Physicochemical, total phenols and pectin methylesterase changes on quality maintenance on guava fruit (Psidium guajava L.) coated with candeuba wax solid lipid nanoparticles-xanthan gum.

The objective of this work was to evaluate the application of candeuba wax solid lipid nanoparticles (SLN) and xanthan gum (XG) as coatings on guava, and their effect on the fruit's physicochemical and nutritional parameters, complementing a previous publication carried out by Zambrano-Zaragoza et al. (2013). The concentrations of SLN were selected according to those reported as the most (65g/L) and least (75g/L) efficient in post-harvest life preservation, and were compared to a coating of XG and untreated control samples. According to results, the submicron-sized systems used in the coatings with a particle size range of 267-344nm, a polydispersity index <0.2, and zeta potential of -22.8 to -30mV remained stable during 8weeks of storage. The best results were from the fruits coated with 65g/L of SLN and stored at 10°C, as they showed the lowest O2 and CO2 respiration rates and, consequently, less weight loss. They also had the best retention of ascorbic acid and total phenol content, with less change in fruit color compared to the control guava and those coated only with XG. These findings indicate that this batch continued their natural maturation process, but at a slower rate than the other samples. The firmness was affected by the activity of the enzyme pectin methylesterase, but results show that the 65g/L coating was efficient in maintaining fruit texture. In contrast, the 75g/L coating produced epoxy in the fruit, causing physiological damage. Finally, the guava coated with XG only had a maturation rate similar to that of the control fruit.

Directed evolution of a 13-hydroperoxide lyase (CYP74B) for improved process performance.

The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand.

Productividad y selectividad del medio de cultivo a partir de guayaba agria (psidium araca) en el crecimiento de levaduras nativas del género candida sp / Productivity and selectivity of culture medium from the sour guava (psidium araca) in the growth of native yeast candida sp

El presente trabajo se llevó a cabo para evaluar la eficiencia del medio de cultivo a partir de guayaba agria (Psidium araca) frente a medios comerciales en el crecimiento de tres cepas nativas: Candida guillermondii, Candida famita y Candida sp. Se evaluó el crecimiento microbiano a diferentes concentraciones de fruta, 5, 10, 25 y 50% p/v, tomando como control los medios comerciales: Malta, Sabouraud y agar papa dextrosa (PDA). La productividad y selectividad del medio de guayaba agria fue determinada mediante el método Ecométrico en un tiempo de 48 horas. Los análisis estadísticos aplicados para evaluar y comparar el crecimiento de las cepas en los medios comerciales y en el medio de guayaba agria a diferentes concentraciones demostraron lo siguiente: Candida guillermondii presentó crecimiento mayor o igual a 25 y 50% p/v comparado con los medios comerciales; Candida famata y Candida sp presentaron mejores crecimientos al 5% p/v, con respecto a los diferentes medios comerciales. Los resultados demostraron que el medio de cultivo es altamente productivo y no selectivo, lo que representa una alternativa en la conservación, el mantenimiento y el desarrollo de las levaduras estudiadas.

This work was carried out to evaluate the efficiency of the culture medium from sour guava (Psidium araca) against commercial media in the growth of three native strains: Candida guillermondii, Candida famata and Candida sp. Microbial growth was evaluated at different concentrations of fruit, 5, 10, 25, 50% w /v, using as control the commercial media: Malta, Sabouraud and PDA (Potato Dextrose Agar). The productivity and selectivity of the sour guava medium was determined by the Ecometric method in a time of 48 hours. The applied statistical analysis to evaluate and compare growth of strains in commercial culture medium and in the medium from sour guava at different concentrations showed: Candida guillermondii grew greater than or equal to 25 and 50% w / v compared with commercial medium, Candida famata and Candida sp showed better growth at 5% w / v, with respect to commercial medium. The results showed that the medium is highly productive and non-selective representing an alternative to the conservation, maintenance and development of the yeasts.

Evaluation of guava during different phases of the industrial processing.

In this work we studied the contents of pectin and protein, pectinmethylesterase (PME) activity, and PME stability in various stages of industrial processing, due to the implication of these values on the quality of the final product. The results of the PME stability at different values of pH showed residual PME activity at alkaline pH (7.0, 8.0, 8.5 and 9.5) and high stability at pH 4.0. These results show that pH treatment is not an efficient method to inactivate the PME enzyme. The presence of residual PME activity in all steps of industrial processing was also verified, showing that PME can change the quality of the pulp during storage.

Pectin methylesterase activity and ascorbic acid content from guava fruit, cv. Predilecta, in different phases of development.

The guava pectin methylesterase (PME) specific activity and vitamin C were assayed in samples from different phases of guava fruit development. The PME enzyme from guava was extracted with borate-acetate buffer, 50 mol/l, pH 8.0, in the presence of NaCl 0.3 mol/l. The results showed PME optimum activity at pH 9 and 95 degrees C, and it is a thermostable enzyme. Guava PME retained 96.8% of activity after 300 min in 90 degrees C. Electrophoresis showed that guava PME contained two isoforms, one with 57 kDa molecular mass. The analyses of the different phases of guava maturation showed that ascorbic acid decreases during the maturation process, but PME activity increases with maturation.

Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of fatty acid hydroperoxides into hemiacetals.

The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon (Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short (5 s-1 min) incubations at 0 degrees C followed by rapid extraction and trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s) product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic acid. Identification was performed by GC-MS analysis and substantiated by the formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of [(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their trimethylsilyl derivatives. The kinetics of (18)O incorporation from [(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava (Psidium guajava), and in each case hemiacetals and enols were detectable by the trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs act as isomerases performing a homolytic rearrangement of fatty acid hydroperoxides into short-lived hemiacetals which upon decomposition produce 3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.

Novel 2D maps and coupling numbers for protein sequences. The first QSAR study of polygalacturonases; isolation and prediction of a novel sequence from Psidium guajava L.

The development of 2D graph-theoretic representations for DNA sequences was very important for qualitative and quantitative comparison of sequences. Calculation of numeric features for these representations is useful for DNA-QSAR studies. Most of all graph-theoretic representations identify each one of the four bases with a unitary walk in one axe direction in the 2D space. In the case of proteins, twenty amino acids instead of four bases have to be considered. This fact has limited the introduction of useful 2D Cartesian representations and the corresponding sequences descriptors to encode protein sequence information. In this study, we overcome this problem grouping amino acids into four groups: acid, basic, polar and non-polar amino acids. The identification of each group with one of the four axis directions determines a novel 2D representation and numeric descriptors for proteins sequences. Afterwards, a Markov model has been used to calculate new numeric descriptors of the protein sequence. These descriptors are called herein the sequence 2D coupling numbers (zeta(k)). In this work, we calculated the zeta(k) values for 108 sequences of different polygalacturonases (PGs) and for 100 sequences of other proteins. A Linear Discriminant Analysis model derived here (PG=5.36.zeta1-3.98.zeta3-42.21) successfully discriminates between PGs and other proteins. The model correctly classified 100% of a subset of 81 PGs and 75 non-PG proteins sequences used to train the model. The model also correctly classified 51 out of 52 (98.07%) of proteins sequences used as external validation series. The uses of different group of amino acids and/or axes orientation give different results, so it is suggested to be explored for other databases. Finally, to illustrates the use of the model we report the isolation and prediction of the PG action for a novel sequence (AY908988) isolated by our group from Psidium guajava L. This prediction coincides very well with sequence alignment results found by the BLAST methodology. These findings illustrate the possibilities of the sequence descriptors derived for this novel 2D sequence representation in proteins sequence QSAR studies.

2D RNA-QSAR: assigning ACC oxidase family membership with stochastic molecular descriptors; isolation and prediction of a sequence from Psidium guajava L.

Quantitative structure-activity relationship (QSAR) techniques for small molecules could be applied to nucleic acids. Unfortunately, almost all molecular descriptors are more successful at encoding branching information than sequences and/or cannot be back-projected. A solution for scaling the QSAR problem up to RNA may be to transform sequences into secondary structures first. Our group has used Markovian negentropies as molecular descriptors for drug design with preliminary results in bioinformatics [Bioinformatics 2003, 19, 2079]. However, RNA-QSAR studies on RNA molecules have not been described to date. Novel Markovian negentropies have been introduced here as molecular descriptors for 2D-RNA structures. An RNA-QSAR study of the ACC proteins from different plants has been carried out. The QSAR recognizes 19/20 sequences (95.0%) within the ACC family and 12/17 (70.6%) of the control group sequences. The model has a high Matthews' regression coefficient (C = 0.68). Overall cross-validation average accuracies were 14 out of 15 for ACC sequences (93.3%) and 10 out of 13 for control sequences (76.9%). Finally, ACC oxidase family membership was assigned to a new sequence isolated for the first time in this work from Psidium guajava L. A backprojection map for this sequence identifies the left stem (40%) and the main stem (45%) as highly important substructures. Results of an nBLAST experiment are consistent with this finding and indicate a high conservation score (>70) for left stem and main stem; whereas major loop, right stem, cap and major loop right half were hardly conserved.

Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction.

Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0 degrees C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography-mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant (14)C-labelled compound, identified by its (1)H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.

Biochemistry of fruit ripening of guava (Psidium guajava L.): compositional and enzymatic changes.

Changes in chemical composition and the activities of hydrolytic enzymes during four different stages of maturity, viz. mature green (MG), color turning (CT), ripe (R), and overripe (OR), have been studied in guava fruits cv. Banarsi Surkha. Chlorophyll content decreased while carotenoid content increased during ripening. Starch content decreased with concomitant increase in alcohol-soluble sugars. Cellulose, hemicellulose, and lignin also decreased up to ripe stage, while pectin continued to decrease up to OR stage. PG (polygalacturonase) and cellulase exhibited progressive increase in activity throughout ripening, whereas pectin methyl esterase (PME) activity increased up to CT stage and decreased at R stage. The activities of alpha-amylase and beta-amylase decreased significantly with ripening. The most notable metabolic changes occurred between MG and CT stage, implying that for improved postharvest handling, guava fruits may be harvested at CT stage.