Catalytic Amyloids as Novel Synthetic Hydrolases

Amyloids are supramolecular assemblies composed of polypeptides stabilized by an intermolecular beta-sheet core. These misfolded conformations have been traditionally associated with pathological conditions such as Alzheimer’s and Parkinson´s diseases. However, this classical paradigm has changed in the last decade since the discovery that the amyloid state represents a universal alternative fold accessible to virtually any polypeptide chain. Moreover, recent findings have demonstrated that the amyloid fold can serve as catalytic scaffolds, creating new opportunities for the design of novel active bionanomaterials. Here, we review the latest advances in this area, with particular emphasis on the design and development of catalytic amyloids that exhibit hydrolytic activities. To date, three different types of activities have been demonstrated: esterase, phosphoesterase and di-phosphohydrolase. These artificial hydrolases emerge upon the self-assembly of small peptides into amyloids, giving rise to catalytically active surfaces. The highly stable nature of the amyloid fold can provide an attractive alternative for the design of future synthetic hydrolases with diverse applications in the industry, such as the in situ decontamination of xenobiotics.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.     

Effect of carbendazim and physicochemical factors on the growth and ochratoxin A production of Aspergillus carbonarius isolated from grapes

Carbendazim is a systemic fungicide that is commonly used on several crops (tobacco, fruit, vegetables, cereals, etc.). This fungicide is used to control fungal infections in vineyards. It is indicated against Botrytis cinerea, Uncinula necator, Plasmopara viticola and other fungi and can be used either alone or coupled with other fungicides. However, there is a lack of in-depth studies to evaluate its effectiveness against growth of Aspergillus carbonarius isolated from grapes and OTA production. A medium based on red grape juice was used in this study. Preliminary studies were performed at 0.98 a(w) and 25 degrees C using carbendazim concentrations over a wide range (1-2000 ng/ml medium) to control both growth of a strain of A. carbonarius isolated from grape and its ability to produce OTA. As the lag phase increased considerably at levels > 1000 ng/ml of medium, detailed studies were carried out in the range 50-450 ng/ml of medium at 0.98-0.94 a(w) and 20-28 degrees C. Statistical analysis (multifactor ANOVA) of the data revealed that the three factors assayed and the interactions a(w)-carbendazim concentration and a(w)-temperature had significant effects on lag phase duration. The highest lag-times were observed at 0.94 a(w,) 20 degrees C, and with 450 ng carbendazim/ml. The three factors also had significant effects of the growth rate and there was an interaction between a(w) and temperature. The growth rate of A. carbonarius in these cultures is favoured by high water availability and relatively high temperatures. However, addition of carbendazim at the assayed levels did not significantly influenced fungal growth rate. Accumulation of OTA was studied as a function of four factors (the three previously considered, and time). All factors had significant effects on the accumulation of OTA. There were also two significant interactions (a(w)-temperature and temperature-time). On the basis of the results obtained, carbendazim does not increase the lag phase of A. carbonarius except at relatively low a(w) and temperatures. It does not substantially decrease fungal growth rate once growth is apparent but it appears to cause an increase in OTA accumulation in the medium at the doses assayed. Carbendazim, which is widely used against fungal infections in grape, can positively influence OTA production by A. carbonarius in field, which can increase OTA content in grape juices and wines.     

Quantifying the efficiency and biases of forest Saccharomyces sampling strategies

Saccharomyces yeasts are emerging as model organisms for ecology and evolution, and researchers need environmental Saccharomyces isolates to test ecological and evolutionary hypotheses. However, methods for isolating Saccharomyces from nature have not been standardized, and isolation methods may influence the genotypes and phenotypes of studied strains. We compared the effectiveness and potential biases of an established enrichment culturing method against a newly developed direct plating method for isolating forest floor Saccharomyces spp. In a European forest, enrichment culturing was both less successful at isolating Saccharomyces paradoxus per sample collected and less labour intensive per isolated S. paradoxus colony than direct isolation. The two methods sampled similar S. paradoxus diversity: The number of unique genotypes sampled (i.e., genotypic diversity) per S. paradoxus isolate and average growth rates of S. paradoxus isolates did not differ between the two methods, and growth rate variances (i.e., phenotypic diversity) only differed in one of three tested environments. However, enrichment culturing did detect rare Saccharomyces cerevisiae in the forest habitat and also found two S. paradoxus isolates with outlier phenotypes. Our results validate the historically common method of using enrichment culturing to isolate representative collections of environmental Saccharomyces. We recommend that researchers choose a Saccharomyces sampling method based on resources available for sampling and isolate screening. Researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments may also have more success using enrichment culturing. We include step-by-step sampling protocols in the supplemental materials.     

Survival of Listeria innocua in dry fermented sausages and changes in the typical microbiota and volatile profile as affected by the concentration of nitrate and nitrite

The involvement of nitrate and nitrite in the formation of N-nitrosamines in foods is a matter of great concern. This situation has led to revise the real amount of nitrate and nitrite needed in meat products to exert proper technological and safety activities, and also to extensive research to find alternatives to their use. The present study addresses the possibility of reducing the ingoing amounts of these additives below the legal limits established by the current European regulations. Different concentrations of nitrate and nitrite were tested on Spanish salchichón-type dry fermented sausages concerning their role in the microbiota and volatile profile. Sausages were manufactured with the maximum ingoing amounts established by the EU regulations (150 ppm NaNO₃ and 150 ppm NaNO₂), a 25% reduction and a 50% reduction; control sausages with no nitrate/nitrite addition were also prepared. The mixtures were inoculated with 5 log cfu/g of Listeria innocua as a surrogate for Listeria monocytogenes. L. innocua numbers in the final product were approximately 1.5 log cfu/g lower in the batch with the maximum nitrate/nitrite concentration when compared to 25 and 50% reduced batches, and about 2 log cfu/g in comparison to the control sausages. The final numbers of catalase-positive cocci were 1 log cfu/g higher in the 50% nitrate/nitrite reduced batch and 2 log cfu/g higher in the control sausages, compared to products manufactured with the maximum nitrate/nitrite concentration. This increase was related to a higher amount of volatile compounds derived from carbohydrate fermentation and amino acid degradation. Sausages with no addition of nitrate/nitrite showed higher amount of volatiles from lipid oxidation. Enterobacteriaceae counts reached detectable values (1-2 log cfu/g) in both nitrate/nitrite reduced sausages and in the control batch, while these organisms were not detected in the batch with the maximum ingoing amount. Nitrate and nitrite exerted a significant effect on the typical microbiota of dry fermented sausages and effectively contributed to control Listeria. These considerations should be taken into account in view of a future restriction in the use of these curing additives.     

Novel Foams Based on Freeze‐Dried Renewable Vital Wheat Gluten

A new way of producing rigid or semi‐rigid foams from vital wheat gluten using a freeze‐drying process is reported. Water/gluten‐based mixtures were frozen and freeze‐dried. Different foam structures were obtained by varying the mixing process and wheat gluten concentration, or by adding glycerol or bacterial cellulose nanofibers. MIP revealed that the foams had mainly an open porosity peaking at 93%. The average pore diameter ranged between 20 and 73 µm; the sample with the highest wheat gluten concentration and no plasticizer had the smallest pores. Immersion tests with limonene revealed that the foams rapidly soaked up the liquid. An especially interesting feature of the low‐wheat‐concentration foams was the “in situ” created soft‐top‐rigid‐bottom foams.

     

Current Trends in Pretreatment and Fractionation of Lignocellulose as Reflected in Industrial Patent Activities

The pretreatment process to disintegrate lignocellulose and to fractionate its three main components hemicellulose, cellulose, and lignin, is a crucial step to enable sustainable and economic value chains based on biomass feedstock. This review provides an overview of the recent patenting activities on pretreatment. Most of these activities focus on optimization of different known processes to improve economics, such as increased catalyst efficiency, effluents recirculation, or lignin valorization. However, also a number of patents and demonstration activities based on emerging concepts for pretreatments are observed.