Search

Abstract Isolation of extracellular vesicles (EV) has been developing rapidly in parallel with the interest in EVs. However, commonly utilized protocols may not suit more challenging sample matrixes and could potentially yield suboptimal results. Knowing and assessing the pitfalls of isolation procedure to be used, should be involved to some extent for EV analytics. EVs in cow milk are of great interest due to their abundancy and large‐scale availability as well as their cross‐species bioavailability and possible use as drug carriers. However, the characteristics of milk EVs overlap with those of other milk components. This makes it difficult to isolate and study EVs individually. There exists also a lack of consensus for isolation methods. In this study, we demonstrated the differences between various differential centrifugation‐based approaches for isolation of large quantities of EVs from cow milk. Samples were further purified with gradient centrifugation and size exclusion chromatography (SEC) and differences were analyzed. Quality measurements were conducted on multiple independent platforms. Particle analysis, electron microscopy and RNA analysis were used, to comprehensively characterize the isolated samples and to identify the limitations and possible sources of contamination in the EV isolation protocols. Vesicle concentration to protein ratio and RNA to protein ratios were observed to increase as samples were purified, suggesting co‐isolation with major milk proteins in direct differential centrifugation protocols. We demonstrated a novel size assessment of vesicles using a particle mobility analyzer that matched the sizing using electron microscopy in contrast to commonly utilized nanoparticle tracking analysis. Based on the standards of the International Society for Extracellular Vesicles and the quick checklist of EV‐Track.org for EV isolation, we emphasize the need for complete characterization and validation of the isolation protocol with all EV‐related work to ensure the accuracy of results and allow further analytics and experiments.

The importance of cereals and pulses in the diet is widely recognized, and consumers are seeking for ways to balance their diet with plant-based options. However, the presence of antinutritional factors reduces their nutritional value by decreasing the bioavailability of proteins and minerals. This study’s aim was to select microbes and fermentation conditions to affect the nutritional value, taste, and safety of products. Single lactic acid bacteria (LAB) strains that reduce the levels of antinutrients in faba bean and pea were utilized in the selection of microbes for two starter mixtures. They were studied in fermentations of a faba bean–oat mixture at two temperatures for 24, 48, and 72 h. The levels of antinutrients, including galacto-oligosaccharides and pyrimidine glycosides (vicine and convicine), were determined. Furthermore, a sensory evaluation of the fermented product was conducted. Fermentations with selected single strains and microbial mixtures showed a significant reduction in the content of antinutrients, and vicine and convicine decreased by up to 99.7% and 96.1%, respectively. Similarly, the oligosaccharides were almost completely degraded. Selected LAB mixtures were also shown to affect the product’s sensory characteristics. Microbial consortia were shown to perform effectively in the fermentation of protein-rich materials, resulting in products with improved nutritional value and organoleptic properties.

The effect of carbohydrate-hydrolysing enzyme blend with or without supercritical CO2 (SFE) defatting on pretreat hempseed meal, hempseeds, peeled hempseeds, hempseed protein powder, and germinated hempseeds was determined. The raw materials and recovered fractions from the treatments were subjected to gel electrophoresis, and their emulsion capacity, activity, and stability as well as colour (CIE L∗a∗b∗ values) were determined. The highest protein contents, 65% (w/w dm), were detected in soluble fractions prepared from germinated, defatted hempseeds followed by soluble fractions of peeled, defatted hempseed, 55% (w/w dm). The gel electrophoresis showed quite similar protein profiles for all samples; however, the edestin content was lower in the germinated samples than in the others. Enzyme treatment and SFE did not have a significant effect on the emulsion properties. Germinated samples demonstrated a higher ability to stabilise emulsions (15-20%) than other pretreated samples. On the other hand, hempseed meal samples had lower emulsification activity and stability values compared to the other samples. The colour of the sample solutions varied from light to dark with a brown to yellowish colour, and PHS samples showed overall higher L∗ values. In conclusion, germination and peeling in combination with defatting are promising methods to produce functional protein concentrates with efficient emulsion stability and activity as well as a mild colour for food applications.