Francisco J. Cinco-Moroyoqui

α-Amylase Activity of Rhyzopertha dominica (Coleoptera: Bostrichidae) Reared on Several Wheat Varieties and Its Inhibition with Kernel Extracts

Total progeny of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) reared on 10 wheat, Triticum aestivum L., varieties was evaluated. Higher amylase activities were detected in populations with few individuals, whereas the opposite was observed in higher populations. As protein ingested increased, reproductive success increased. However, consumption of wheat protein was inversely correlated with amylase activity levels (r = -0.66). Amylase activity in homogenates of R. dominica populations showed variable inhibition by wheat extracts prepared from wheat varieties on which they were reared. Insect populations with lowest amylase activities were inhibited more by wheat extracts than those with higher amylase activity (r = -0.77). An electrophoretic analysis revealed four phenotypes showing combinations of three isoamylases (Rm 0.70, 0.79, and 0.90) in different populations of R. dominica. Some of the insect progeny that emerged from resistant wheat varieties contained the three isoamylases, whereas progeny that emerged from the most susceptible varieties showed reduced activity of isoamylases 0.70 or 0.90. These results suggest that the alpha-amylase activity levels and the composition of isoamylases in R. dominica populations are modulated by diet and that the alpha-amylase inhibitory activity of the wheat kernels influences these variations.

Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae)

Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 degrees C for 1 h, but at 60 degrees C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. beta-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V(max)/K(m) ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V(max)/K(m)=90.4+/-2.3 and 78.9+/-6.6, respectively) was less efficient than that on amylose (V(max)/K(m)=214+/-23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V(max)/K(m)=260.6+/-12.9 and 326.5+/-9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V(max)/K(m)=202.9+/-5.5 and 215.9+/-6.2, respectively), but amylopectin was a poor substrate (V(max)/K(m)=124.2+/-7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.

Identification of α‐amylase inhibitors in triticale grain

In this study three complete triticales, three substituted (one gene from rye has been replaced by one gene from wheat) triticales, and parental wheat and rye were analysed for α-amylase inhibitory activity to evaluate whether the genetic modification influenced triticale α-amylase inhibitory activity. Gel filtration chromatography and thermostability analyses were performed to partially isolate and characterize α-amylase inhibitors. Results demonstrated that substituted triticales and wheat had higher α-amylase inhibitory activities and higher water-soluble protein contents than complete triticales and rye. Sodium dodecylsulfate-PAGE-electrophoresis showed that all triticales, irrespective of their classification, inherited the water-soluble protein patterns from their parents: wheat and rye. In a substituted triticale (Pony ‘S’), two peaks with α-amylase inhibitory activity were resolved by gel filtration chromatography; they were designated T1 and T2 according to their order of elution. T1 showed a higher inhibitory activity but a lower thermostability at 70 °C than T2; T1 apparently comes from wheat, whereas T2 presumably comes from rye. © 1999 Society of Chemical Industry

Starch Debranching Enzyme Activity and Its Effects on Some Starch Physicochemical Characteristics in Developing Substituted and Complete Triticales (X Triticosecale Wittmack)

The present work was carried out to make a comparison between the starch debranching enzyme activity and the synthesis of total carbohydrates, starch, amylose, and amylopectin in developing kernels of two sets of triticales differing in genome composition (complete and substituted triticales). The results showed that the carbohydrate and starch accumulation observed in both genotypes of triticales increased rapidly at the early stage, reaching the maximum at the medium stage and decreasing slightly or remaining relatively constant at the last stage of kernel development. At the end of the development stage, the mature grains of complete and substituted triticales accumulated 62.5 and 54.6% starch, respectively. Both sets of triticales showed the same amylose accumulation profile as well as degree of polymerization; however, at the maturity stage there was a small but significant difference between the two sets. The final polymerization values of amylose chains in mature seeds of complete and substituted t...

Physicochemical characterization of starch from hexaploid triticale (X Triticosecale Wittmack) genotypes

The starch granules of complete and substituted triticale genotypes were analyzed for morphology and size distribution using scanning electron microscopy (SEM) and laser diffraction, respectively. A quantitative comparison of total carbohydrate, starch, and amylose contents was also performed. The results showed that the complete triticales contained 8.8% more total carbohydrate content and 13.8% more starch content than the substituted ones. No significant differences in the average amylose content (22.8%) were observed between the two sets of triticales. However, the A- and B-type starch granules of the substituted triticales showed significant differences in amylose content. Starch granule distribution profiles showed that the sizes of the A- and B-type starch granules of complete triticales were larger (in the range of 18–41 μm and 2–13 μm, respectively) than those of substituted triticales (in the range of 8–38 μm and 0.5–6 μm, respectively). This study demonstrated that the starch in triticale genotypes differs in physicochemical properties.

Comparison of Protein and Starch Content of Substituted and Complete Triticales (X Triticosecale Wittmack): Contribution to Functional Properties

The starch and protein content of substituted and complete triticales were quantitated and evaluated for their contribution to the functional properties of flours by means of the amylograph, farinograph, and the breadmaking test. Although no clear differences were observed in the starch and protein contents of the two triticale genotypes, a detailed analysis of their components revealed compositional differences between them. The amylose content was lower in the substituted triticales Duron S and Alamos 83 than in the complete types Tarasca 87 and Brumby II (P ≤ 0.05). Among the protein fractions, only the albumin and the total polymeric protein, although with an opposite effect, significantly contributed to the rheological properties of triticale doughs evaluated with the farinograph and in the breadmaking test. The SDS-PAGE test showed that the substituted triticales contained an additional HMW-GS band that was not observed in the complete triticales. Amylograph peak temperatures and maximum viscosities, farinograph development times, and loaf volumes of triticale doughs were higher in substituted triticale flours than those of the complete ones. This study demonstrated that the triticale genotypes can be differentiated not only by quantitating their starch and protein composition, but also by testing their functional dough properties.

Aislamiento y caracterización parcial de miosina del manto de calamar gigante (Dosidicus gigas)

In the present work the myosin molecule from jumbo squid mantle (Dosidicus gigas) was isolated and characterized with the aim to evaluate the influence of ionic strength on its gelling properties. It was found that the myosin molecule possesses different chemical and structural characteristics than other vertebrates and invertebrates species, such as some cephalopods, which might explain differences in gelation in comparison to those from other organisms. A lower content of total sulfhydryl groups (TSH) possibly caused an improvement in the myosin molecule flexibility when the ionic strength increased (p ˂ 0.05). The aforementioned possibly affected (p ˂ 0.05) the enzyme activity, surface hydrophobicity, viscosity and RSH groups. The results demonstrate that the myosin molecule from jumbo squid is structurally different from the rest of the marine species.

Contribution and Interactions of Hydroxycinnamic Acids Found in Bran and Wholegrain Sorghum (Sorghum bicolor L. Moench): Effects on the Antioxidant Capacity and Inhibition of Human Erythrocyte Hemolysis

An imbalance between free radicals and antioxidants is known as oxidative stress, and it promotes cellular aging and the development of chronic noncommunicable diseases. The bioactive compounds present in food play an important role in preventing oxidative stress. The aim of this study was to determine the contributions and interactions of the hydroxycinnamic acids found in the bran and whole grain of sorghum and to evaluate their effects on the antioxidant capacity and inhibition of the hemolysis of human erythrocytes. Results showed that the caffeic acid, p-coumaric acid, and ferulic acid found in sorghum contributed to the scavenging of DPPH and ABTS radicals in various proportions. Ferulic acid, which was present in bound form in the bran and wholegrain sorghum, significantly inhibited the AAPH radical-induced oxidation of the erythrocyte membranes by 78.0 and 4.3%, respectively. Combinations of two, three, or four hydroxycinnamic acids may interact in an antagonistic or synergistic manner, thereby altering each others bioactivities. The various interactions between the different sorghum bioactives can have a significant impact on their potential bioactivities. These results can be useful in the design of functional foods that aim to deliver bioactives to mitigate cellular aging or noncommunicable diseases.

Refolding and activation from bacterial inclusion bodies of trypsin I from sardine (Sardinops sagax caerulea)

BACKGROUND Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.

Potentiation of antifungal effect of a mixture of two antifungal fractions obtained from Baccharis glutinosa and Jacquinia macrocarpa plants

ABSTRACT The aim of the present work was to evaluate the effect of mixtures of antifungal fractions extracted from Baccharis glutinosa and Jacquinia macrocarpa plants on the development of the filamentous fungi Aspergillus flavus and Fusarium verticillioides. The minimal inhibitory concentration that inhibited 50% of growth (MIC50) of each plant antifungal fraction was determined from the percentage radial growth inhibition of both fungi. Binomial mixtures made with both plant fractions were used at their MIC50 to determine the Fractional Inhibitory Concentration index (FIC index) for each fungus in order to evaluate their synergistic effect. Each synergistic mixture was analyzed in their effect on spore germination, spore size, spore viability, mitotic divisions, hyphal diameter and length, and number of septa per hypha. Some antifungal mixtures, even at low concentrations, showed higher antifungal effect than those of the individual antifungal fraction. The FIC indices of mixtures that showed the highest antifungal activity against A. flavus and F. verticillioides were 0.5272 and 0.4577, respectively, indicating a synergistic effect against both fungi. Only 12% and 8% of the spores of A. flavus and F. verticillioides, respectively, treated with the synergistic mixtures, were able to germinate, although their viability was not affected. An increase in the number of septa per hypha of both fungi was observed. The results indicated that the synergistic mixtures strongly affected the fungal growth even at lower concentrations than those of the individual plant fractions.