Influence of the Nucleophile on the Candida antarctica Lipase B‐Catalysed Resolution of a Chiral Acyl Donor

The resolution of methyl (±)‐3‐hydroxypentanoate catalysed by Candida antarctica lipase B has been performed by using ammonia and benzyl amine as nucleophiles. In all cases, the lipase reacts faster with the R enantiomer of the ester, but when benzyl amine is used, the enantiomeric ratio is approximately three times as high as that measured for ammonia. The analysis of the molecular dynamics simulations carried out over the corresponding deacylation transition state analogues indicated specular binding modes between enantiomers that vary greatly upon the nucleophile used. For the case of ammonia, an intramolecular hydrogen bond between the β‐hydroxyl group and the protons of the nucleophile is established. However, the presence of the substituent in benzyl amine disrupts this interaction. Instead, the acyl chain binds to a more restrictive area of the protein where the higher number of contacts established with the side chains of Thr40, Gln157 and Ile189 have been identified as the reason for the higher enantioselectivity observed in the aminolysis reaction.     

Effect of high pressure homogenization on lactic acid bacteria phages and probiotic bacteria phages

To investigate the effect of high pressure homogenization on virus inactivation, phages specific for Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus paracasei and Lactobacillus plantarum were studied. The influence of pressure, number of passes, suspension medium and phage concentration were studied at 25 °C. Reductions in viability were proportional to pressure and number of passes, though the inactivation extent was phage-dependent. At 100 MPa, some bacteriophages were completely inactivated (6 log₁₀ reduction) after 3 or 5 passes, while others remained infective after 8 passes. For all phages, treatment at 60 MPa was insufficient for complete inactivation, even after 8 passes. No clear influence of suspending medium was observed. Inactivation seems to depend on phage concentration; the higher the initial load, the bigger the reduction achieved. Although these results showed that several phages studied are resistant to high-pressure homogenization, this strategy could be combined with others to control their presence in raw milk.     

Effect of ester group bulkiness of polyacrylates on miscibility with poly(vinyl acetate)

BACKGROUND: The miscibility of poly(vinyl acetate) (PVAc) with a series of polyacrylates, namely poly(methyl acrylate) (PMA), poly(ethyl acrylate) (PEA) and poly(butyl acrylate) (PBA), with ester chains of varying bulkiness was studied from both qualitative and quantitative points of view. RESULTS: The evidence from thermal (differential scanning calorimetry) and Fourier transform infrared analyses demonstrates that the miscibility of PVAc with the polyacrylates is rapidly reduced on increasing the bulkiness of their ester chains. Miscibility is quantitatively discussed in the framework of the corresponding states theory applying the approximate Patterson form. For this reason the characteristic values of volume, temperature and pressure were preliminarily evaluated both experimentally, through dilatometric measurements, and by calculations. At the same time, calorimetric measurements allowed the evaluation of the enthalpies of mixing of the components. CONCLUSION: The conclusions reached with this approach are that polar interactions are active among the repeat units of the blend components, but the intensities of these forces are progressively reduced by the shielding of the apolar groups of the polyacrylates. The steric hindrance of the hydrocarbon groups shields the interactions of the polar groups of PVAc with the COOR moieties of the acrylates, so that the miscibility progressively decreases from PMA to PEA to PBA. Copyright © 2009 Society of Chemical Industry     

β-SiALON obtained from carbothermal reduction of kaolinite employing sample controlled reaction temperature (SCRT)

β-SiALON has been obtained from carbothermal reduction of kaolinite by applying the sample controlled reaction temperature (SCRT) method. By using this technique for the synthesis it has been possible to obtain composites with different percentages of SiALON and with different microstructures.     

Ion mobility-mass spectrometry reveals the influence of subunit packing and charge on the dissociation of multiprotein complexes

The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments.     

Vitamin D3 Enriches Ceramide Content in Exosomes Released by Embryonic Hippocampal Cells

The release of exosomes can lead to cell–cell communication. Nutrients such as vitamin D3 and sphingolipids have important roles in many cellular functions, including proliferation, differentiation, senescence, and cancer. However, the specific composition of sphingolipids in exosomes and their changes induced by vitamin D3 treatment have not been elucidated. Here, we initially observed neutral sphingomyelinase and vitamin D receptors in exosomes released from HN9.10 embryonic hippocampal cells. Using ultrafast liquid chromatography tandem mass spectrometry, we showed that exosomes are rich in sphingomyelin species compared to whole cells. To interrogate the possible functions of vitamin D3, we established the optimal conditions of cell treatment and we analyzed exosome composition. Vitamin D3 was identified as responsible for the vitamin D receptor loss, for the increase in neutral sphingomyelinase content and sphingomyelin changes. As a consequence, the generation of ceramide upon vitamin D3 treatment was evident. Incubation of the cells with neutral sphingomyelinase, or the same concentration of ceramide produced in exosomes was necessary and sufficient to stimulate embryonic hippocampal cell differentiation, as vitamin D3. This is the first time that exosome ceramide is interrogated for mediate the effect of vitamin D3 in inducing cell differentiation.     

Partial Characterization of Nine Bacteriocins Produced by Lactic Acid Bacteria Isolated from Cold-Smoked Salmon with Activity against Listeria monocytogenes

Nine LAB bacteriocin-producers, isolated from vacuum-packaged cold-smoked salmon (CSS), were phenotypically and genotypically identified as Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Enterococcus faecium, and Pediococcus acidilactici. Their bacteriocins were partially characterized. The antimicrobial spectrum was determined against Listeria monocytogenes, E. faecalis, E. faecium, and Staphylococcus aureus. The molecular size of bacteriocins ranged from 2.8 to 4.5 kDa. They were inactivated by treatment with proteolytic enzymes but not by lipolytic or glycolytic enzymes. Maximal activity against L. monocytogenes ranged between 800 and 10000 AU/mL at pH 6.5. Most of the bacteriocins maintained full activity in a pH range of 2.0 to 8.0 but were partially or completely inactivated at pH 10.0. After heating at 60°C and 100°C, only two bacteriocins from Lb. curvatus strains partially lost activity. All bacteriocins showed a narrow spectrum of activity and a high anti-listerial activity, which is characteristic of the class IIa bacteriocins. Isolated bacteriocin-producing LAB could be used successfully in the bio-preservation of CSS and development of new potential bio-preservatives for CSS active against L. monocytogenes.     

Chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil

The anti-yeast activity of lemon grass oil was evaluated against several food spoiling yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailii, Aureobasidium pullulans, Candida diversa, Pichia fermentans, Pichia kluyveri, Pichia anomala and Hansenula polymorpha) through disc diffusion and microbroth dilution method. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) varied from 0.28 to 1.3 mg/ml and 0.56 to 4.5 mg/ml, respectively, where highest MIC (2.25 mg/ml) was shown by A. pullulans and lowest MIC (0.28 mg/ml) was shown by C. diversa and P. anomala. Kill time assay was conducted for selected three yeast strains where S. cerevisiae showed highest reduction (3 log cfu) in viability within 24 h exposure of lemon grass oil at MFC level. Further, the anti-yeast efficacy of lemon grass oil alone and in combination with thermal treatment was evaluated in real food system i.e. mixed fruit juices. Chemical composition analysis of lemon grass oil by gas chromatography–mass spectrometry (GC–MS) revealed that the dominant compounds were geranial (40.5%), neral (30.7%), geranyl acetate (5.1%), caryophyllene (2.5%). Present results established the superior performance of integrated (thermal–lemon grass oil) treatment over the individual exposure (lemon grass oil or thermal treatment alone) for fruit juice preservation.     

Rapid Quantification of Soyasaponins I and βg in Italian Lentils by High-Performance Liquid Chromatography (HPLC)–Tandem Mass Spectrometry (MS/MS)

In this work, an innovative and fast analytical method for the quantification of soyasaponins I and βg in lentils has been developed. Samples were extracted using 70 % aqueous ethanol at room temperature and then injected into a high-performance liquid chromatography–tandem mass spectrometry system. The correlation coefficients of calibration curves of the analyzed compounds were ≥0.9997. The recoveries obtained by spiking the lentil samples with a standard mixture of soyasaponins I and βg at 50 and 100 mg l^?1 were in the range of 96–101 and 98–103 %, respectively. The validated method was applied to the analysis of 30 lentil samples from central Italy. Soyasaponins I and βg were present in these lentils in concentrations that ranged from 54 to 226 mg kg^?1 and from 436 to 1,272 mg kg^?1, respectively. Our data indicated that lentils cultivated in fields at intermediate altitudes (1,142–1,387 m) showed the highest levels of soyasaponins, a finding confirmed by principal component analysis.