Isolation, morphological and molecular characterization of phytate-hydrolysing fungi by 18S rDNA sequence analysis

Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to efficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing fungi were isolated using phytate specific medium. Fungal isolates were selected according to their higher phytase activities. These isolates were further characterized and identified by morphological and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers. This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed additive in poultry and swine industries.

Fósforo fítico y actividad de fitasa en fórmulas infantiles basadas en cereales

El ácido fítico es un ácido orgánico presente en granos de cereales. El fósforo contenido en su molécula (PFi) no es absorbible por una baja solubilidad, aunque su biodisponibilidad puede incrementarse por la actividad de enzimas fitasas (AFi). Con el objetivo de cuantificar el contenido de PFi y AFi en suplementos elaborados a partir de cereales y destinados a infantes lactantes, se seleccionaron cinco fórmulas de amplia distribución en mercados locales,identificadas de acuerdo a los cereales base de su composición como: A (arroz), T (trigo), ATS (arroz, trigo y aislado de soya), ATM (arroz, trigo y maíz) y ATMS (arroz, trigo, maíz y aislado de soya). Se tomaron cinco muestras por fórmula, cada una correspondiente a un envase (400 a 500 g) elaborado en lotes diferentes y con fecha de vencimiento posterior al muestreo. La proteína cruda varió de 16,8 a 7,2%, con el mayor valor para ATS, y los menores para T y ATM (P< 0,01). El extracto etéreo mostró un rango de 0,31 a 0,75%, mientras la relación calcio:fósforo entre 1,6 en ATM y 1,1 para el resto. El PFi representó el 61,5% del fósforo total en T, con diferencias (P< 0,01) respecto al resto (39,9 ± 6,8%). Sólo se detectó AFi en ATM, T y ATS (570, 300 y 152 U/kg, respectivamente). Se concluye que la composición química se ajustó a lo reportado por los fabricantes, con un elevado contenido de PFi y una AFi fundamentalmente asociados a la presencia de trigo en las fórmulas.

Phytic phosphorus and phytase activity in cerealbased infant formulas. Phytic acid is an organic acid present in cereal grains. The phosphorus inside its molecule (PPhy) is not available because of its low solubility, though the bioavailability could be increased by the activity of phytase enzymes (PhyA). With the purpose of quantifying the PPhy and PhyA in supplements manufactured from cereals and intended for lactating infants, five formulas of wide distribution in local markets were selected and identified depending on the main vegetable ingredients as A (rice), T (wheat), ATS (rice, wheat and soy isolate), ATM (rice, wheat and maize) and ATMS (rice, wheat, maize and soy isolate). Five samples were taken from each formula, each one corresponded to a commercial brand (400 to 500 g), coming from different batches and before their expiration date. The crude protein ranged from 7.2 to 16.8%, with de highest value for ATS and the lowest for T and ATM (P< 0.01). Ether extract varied from 0.31 to 0.75%, while the calcium: phosphorus ratio from 1.6 for ATM, to 1.1 for the rest. The PPhy was 61.5% of the total phosphorus in T, with differences (P<0.01) compared to the other formulas (39.9 ± 6.8%).PhyA was only detected in ATS, T and ATM (152,300 and 570 U/kg, respectively). The chemical composition complied with the manufacturer report, with a high content of PPhy and PhyA associated to the wheat presence in the formula.

Determination of plasma oxalate with chloride ion insensitive oxalate oxidase.

A simple colorimetric method has been developed for determination of oxalate in plasma using a C1-insensitive oxalate oxidase purified from grain sorghum leaves. The ultrafiltered plasma collected in 3.5 N HC1 was pretreated with NaNO2 to avoid possible interference by ascorbate. The minimum detection limit of the method is 0.225 mg/l. The % recovery of the added oxalate was 91.5 +/- 5.0 (mean +/- S.D., n = 15). The coefficient of variation within and between batch were < 3 and < 5 respectively. The mean plasma oxalate concentration in healthy subjects was 0.29 mg/l. The method has the advantage over other enzymic methods that it doesn't require the removal of C1- prior to oxalate assay.

Purification and characterization of ribulose-1,5-bisphosphate carboxylase from triticale.

Ribulose-1,5-bisphosphate carboxylase has been isolated from a synthetic cereal triticale and purified using a newly developed rapid procedure involving precipitation with ammonium sulphate (35-55% saturation), DEAE-cellulose (DE-52) chromatography and filtration through Sepharose CL-68. Molecular weights of the enzyme subunits are 15.5 and 52 kDa which corresponds to 540 kDa for the hexadecameric holoenzyme. Isoelectric focussing showed that the enzyme has a pI of 4.2. Various kinetic constants determined under aerobic conditions are: Km (CO2), 118 microM; Km (RuBP), 220 microM (at 20 mM NaHCO3) and Vmax, 690 nmole CO2 fixed/mg enzyme/min.

Soybean varietal tolerance to inter cropping with semi-dwarf grain sorghum

Two soybean [Glycine max [L. Merrill] varieties; viz. Clark-63 and Cuttler-71 were intercropped with the grain sorghum [Sorghum bicolor Moenc] variety Dorado in a 2 + 2 row intercropping pattern. Both soybean varieties and the grain sorghum variety were also separately planted with the solid recommended plant densities [Solid I] as well as in comparative treatments where the same plant densities were applied under intercropping [Solid II]. Sorghum grain yield per plant was increased by 47 and 35 percent from the comparative monoculture treatment yield due to the intercropping with Cuttler and Clark varieties respectively. On the other hand, soybean pod-set, seed yield per plant and seed index were reduced under intercropping, the reduction in these yield components was greater for Cuttler than for Clark variety. Land Equivalent Ratio [LER] values increased in efficiency by 17 and 7 percent when grain sorghum was intercropped with Clark and Cuttler varieties, respectively. The competitive Ratio [CR] values indicated that one grain sorghum plant was equivalent to 2.45 and 1.60 soybean plants of Cuttler and Clark varieties, respectively. Choice of a suitable soybean variety under this intercropping pattern may play an important role for increasing land equivalent ratio under conditions of the experiment