Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Anisotropic mesh optimization and its application in adaptivity

The construction of solution-adapted meshes is addressed within an optimization framework. An approximation of the second spatial derivative of the solution is used to get a suitable metric in the computational domain. A mesh quality is proposed and optimized under this metric, accounting for both the shape and the size of the elements. For this purpose, a topological and geometrical mesh improvement method of high generality is introduced. It is shown that the adaptive algorithm that results recovers optimal convergence rates in singular problems, and that it captures boundary and internal layers in convection-dominated problems. Several important implementation issues are discussed. © 1997 John Wiley & Sons, Ltd.     

Development and immunobiological evaluation of nanoparticles containing an immunodominant epitope of herpes simplex virus

Herpes simplex virus (HSV) 1 and 2 are viruses that infect individuals worldwide and for which there is no cure or vaccine available. The protective response against herpes is mostly mediated by CD8 T lymphocytes that respond to the immunodominant SSIEFARL epitope. However, there are some obstacles concerning the use of free SSIEFARL for vaccine or immunotherapy. The aim of this study was to evaluate the feasibility of nanoencapsulation of SSIEFARL and its immunostimulatory properties. Nano/SSIEFARL was produced by interfacial polymerization in methylmetacrylate, and the physico‐chemical properties, morphology and immunobiological parameters were evaluated. To evaluate the ex vivo capacity of Nano/SSIEFARL, we used splenocytes from HSV‐1‐infected mice to enhance the frequency of SSIEFARL‐specific CD8 T lymphocytes. The results indicate that Nano/SSIEFARL has a spherical shape, an average diameter of 352 ± 22 nm, the PDI was 0.361 ± 0.009 and is negatively charged (−26.30 ± 35). The stability at 4°C was 28 days. Also, Nano/SSIEFARL is not toxic for cells at low concentrations in vitro and it is taken up by JAWS II dendritic cells. No histopathological changes were observed in kidneys, liver and lymph nodes of animals treated with Nano/SSIEFARL. Nan/SSIEFARL increased the production of IL‐1β, TNF‐α and IL‐12 by the dendritic cells. Finally, Nano/SSIEFARL expanded the frequency of SSIEFARL‐specific CD8+T lymphocytes at the same rate as free SSIEFARL. In conclusion all data together indicate that SSIEFARL is suitable for nanoencapsulation, and the system produced presents some immunoadjuvant properties that can be used to improve the immune response against herpes.     

Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis

The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined. The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases. Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus. The nucleotide sequence of CHS2 and its flanking regions has been submitted to GenBank under Accession Number Y09231. © 1998 John Wiley & Sons, Ltd.     

Effect of chitosan coating in preventing deterioration and preserving the quality of fresh‐cut papaya ‘Maradol’

BACKGROUND: Chitosan can form antimicrobial, semi‐permeable barriers that limit gas exchange and reduces water loss in fruits. Consumer interest in fresh‐cut papaya fruit is leading to increasing demand because of its sensorial and antioxidant properties. However, papaya is a highly perishable product that is prone to loss of weight, loss of firmness and microbial attack. The aim of this study was to evaluate the effect of chitosan coatings on the overall quality of fresh‐cut papaya. Chitosan coatings of low (LMWC), medium (MMWC) and high (HMWC) molecular weights, at concentrations of 0.01 and 0.02 g mL^?1, were applied to fresh‐cut papaya cubes. The treated cubes were stored at 5 °C and changes in quality were evaluated. RESULTS: MMWC maintained the highest color values (L* and b*) and firmness. Chitosan coatings suppressed mesophilic plate count, and the growth of molds and yeast, compared to controls. The MMWC coatings at 0.02 g mL^?1 resulted in the highest antimicrobial activity and decreased the activity of the enzymes polygalacturonase and pectin methylesterase, followed by low and high MW chitosan coatings at 0.02 g mL^?1. CONCLUSION: The application of the MMWC treatment at 0.02 g mL^?1 could be used to reduce deteriorative processes, maintain quality and increase the shelf life of fresh‐cut papaya stored at 5 °C. Copyright © 2008 Society of Chemical Industry     

Effect of Lactobacillus plantarum and chitosan in the reduction of browning of pericarp Rambutan (Nephelium lappaceum)

The effects of Lactobacillus plantarum alone or in combination with chitosan were evaluated on quality and color retention in rambutan fruits (Nephelium lappaceum) stored at 25 °C and 10 °C with 75 ± 2.5% of relative humidity for 10 and 15 days, respectively. The development of the microorganisms was evidenced by viability analyses and lactic acid production. The application of L. plantarum significantly improved color retention (a* and L*), and reduced weight losses. The lactobacilli, alone or in combination with chitosan, preserved fruit quality characteristics such as firmness, total soluble solids and titratable acidity. The lactobacilli application on rambutan pericarp produced acidification of pericarp and avoided the browning; thereby desiccation was prevented due to biofilm formation.     

Microscopic and spectroscopic characterization of paintbrush-like single-walled carbon nanotubes

Understanding and controlling the chemical reactivity of carbon nanotubes (CNTs) is a fundamental requisite to prepare novel nanoscopic structures with practical uses in materials applications. Here, we present a comprehensive microscopic and spectroscopic characterization of carbon nanotubes which have been chemically modified. Specifically, scanning tunneling microscopy (STM) investigations of short-oxidized single-walled carbon nanotubes (SWNTs) functionalized with aliphatic chains via amide reaction reveal the presence of bright lumps both on the sidewalls and at the tips. The functionalization pattern is consistent with the oxidation reaction which mainly occurs at the nanotube tips. Thermogravimetric analysis (TGA), steady-state electronic absorption (UV-vis-NIR), and Raman spectroscopic studies confirm the STM observations.     

Influence of high hydrostatic pressure on the proteolysis of beta-lactoglobulin A by trypsin

This work describes the effect of the hydrolysis time and pressure (0.1-400 MPa) on the proteolysis of beta-lactoglobulin A (beta-lg A) with trypsin, either conducting hydrolysis of beta-lg under pressure or hydrolysing beta-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of beta-lg. Trypsin degraded pressure-modified beta-lg and pressure-induced beta-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val(15)-Arg(40)), (Val(41)-Lys(69))S-S(Leu(149)-Ile(162)) and (Val(41)-Lys(70))S-S(Leu(149)-Ile(162)). These were further cleaved at the later stages of proteolysis to yield: (Val(15)-Tyr(20)), (Ser(21)-Arg(40)), (Val(41)-Tyr(60)), (Trp(61)-Lys(69))S-S(Leu(149)-Ile(162)) and (Trp(61)-Lys(70))S-S(Leu(149)-Ile(162)). Particularly, in the tryptic hydrolysates of pre-pressurized beta-lg, two other fragments linked by disulphide bonds: (Lys(101)-Arg(124))S-S(Leu(149)-Ile(162)) and (Tyr(102)-Arg(124))S-S(Leu(149)-Ile(162)), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys(121) and Cys(160), that normally forms the disulphide bond Cys(66)-Cys(160). In the light of these results, structural modifications of beta-lg under high pressure are discussed.