Protein Recovery Research Articles

Heat-induced pressed gels from canola press cakes: Exploring the impact of starting materials, stirring conditions, and carbohydrase pretreatment

Alternative plant protein sources offer excellent solutions for tackling the current challenge of food insecurity and sustainability. Inspired by soy tofu, pressed gels represent a robust and versatile way to create protein-enriched plant products. Here, production of heat-induced pressed gels from canola cold-pressed cakes (CPC) and hot-pressed cakes (HPC) was investigated under varied stirring conditions. Pressed gels prepared from CPC resulted in a greater yield and protein recovery than that of HPC. While using carbohydrases as a pretreatment was ineffective in improving yield and protein recovery, applying a stirring condition during heating increased the protein recovery up to 38.3%. Also, stirring condition was proved to be able to modulate the textural properties by controlling the compactness and the size of aggregates. It is revealed that pressed gels are stabilized through a combination of hydrogen bonds, hydrophobic interactions, and disulfide bonds. In comparison to canola press cake, the pressed gels contained less glucosinolates and phenolic compounds, but more phytic acid. A mechanism of formation has been hypothesized based on the nucleation-growth mechanism, and a shift was proposed from diffusion-limited processes in non-stirred pressed gels to reaction-limited process in stirred pressed gels. In conclusion, the potential of canola heat-induced pressed gels was demonstrated both as a stand-alone product and a micro-structured protein extract.

Recovery of Proteins from Sweet Potato Cell Liquid by Acidification <i>via</i> Inoculation-Enhanced Fermentation and Determination of Functional Properties of Protein Products

Recovery of Proteins from Sweet Potato Cell Liquid by Acidification <i>via</i> Inoculation-Enhanced Fermentation and Determination of Functional Properties of Protein Products

Influence of Different Drying Processes on the Chemical and Texture Profile of Cucurbita maxima Pulp.

The effects of hot air (HAD), vacuum (VAD) and conductive (CD) drying on the chemical and textural profiles of Cucurbita maxima pulp were investigated to find suitable drying conditions to avoid postharvest losses of pumpkin. The results showed that the drying methods had a significant effect (p < 0.05) on the chemical and textural profiles of pumpkin pulp. The ash content was lower in VAD (up to 7.65%) than in HAD (up to 9.88%) and CD pulp (up to 9.21%). The samples of HAD, CD and VAD had a higher fat content, up to 3.07, 2.66 and 2.51%, respectively, than fresh pulp (1.55%). The total fibre content is lower for VAD (up to 8.78%) than for HAD (up to 15.43%) and CD pulp (13.94%). HAD pulp at 70 °C (~15.51%) and VAD and CD pulp processed between 50 and 60 °C (~22%) are good sources of protein. HAD and CD pulp at 70 °C and VAD at 50 °C resulted in a high sugar content (up to 83.23%). In addition to drying, the extraction time of 40 min used in ultrasound-assisted extraction is optimal, especially for protein and sugar recovery in dried samples. Drying also led to strong changes in the textural properties of the pulp, so that an excellent dried intermediate product is the one obtained using HAD at a temperature of 70 °C and an airflow of 0.5 m/s.

Use of high‐intensity ultrasound and micellar casein concentrate addition for improving whey Ricotta cheese production

The influence of high‐intensity ultrasound (E = 42.2 W*s/mL) and coprecipitation processes with micellar casein concentrate (0.5, 1.5% w/w) on improving whey protein recovery during Ricotta cheese elaboration was evaluated. Acid and sweet whey were used. Whey treatment (control, ultrasonicated whey, coprecipitation process and ultrasonication + coprecipitation) was used as the independent variable in a completely randomised design. Since neither acidification nor pH adjustment was required, sonication reduced processing time (~6.6%). Coprecipitation (1.5%) enhanced the cheese yield generating textural defects. Softer textures were obtained by using ultrasonication and coprecipitation, suggesting the possibility of producing ricotta cheeses with higher yields and unique technological features.

Pulsed Electric Field Technology for Recovery of Proteins from Waste Plant Resources and Deformed Mushrooms: A Review

Proteins are complex molecules, which play a vital role in our body’s function, the building of tissues, and the regulation of metabolic activity. They are crucial to children’s growth and serve as a key component in the body’s process of distributing oxygen. Proteins fuel the body by supplying the required nutrition and energy. Currently, there is an increasing demand for proteins on large scales with no detrimental effects. The adverse health effects of animal proteins have resulted in a growing preference for plant-based proteins, which offer a healthier daily dosage. Valuable proteins can be extracted from various parts of the plant, including stems, leaves, seeds, fruits, vegetables, and roots. Notably, protein extraction from waste plant and mushroom parts minimizes the product wastage and improves the overall production to support economic sustainability. There are several protein extraction techniques available, where the replacement of non-thermal methods with thermal ones is promising nowadays due to the appreciable retainment of protein quality. Pulsed Electric Field (PEF) technology is one of the most efficient non-thermal tools used to assist with extracting these proteins at the minimum processing time and energy consumption when compared with thermal techniques. It relies on the application of a high-voltage pulse between two electrodes to treat samples inside the treatment chamber. While electrode shapes and treatment chamber designs primarily govern the electric field’s application, optimizing process parameters such as electric field strength, pulse width, number of pulses, and pulse waveshape assists in obtaining a desirable enhancement in the protein yield. The primary objective of this review is to explain the PEF-assisted protein extraction process applicable to waste plant parts and deformed mushrooms. While PEF is not a novel concept, utilizing it as a pre-extraction treatment to the aforementioned waste resources would aid in improving the production of value-added protein products economically. So far, PEF has shown immense promise in assisting with protein extraction studies, but requires further research in order to establish this area for large-scale industrial applications.

Enhanced Electrophoretic Depletion of Sodium Dodecyl Sulfate with Methanol for Membrane Proteome Analysis by Mass Spectrometry.

Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization.

Proteolytic Activity of Silkworm Thorn (Cudrania tricuspidata) Fruit for Enzymatic Hydrolysis of Food Proteins.

This study aimed to isolate the proteolytic fraction from the silkworm thorn fruit (Cudrania tricuspidata) through ethanol precipitation at different ratios, and to determine its proteolytic activity and optimal activity conditions. Furthermore, the hydrolysis characteristics and antioxidant activity of soy protein isolate (SPI) and whey protein concentrate (WPC) hydrolyzates obtained through the enzymatic hydrolysis of freeze-dried silkworm thorn fruit powder (SF) were evaluated. For isolation and partial purification of proteolytic fraction, the water-solubilized fraction of the silkworm thorn fruit was purified through ethanol precipitation at four different ratios of 1:1, 1:2, 1:4, and 1:6 (v/v). The protein recovery rate, caseinolytic activity, protein pattern, and optimal activity (pH, temperature, and inhibitors) of fractional ethanol precipitate obtained from the silkworm thorn fruit (ESF) were evaluated. The proteolytic fraction obtained from silkworm thorn fruit exhibited a major protein band around 65-70 kDa and showed the highest proteolytic activity at a 1:4 ratio of ethanol precipitation (p < 0.05). The optimal activity of the measured enzyme fraction was determined to be at pH 9.0 and 50 °C, and the proteolytic activity of ESF was almost inhibited by phenyl methyl sulphonyl fluoride (PMSF, 2 mM), a serine protease inhibitor. Compared to Alcalase and papain, extensively used as commercial enzymes, the silkworm thorn fruit powder was less effective in hydrolyzing SPI and WPC. Nevertheless, SPI and WPC hydrolyzates mediated with silkworm thorn fruit powder showed even better antioxidant activities than those mediated with Alcalase and papain. Thus, our results show the potential application of silkworm thorn fruit as a novel source of plant protease for producing human-grade protein hydrolyzates.

Assessing the precision of a detergent-assisted cartridge precipitation workflow for non-targeted quantitative proteomics.

Detergent-based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent-based workflow for quantitative proteomics, we herein evaluate the precision of a bottom-up sample preparation strategy incorporating cartridge-based protein precipitation with organic solvent to deplete SDS. The variance of data-independent acquisition (SWATH-MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS-assisted cartridge workflow yield a coefficient of variance (CV) of 13%-14%. By comparison, conventional (detergent-free) in-solution digestion increased the CV to 50%; in-gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent-based precipitation workflow is a reliable approach for in depth, label-free quantitative proteome analysis.

Compound mutation by ultraviolet and diethyl sulfate of protease producing thermophilic bacteria to hydrolyze excess sludge

Excess sludge (ES), a resource-rich organic waste, can be solubilized by thermophilic enzymes to extract proteins for sludge reduction and resources recovery. To solve the problems of low hydrolysis effect of ES and low enzyme producing ability of wild thermophilic bacteria, ultraviolet and diethyl sulfate (UV-DES) were adopted to mutate thermophilic bacteria in this study. Mutation sites were detected and annotated by whole genome sequencing analysis. The results showed that UV-DES mutagenesis could effectively improve enzyme-producing capacity of thermophilic bacteria and promote the hydrolysis of ES. The protease activity of the mutant strain KT16 was 46.7 % higher than that of the original strain DC8. The protein extraction rate with enzyme produced by KT16 reached 83.3 %. The total content of proteins recycled through KT16 enzyme solution was 3539.6 mg·L-1, 18.4 % higher than that of DC8. This work provided a theoretical idea and technical guidance for the protein recovery from ES.

Effect of ultrasound on physicochemical, functional and antioxidant properties of red kidney bean (Phaseolus vulgaris L.) proteins extract

A substantial quantity of red kidney bean (RKB) by-products (inferior size beans) is discarded during its processing. This study aimed at valorization of RKB side stream through the recovery of proteins and antioxidant compounds by ultrasound (US) treatment. Impact of US on RKB proteins characteristics was conducted at varying power (300–500 W) and time (5–20 min). The US induced changes were assessed in terms of proteins recovery, physico-chemical, functional, in-vitro proteins digestibility and antioxidant activity. The structural, molecular and thermal properties of the US extracted proteins were analyzed. A highest proteins recovery (67.76 ± 1.29 %) was observed at 450 W and 15 min of ultrasonication treatment. Due to high shear, mechanical, energy and cavitation effect of ultrasonication, some properties of RKB proteins extract significantly (p < 0.05) increased with higher ultrasound power and time. Among those the highest values measured were solubility (98.72 ± 0.87%), emulsification ability (111.85 ± 3.34m2/g), and foaming ability (178.88 ± 7.28%), proteins in-vitro digestibility (87.08 ± 0.1%), DPPH radical scavenging activity (57.53 ± 3.3%) and ferric reducing antioxidant power (64.03 ± 2.4 μg BHT/mL) antioxidant potential, and total phenol content (75.60 ± 2.39141 μg GAE/mL). The solubility and free sulfhydryl group content was significantly (p < 0.01) affected by the interaction between ultrasonication power and time. Hence, high US power and time (10–15 min) could be a suitable technology to improve the properties of RKB proteins extract for food processing applications.

Recovery of soy whey protein from soy whey wastewater at various cavitation jet pretreatment time and their structural and emulsifying properties

Protein-polysaccharide composite is of great significance for the development of soluble protein recovery process. This study investigated the effects of cavitation jet (CJ) pretreatment at different time (0, 60, 120, 180, 240, 300 s) intervals on the recovery of soy whey protein (SWP) from soy whey wastewater using chitosan (CH). In addition, the structure and properties of the SWP/CH complexes were examined. The results showed that the recovery yield of SWP reached 84.44 % when the CJ pretreatment time was 180 s, and the EAI and ESI values of the SWP/CH complex increased from 32.39 m2/g and 21 min to 48.47 m2/g and 32 min, respectively. In the CJ pretreatment process, SWP promotes the recombination with chitosan through electrostatic interaction and hydrogen bond, while hydrophobic interaction is also involved. This study has guiding significance for CJ technology in the recovery and utilization of protein in industrial wastewater.

Analytical figures of merit of a dual-wavelength absorbance approach for real-time broad protein content monitoring for biomanufacturing

Real-time in-line broad protein content monitoring in biomanufacturing downstream unit operations enables the ability to optimize and afford consistent protein recovery. Protein determination from 2 to 400 mg/mL is demonstrated herein via real-time dual-wavelength LED photometric sensor configured at 280 and 310 nm. The figures of merit of this approach include measurement accuracy within the common acceptance criteria of 100 % ± 5 with negligible bias across the linear dynamic ranges. This work expands the utility of an LED based photometric sensor for biopharmaceutical process analytical technology (PAT) applications. It is also congruent with process digitalization and automation industry 4.0 concepts underpinned by Quality by Design (QbD) principles.

Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.

Chitosan-based matrixes for recombinant protein purification from LPS

In this study, the chitosan-based matrixes (granules and sponge) were synthesized and characterized as potential materials for protein purification from lipopolysaccharide (LPS) content. Two phage-borne enzymes with medical potential (endolysin 15 kDa and depolymerase 200 kDa) were used as model proteins for purification efficiency measurement. The endolysin purification by the chitosan-based matrix in granule form enables high protein recovery (99%) but with a low level of LPS removal (4%). In contrast, the sponge form exhibited higher efficacy in LPS elimination (64%) and it was accompanied by the loss of ∼28% of the endolysin content. The LPS removal from depolymerase samples was similar for both matrixes forms at the levels of 72% and 78% with a superior capacity over commercially available endotoxin removal resin (62%). The active depolymerase recovery was around 89% and 76% for granules and sponges, respectively. No proteolytic effect and a slight decrease in enzyme activity were observed. In conclusion, the purification efficiency of chitosan-based matrixes from LPS was dependent on the morphology of the matrixes but not on the size of the protein. Our study proved that chitosan-based matrixes are attractive options for recombinant protein pre-clean purposes.

Coupling protein recovery to methane and lactic acid production to valorize duckweed used in selenate remediation

Duckweed (DW) can be used for metal bioremediation, but can also serve as a renewable feedstock for various processes such as protein production, acidogenesis and methanogenesis. In this study, a selenate (3 mg L−1) supplemented modified Steinberg medium was used as a feed for DW cultivation, which gave a selenate removal efficiency of 55%. Thereafter, an optimized protein extraction protocol was developed yielding a∼20% protein extraction efficiency from both the control and selenate-enriched DW. The solid fraction obtained during protein extraction was utilised for lactic acid (LA) production (upto 5 g L-1), whereas the liquid fraction (after protein extraction) was used as the substrate for methane production (∼150 mL gCOD−1). Selenate enriched DW achieved faster LA production and showed better methane production kinetics when compared to the control DW. This study shows that bioenergy and biocommodity production can be efficiently coupled to selenium removal from polluted wastewaters using DW.

Retraction: Photo-responsive hole formation in the monolayer membrane wall of a supramolecular nanotube for quick recovery of encapsulated protein.

[This retracts the article DOI: 10.1039/D2NA00035K.].

Purification and Quality Control of Recombinant Proteins Expressed in Mammalian Cells: A Practical Review.

In the recent years, there has been a rapid development of new technologies and strategies when it comes to protein purification and quality control (QC), but the basic technologies for these processes go back a long way, with many improvements over the past few decades. The purpose of this chapter is to review these approaches, as well as some other topics such as the advantages and disadvantages of various purification methods for intracellular or extracellular proteins, the most effective and widely used genetically engineered affinity tags, solubility-enhancing tags, and specific proteases for removal of nontarget sequences. Affinity chromatography (AC), like Protein A or G resins for the recovery of antibodies or Fc fusion proteins or immobilized metals for the recovery of histidine-tagged proteins, will be discussed along with other conventional chromatography techniques: ion exchange (IEC), hydrophobic exchange (HEC), mixed mode (MMC), size exclusion (SEC), and ultrafiltration (UF) systems. How to select and combine these different technologies for the purification of any given protein and the minimal criteria for QC characterization of the purity, homogeneity, identity, and integrity of the final product will be presented.

Optimization of Ultrasound-Assisted Protein Extraction from Watermelon Seeds: Taguchi Approach

Nowadays, there has been a growing interest in finding alternative protein sources for both the food industry and nutritional purposes. Protein experts have recently focused on investigating watermelon seeds, which are not only a food processing waste but also contain high-quality proteins. Therefore, this study aimed to achieve maximum protein extraction from watermelon seeds using an ultrasound-assisted extraction process. The study investigated the effects of pH (A; 7─11), sonication temperature (B; 30─60 °C), and sonication time (C; 5─15 min) on protein recovery to develop a Taguchi model. Through optimization, the optimal conditions for maximum protein recovery (85.81%) within the range of process variables were found to be 11 pH, 45 °C sonication temperature, and 10 min sonication time (A3B2C2). An analysis of variance (ANOVA) revealed that pH and sonication temperature significantly influenced the protein extraction process (P

Persian everlasting pea (Lathyrus rotundifolius L.) protein isolate as a potential protein source for food application: Effect of ultrasound‐assisted extraction method on the properties of the protein isolates

AbstractFinding new protein sources and processes comprising targeted functional properties is crucial for the increasing global food demand. In the current paper, a new protein isolate is introduced with potential benefits based on the seeds of Persian everlasting pea (Lathyrus rotundifolius L.). Conventional aqueous extraction for the preparation of protein isolates (CPEP) was compared with pre‐ultrasonic (PUPEP) and ultrasound‐assisted (UAPEP) extractions, and the physicochemical and functional properties of the proteins were investigated. By the implication of ultrasound trials, protein recovery was increased from 49.88% for CPEP to 55.02% and 56.11%, for PUPEP and UAPEP, respectively. According to gel electrophoresis, no major distinction in molecular weight between protein fractions of different samples was perceived and sonication practice did not convert the primary structures of proteins. FTIR results uncovered changes in different extraction modes, implying that ultrasound could transform the secondary structures of the protein. Thermal properties and surface tension were decreased during sonication due to the conformational changes. The results of this study indicated that Persian everlasting pea protein isolate could be considered as a novel source of valuable protein and functional ingredient in the food industry.Practical applicationsThe utilization of plant‐based protein products as sustainable alternatives to animal proteins has been increasing recently. However, compared with animal proteins, plant‐based proteins reveal relatively weak functional properties such as water‐holding capacity and gelation. Therefore, they need to be modified before being incorporated into food products. Ultrasonic treatment is an emerging technology that has many applications in food science and technology, such as increasing the extraction efficiency and improving the physicochemical and functional properties of food ingredients. Lathyrus rotundifolius seeds contain high amounts of protein and can be used as a source of plant‐based protein. The effect of ultrasonic treatment in the study was investigated and confirmed to improve the extraction efficiency and functional properties of Persian everlasting pea protein, which can be used in the food industry.

The effects of combined co-expression of groel/es and trigger factor chaperone towards Thermus thermophilus DNA polymerase enzyme expression on Escherichia coli BL21(DE3)

The Thermus thermophilus (Tth) DNA polymerase enzyme, originating from the thermophilic bacterium T. thermophilus, exhibits bifunctionally as both a DNA polymerase and a reverse transcriptase. When expressed in Escherichia coli, the T. thermophilus DNA polymerase enzyme frequently results in the formation of inclusion bodies. This occurrence is attributed to the rapid rate of protein expression in E. coli which surpasses the availability of chaperone proteins and reduced cytoplasmic conditions. The formation of inclusion bodies can reduce the recovery of soluble protein. A combination of GroEL/ES and trigger factor chaperone co-expression was used to overcome this deficiency. Thermus thermophilus gene DNA Pol/pG-Tf3 was transformed into the host E. coli BL21(DE3) and expressed with and without chaperone co-expression. Utilizing co-expression of the chaperone combination GroEL/ ES and trigger factor can reduce the formation of inclusion bodies or decrease the insoluble fraction in the expression of the T. thermophilus DNA polymerase enzyme, resulting in a co-expression total protein content of 3.9258 mg/ml.