Review: current trends in oat protein recovery and utilization in aqueous food systems

Oat protein itself, as a substance, has extensively been studied providing information on its nutritional value, some functional properties and possible applicability in food systems. Chosen protein isolation methods and technological aspects define final composition of obtained oat protein product, its concentration, nutrition value and its functionality in food industry. Scientific data on oat protein recovery methods, typically relying on protein solubility or dry fractionation, provides an insufficient knowledge about the success in commercialization of oat protein recovery technologies and their derivatives in form of oat protein. The aim of the study was to analyse and summarize the research findings on oat protein extraction methods and functional properties of oat protein. Semi-systematic, monographic methods were used to analyse the oat protein isolation techniques, functional properties of oat protein in aqueous food systems, covering the latest information on oat protein extraction methods. Wet and dry isolation methods were demonstrated as main methods in oat protein extraction. Functional properties of oat protein, such as thermal stability, solubility, emulsification, water hydration capacity and foaming were reviewed and evaluated, identifying limitations and protein alterations which occur through the oat protein extraction process. The study provides recent trends in oat protein recovery technologies, along with an overview of current and potential oat protein utilization in food systems.

High throughput method development and optimised production of leaf protein concentrates with potential to support the agri-industry

Invasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.

Single-sample proteome enrichment enables missing protein recovery and phenotype association

Proteomic studies characterize the protein composition of complex biological samples. Despite recent developments in mass spectrometry instrumentation and computational tools, low proteome coverage remains a challenge. To address this, we present Proteome Support Vector Enrichment (PROSE), a fast, scalable, and effective pipeline for scoring protein identifications based on gene co-expression matrices. Using a simple set of observed proteins as input, PROSE gauges the relative importance of proteins in the phenotype. The resultant enrichment scores are interpretable and stable, corresponding well to the source phenotype, thus enabling reproducible recovery of missing proteins. We further demonstrate its utility via reanalysis of the Cancer Cell Line Encyclopedia (CCLE) proteomic data, with prediction of oncogenic dependencies and identification of well-defined regulatory modules. PROSE is available as a user-friendly Python module from https://github.com/bwbio/PROSE.

Latest Advances in Protein-Recovery Technologies from Agricultural Waste

In recent years, downstream bioprocessing industries are venturing into less tedious, simple, and high-efficiency separation by implementing advanced purification and extraction methods. This review discusses the separation of proteins, with the main focus on amylase as an enzyme from agricultural waste using conventional and advanced techniques of extraction and purification via a liquid biphasic system (LBS). In comparison to other methods, such as membrane extraction, precipitation, ultrasonication, and chromatography, the LBS stands out as an efficient, cost-effective, and adaptable developing method for protein recovery. The two-phase separation method can be water-soluble polymers, or polymer and salt, or alcohol and salt, which is a simpler and lower-cost method that can be used at a larger purification scale. The comparison of different approaches in LBS for amylase purification from agricultural waste is also included. Current technology has evolved from a simple LBS into microwave-assisted LBS, liquid biphasic flotation (LBF), thermoseparation (TMP), three-phase partitioning (TPP), ultrasound-assisted LBS, and electrically assisted LBS. pH, time, temperature, and concentration are some of the significant research parameters considered in the review of advanced techniques.

Quantification of Soy-Derived Ingredients in Model Bread and Frankfurter Matrices with an Optimized LC-MS/MS External Standard Calibration Workflow

The detection and quantification of soy protein is important for food allergen management and identifying the presence of undeclared soy proteins. Heat processing and matrix interactions can affect the accuracy of allergen detection methods. The sensitivity of ELISA methods can be compromised if protein epitopes are modified during processing. Therefore, an MS-based method was evaluated for the recovery of total soy protein in incurred matrices. MS-based quantification of total soy protein was assessed using a combination of external and internal standards. The reproducibility of the standard curves was investigated by comparing within-day and among among-day variation. Incurred samples were prepared using bread and frankfurters as model food matrices. Several soy-derived ingredients were used to prepare the matrices with varying levels of soy protein (1, 10, 50, or 100 ppm total soy protein). A pooled standard curve was used to estimate the total soy protein concentration of the incurred food matrices and the percent total protein recovery. The variation of replicate standard curves between days and among all days was not significant. The differences in slopes obtained from replicate standards run on different days were minimal. The most influential factor on the quantitative protein recovery in incurred samples was the effect of the physical matrix structure on protein extraction. The lowest percent protein recoveries, less than 50%, were calculated for uncooked matrices. The cooked matrices had percent recoveries between 50-150% for all total soy protein levels. Other factors, such as type of ingredient, were determined to be not as impactful on recovery. The MS method described in this study was able to provide sensitive detection and accurate quantification of total soy protein from various soy-derived ingredients present in processed food matrices.

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

Pre-Processing Unsellable Tomatoes into Separated Streams with the Intention of Recovering Protein

PurposeA large proportion of the European Union’s tomato crop is discarded during harvesting and there is a valorisation potential to recover proteins from this waste. MethodsCherry tomatoes were segregated into three separate components: juice, pomace (peels and skins), and seeds. The peels and skins, and seeds were separately hydrolyzed with carbohydrases to determine whether protein recovery could be increased. In addition, a strategy to fractionate the seeds using sequential washing of milled tomato seeds followed by low-speed centrifugation to remove the denser seed hulls and to collect the protein rich kernels remaining in suspension. ResultsThe protein content of the seeds was highest with 27.4% while the peels and skins contained 7.6%. Carbohydrase mediated hydrolysis revealed a minor increase in protein recovery from seeds by 10% using Filta 02L (cellulase, xylananse and β-glucanase), and the quantity of protein recovered from peels and skins increased by 210% using Tail 157 (pectinase, hemicellulase). The strategy to separate the seeds into two fractions, revealed that a higher proportion of the fibre (65%) was associated with the hull fraction compared with the original seeds (47%). A significant proportion of the fibre in this fraction was composed lignin although the protein contents between both fractions was similar ranging from 27.4% to 29.9%. ConclusionsThese results reveal that carbohydrases were quite effective in protein extraction from peels and skins, but not from seeds. An alternative strategy was developed to remove the seed hulls from the milled seeds and to collect a crude protein fraction where the protein content could be further improved.

High-Performance Passive Plasma Separation on OSTE Pillar Forest

Plasma separation is of high interest for lateral flow tests using whole blood as sample liquids. Here, we built a passive microfluidic device for plasma separation with high performance. This device was made by blood filtration membrane and off-stoichiometry thiol–ene (OSTE) pillar forest. OSTE pillar forest was fabricated by double replica moldings of a laser-cut polymethylmethacrylate (PMMA) mold, which has a uniform microstructure. This device utilized a filtration membrane to separate plasma from whole blood samples and used hydrophilic OSTE pillar forest as the capillary pump to propel the plasma. The device can be used to separate blood plasma with high purity for later use in lateral flow tests. The device can process 45 μL of whole blood in 72 s and achieves a plasma separation yield as high as 60.0%. The protein recovery rate of separated plasma is 85.5%, which is on par with state-of-the-art technologies. This device can be further developed into lateral flow tests for biomarker detection in whole blood.