter (PAOX1) was compared with the constitutive promoter (PGAP). The intracellular yield of rLlALP2 was about four-fold greater under the control of PGAP compared to PAOX1 and extracellular expression level of rLlALP2 was around eight-fold (75-100 mg/L) greater. The successful production of active rLlALP2 in P. pastoris will allow us to conduct the animal feed supplementation studies and structural studies.