Protein Recovery Research Articles

Assessing protein and albumin recovery rates in different ascites filtration membrane washing methods for cell-free and concentrated ascites reinfusion therapy.

In cell-free and concentrated ascites reinfusion therapy (CART), the protein recovery rate decreases when the filtration membrane gets clogged. Employing a device with a filtration membrane washing feature prevents clogging, but it leads to the loss of ascites within the filter, resulting in reduced protein recovery. This study employed a device with a membrane washing function to investigate the relationship between protein recovery rate and the quantity of washing solution used, depending on the selected washing method. We analyzed cases of CART conducted at Fujita Health University Hospital between May 2021 and November 2022. The cases were divided and compared between two groups: one using flush and rinse as the washing method (flush+rinse group) and another using only flushing (flush group). We identified nine cases and 16 sessions. In the flush+rinse group, the median amount of washing solution used per membrane washing was 259mL per cycle, whereas it was 54mL per cycle in the flush group. This difference was statistically significant (p<0.0001). The median total protein recovery rate was 53.8% for the flush+rinse group and 78.8% for the flush group, with the latter showing a significantly higher value (p=0.0199). In CART using a membrane washing function, adopting a washing method that reduces the amount of washing solution leads to an increase in the total protein recovery rate.

Engineered polymer nanoparticles as artificial chaperones facilitating the selective refolding of denatured enzymes

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.

How processing affects marker peptide quantification – A comprehensive estimation on bovine material relevant for food and feed control

Processing food and feed challenges official control e.g. by modifying proteins, which leads to significant underestimation in targeted, MS-based protein quantification. Whereas numerous studies identified processing-induced changes on proteins in various combinations of matrices and processing conditions, studying their impact semi-quantitatively on specific protein sequences might unveil approaches to improve protein quantification accuracy. Thus, 335 post-translational modifications (e.g. oxidation, deamidation, carboxymethylation, Amadori, acrolein adduction) were identified by bottom-up proteomic analysis of 37 bovine materials relevant in food and feed (meat, bone, blood, milk) with varying processing degrees (raw, spray-dried, pressure-sterilized). To mimic protein recovery in a targeted analysis, peak areas of marker and reference peptides were compared to those of their modified versions, which revealed peptide-specific recoveries and variances across all samples. Detailed analysis suggests that incorporating two modified versions additionally to the unmodified marker may significantly improve quantification accuracy in targeted MS-based food and feed control in processed matrices.

An Ionic Liquid-Based Biorefinery Approach for Duckweed Utilization.

This study establishes a foundation for the ionic liquid (IL) pretreatment of duckweed biomass. An optimized IL-based process was designed to exploit the unique properties of duckweed including efficient metal removal, potential starch accumulation, and protein accumulation. Two ILs, namely, dimethylethanolammonium formate ([DMEtA][HCOO]) and N,N-dimethylbutylammonium hydrogen sulfate ([DMBA][HSO4]), were investigated for the pretreatment of two duckweed species (Spirodela polyrhiza and Lemna minor). The evaluation focused on starch recovery, sugar release, protein recovery, and metal extraction capabilities. [DMEtA][HCOO] demonstrated near-quantitative starch recoveries at 120 °C, while [DMBA][HSO4] showed similar performance at 90 °C within a reaction time of 2 h. Saccharification yields for most pulps exceeded 90% after 8 h of hydrolysis, outperforming "traditional" lignocellulosic biomasses such as miscanthus or sugarcane bagasse. Approximately 50 and 80 wt % of the protein were solubilized in [DMEtA][HCOO] and [DMBA][HSO4], respectively, while the remaining protein distributed between the pulp and lignin. However, the solubilized protein in the IL could not be recovered due to its low molecular weight. Regarding metal extraction, [DMEtA][HCOO] demonstrated higher efficiency, achieving 81% removal of Ni from Lemna minor's pulps, whereas [DMBA][HSO4] extracted only 28% of Ni with slightly higher pulp concentrations. These findings indicate the need for further optimization in concurrent metal extraction using ILs.

Comparative studies on enhancing pea protein extraction recovery rates and structural integrity using ultrasonic and hydrodynamic cavitation technologies

This study explored the efficacy of various cavitation technologies, including ultrasonic bath (USB), ultrasonic plate (US-plate), ultrasonic probe (US-probe), and hydrodynamic cavitation (HDC), in extracting proteins from peas. US-probe showed the highest protein recovery rate (52.53 g/hg protein in pea powder) among all lab-scale cavitation equipment while HDC demonstrated significant potential for scaling up, notably improving both the purity (80.35 g/hg dried precipitate) and recovery rate (56.85 g/hg) of pea protein isolate (PPI) compared to conventional extraction (CE). SDS-PAGE, LC-MS/MS, FTIR and Fluorescence analysis were used to analyse the impact of these cavitation technologies on the structures of pea protein. The results confirmed that cavitation preserved PPI's primary structure while altering its secondary and tertiary structures, particularly under US-probe treatment, which significantly unfolded proteins. The SEM results revealed a marked reduction in protein bodies adhering to starch granules in residues from US-probe and HDC treatments compared to CE, correlating with their higher protein recovery rates.

Development of a CRISPR/Cas12a-mediated aptasensor for Mpox virus antigen detection

The emergence and rapid spread of Mpox (formerly monkeypox) have caused significant societal challenges. Adequate and appropriate diagnostics procedures are an urgent necessity. Herein, we discover a pair of aptamers through the systematic evolution of ligands by exponential enrichment (SELEX) that exhibit high affinity and bind to different sites towards the A29 protein of the Mpox virus. Subsequently, we propose a facile, sensitive, convenient CRISPR/Cas12a-mediated aptasensor for detecting the A29 antigen. The procedure employs the bivalent aptamers recognition, which induces the formation of a proximity switch probe and initiates subsequent cascade strand displacement reactions, then triggers CRISPR/Cas12a DNA trans-cleavage to achieve the sensitive detection of Mpox. Our method enables selective and ultrasensitive evaluation of the A29 protein within the range of 1 ng mL−1 to 1 μg mL−1, with a limit of detection (LOD) at 0.28 ng mL−1. Moreover, spiked A29 protein recovery exceeds 96.9%, while the detection activity remains above 91.9% after six months of storage at 4 °C. This aptasensor provides a novel avenue for exploring clinical diagnosis in cases involving Mpox as facilitating development in various analyte sensors.

Production of soft unripened cheeses using acidic and salty coagulants: Investigation of technological and sensory characteristics.

Soft cheeses are coagulated milk products obtained through acidification or applying a combination of acids and heat. In this research, in order to improve technological characteristics, the effects of different coagulants (salt and acids) and process parameters (temperature and homogenization pressure) on the organoleptic, textural, and functional characteristics of soft (unripened) cheese were investigated. The results revealed significant differences between cheeses coagulated with acid and mineral salt regarding protein recovery, fat content, and moisture content (p < .05). Acidic coagulants (74%-94%) resulted in higher cheese yield compared to mineral salt (66%-88%). Texture analysis indicated that the cheese produced with acetic acid had a firmer texture, while samples treated with citric acid exhibited better cohesiveness. Cheeses produced with minerals displayed more acceptable organoleptic characteristics regarding flavor, odor, and texture. This study offers valuable technological insights into cheese production with the highest yield and maximum acceptability.

Effect of Post-Extraction Ultrasonication on Compositional Features and Antioxidant Activities of Enzymatic/Alkaline Extracts of Palmaria palmata.

Palmaria palmata is a viable source of nutrients with bioactive properties. The present study determined the potential role of post-extraction ultrasonication on some compositional features and antioxidant properties of enzymatic/alkaline extracts of P. palmata (EAEP). No significant difference was detected in terms of protein content and recovery, as well as the amino acid composition of the extracts. The nitrogen-to-protein conversion factor of 5 was found to be too high for the seaweed and EAEP. The extracts sonicated by bath for 10 min and not sonicated showed the highest and lowest total phenolic contents (p < 0.05), respectively. The highest radical scavenging and lowest metal-chelating activities were observed for the non-sonicated sample, as evidenced by IC50 values. The extract sonicated by bath for 10 min showed the most favorable in vitro antioxidant properties since its radical scavenging was not significantly different from that of the not-sonicated sample (p > 0.05). In contrast, its metal-chelating activity was significantly higher (p < 0.05). To conclude, post-extraction ultrasonication by an ultrasonic bath for 10 min is recommended to increase phenolic content and improve the antioxidant properties of EAEP.

Sustainable Valorization of Coffee Silverskin: Extraction of Phenolic Compounds and Proteins for Enzymatic Production of Bioactive Peptides.

Coffee silverskin (CS), a by-product of the coffee roasting process, has high protein content (16.2-19.0%, w/w), making it a potential source for plant protein and bioactive peptide production. This study aims to develop innovative extraction methods for phenolic compounds and proteins from CS. The conditions for hydrothermal (HT) extraction of phenolic compounds from CS were optimized by varying CS loading (2.5-10%, w/v), temperature (110-130 °C), and time (5-30 min) using a one-factor-at-a-time (OFAT) approach. The highest TPC of 55.59 ± 0.12 µmole GAE/g CS was achieved at 5.0% (w/v) CS loading and autoclaving at 125 °C for 25 min. Following hydrothermal extraction, CS protein was extracted from HT-extracted solid fraction by microwave-assisted alkaline extraction (MAE) using 0.2 M NaOH at 90 W for 2 min, resulting in a protein recovery of 12.19 ± 0.39 mg/g CS. The CS protein was then subjected to enzymatic hydrolysis using protease from Bacillus halodurans SE5 (protease_SE5). Protease_SE5-derived CS protein hydrolysate had a peptide concentration of 0.73 ± 0.09 mg/mL, with ABTS, DPPH, and FRAP values of 15.71 ± 0.10, 16.63 ± 0.061, and 6.48 ± 0.01 µmole TE/mL, respectively. Peptide identification by LC-MS/MS revealed several promising biological activities without toxicity or allergenicity concerns. This study's integrated approach offers a sustainable and efficient method for extracting valuable compounds from CS, with potential applications in the food and pharmaceutical industries.

The translational content and expression of intracellular sensors under endoplasmic re-ticulum stress in diabetic rats

Endoplasmic reticulum stress (ER stress) activates the unfolded protein response (UPR) as a defense mechanism for maintaining cellular vital activity. Data on interaction between pathways of UPR-sensors, their role and participation in the recovery of regulatory proteins at constant level are currently being accumulated. An experimental model of type 2 diabetes mellitus (T2DM) was reproduced in male rats fed on 3-month high-fat diet with additional streptozotocin administration. Rats in different groups were treated with metformin, propionate, and their combination for 2 weeks. We analyzed the expression of the GRP78, PERK, IRE-1, and ATF6 genes in gastric glandulocytes using PCR primers. The quantitation of protein content of the GRP78, PERK, IRE-1, and ATF6 genes was performed using western blotting. Statistical intergroup differences were calculated using a one-way ANOVA test followed by Tukey’s post-hoc. Comparison of mRNA and protein contents at rest state, in condition of ER stress and drug treatment, as well as the study of correlations between UPR-sensors, provides a basis for the development of the concept of defense mechanism activation in gastric glandulocytes under T2DM conditions. Under stress, chaperone GRP78 plays a crucial role to prevent protein misfolding: it dissociates from ER, enters cisternae and inactivates misfolded proteins. The ATF6 pathway is activated in all conditions. It probably activates transcription of the corresponding content turnover sites in both GRP78 and ATF6. The IRE-1 and PERK pathways are activated in glandulocytes under different circumstances, and they probably target to different regulatory actions. Combination drug treatment in T2DM conditions induces activation of all signaling sensors in the UPR system with simultaneous maximal downregulation of GRP78, causing a balance in the intracellular homeostasis system.

Statistical optimized production of Phytase from Hanseniaspora guilliermondii S1 and studies on purification, homology modelling and growth promotion effect

This investigation was performed to obtain a promising phytase enzyme producing yeast. In this regard, the PSM was used to isolate the phytase-producing Hanseniaspora guilliermondii S1 (MG663578) from sugarcane juice. The SSF optimum conditions for phytase generation were optimized using (OVAT) one-variable-at-a-time strategy using both Box-Behnken design and shake flask method (g/100 ml: 0.05 yeast extract, 0.15 Peptone, 0.05 malt extract 0.50 dextrose, pH 5.8 and 28ᵒC). The protein model developed was shown to be adequate for phytase production (91% accuracy), with the greatest phytase productivity in shake flask with substrate jack fruit seed powder being 395 ± 0.43 U/ml compared to 365U/ml for the BBD projected value. Crude Phytase was partially purified with a protein recovery of 43%, revealing a molecular weight of 120 kDa. It had an enzyme kinetic value of Km 3.3 mM and a Vmax of 19.1 mol/min. The 3D structure of PhyS1 amino acid sequences (PhyS1. B99990002) was simulated using Modeler 9.23, and the validated result revealed that 86.7% were in the favored region by Ramachandran plot. The SAVES server verified the 3D PDB file as satisfactory, and the model (in.pdb format) was uploaded in the PMDB database with the accession number ID: PM0082974. At the lab level, Hanseniaspora guilliermondii S1 (MG663578) producing phytase exhibited successful plant growth promotion activity in Ragi - CO 19 (Eleusine coracana L.) and Rice -Navarai - IR 64 (Oryza sativa L.). As a result, a phytase-based formulation for sustainable agriculture must be developed and tested on a large scale in diverse geographical areas of agricultural lands to determine its effect and potential on plant development.

Cold‐pressing, ethanol defatting, and saline extraction enhances properties of protein products from new pennycress varieties (covercress™)

AbstractPennycress (Thlaspi arvense L.), a winter oilseed and cover crop, is a source of novel proteins and oil for biofuel. New yellow‐seeded pennycress specialty varieties, named covercress™, were developed via conventional breeding and gene editing, but protein characteristics are still unknown. This work evaluated two covercress lines, TT8 and Y1126, for protein extraction and functionality, and the combination of cold‐pressing, alcohol‐defatting, and saline extraction to produce a protein that could align better with clean labeling. Seeds were first cold‐pressed in a tubular radial expeller then defatted at 60°C with anhydrous ethanol (‐Eth) or hexane (‐Hex) until ≤0.8% residual oil. Protein was extracted from defatted press cake (PC) using the saline method (0.1 M NaCl, 1: 10 w/v, 50°C, 2 h) for hexane‐defatted wild‐type pennycress (WTP) seed meal and PC. TT8 PC had 33%–67% more globulin and glutelin than WTP PC, but ethanol defatting reduced albumin + globulin amounts by 14%–32%. TT8 PC protein recoveries (63% and 40%) were 2‐ to 3‐fold greater than that of Y1126 PC‐Eth and their extracts had greater purity (85%–91% vs. 70% protein). Y1126 PC‐Eth protein showed a nearly constant 68%–70% solubility from pH 2 to 10, but TT8 PC‐Eth protein was more soluble in acidic pH (87%–90% solubility). Y1126 PC‐Eth protein showed greater foaming capacity (146–150 mL), while TT8 PC‐Eth protein had superior emulsifying properties (EAI 141–236 m2/g protein and ESI 13–26 min). This work demonstrated the enhanced functionality of TT8 and Y1126 proteins produced from an environmentally friendly and industrially relevant method.

Hollow anion-Janus-modified poly(vinylidene fluoride) (PVDF) fiber membranes for nanosized virus removal

In this study, a hollow polyvinylidene fluoride (PVDF) fiber was developed and modified into a Janus membrane (Janus-VM) and an entirely hydrophobic membrane (Hydrophobic-VM) for removing 20-nm-scale viruses during biopharmaceutical processing. The Janus-VM has anionic properties on the outer and feed hydrophilic surfaces, and the inner pore surface (I.P.S) maintains the anionic-hydrophobic properties of the PVDF. Theoretically, an electrostatic repulsion force might be formed between the surface of the strong anionic membrane and anionic bacteriophages, preventing bacteriophages from adsorbing to the membrane surface. As a result, water flux can be increased through the reduction of membrane fouling. The PVDF membrane was modified through UV-irradiation, which causes –OH radicals to form on the membrane surface, and chemical grafting with anionic-hydrophobic/anionic-hydrophilic modifiers, which enhances the membrane’s anion characteristics (−27, −48 mV). Additionally, the average pore size was reduced from 34 nm to less than 20 nm through chemical surface modification, after which the Janus membrane showed an excellent initial water flux, with the protein recovery ratio (PRR) approaching 98.9 %. UV absorbance analysis revealed 100 % removal efficiency for 20- and 40-nm gold particles on the membranes. Furthermore, the initial log reduction value (LRV) was observed through flux performance using MS2 and PP7 bacterial phages, for which the Janus membrane showed an excellent virus removal performance (LRV > 6.8 and 6.5).

Refolding SDS-mediated partially-unfolded proteins: A distinguishing role of tetraalkylammonium ionic liquids via mixed micellar aggregation

The current study reports a new strategy for ionic liquid (ILs) assisted refolding of sodium dodecyl sulfate (SDS)-induced partially unfolded protein via mixed micellar aggregate formation between SDS and the ionic liquid. This approach can indeed be utilized to keep many essential and sensitive proteins as active and prolong their shelf life. For SDS-induced partially unfolded proteins, the ionic liquid has been identified to function as a refolding agent. The phenomenon has been observed by us for the first time and we have thoroughly investigated the same utilizing a variety of spectroscopic, microscopic approaches, and attempted to rationalize from computational techniques as well. The steady-state fluorescence emission spectra and time-resolved fluorescence decay show that both the intrinsic emission intensity and the average lifetime, respectively, of the surfactant-induced partially-unfolded protein increase in the presence of ionic liquid, indicating a possible reversal of unfolding (refolding) of the protein (up to ∼95 % revival of luminescence intensity and average lifetime with respect to the native protein). The CD and FTIR results also confirmed that the α-helical content of the pre-unfolded protein reverts substantially and is very close to that of the native protein. To understand the refolding process of SDS-induced partially-unfolded protein, we have further studied the structural recovery of urea-induced pre-denatured protein utilizing the same ionic liquid. Interestingly, no such refolding was observed in this case. From the experimental results, it appears that ionic liquid is involved in scavenging the unfolding surfactant monomers by forming mixed-micellar aggregates where the ion-ion attachment is likely to have impact on the said process.

Identification of Fouling Occurring during Coupled Electrodialysis and Bipolar Membrane Electrodialysis Treatment for Tofu Whey Protein Recovery.

Tofu whey, a by-product of tofu production, is rich in nutrients such as proteins, minerals, fats, sugars and polyphenols. In a previous work, protein recovery from tofu whey was studied by using a coupled environmental process of ED + EDBM to valorize this by-product. This process allowed protein recovery by reducing the ionic strength of tofu whey during the ED process and acidifying the proteins to their isoelectric point during EDBM. However, membrane fouling was not investigated. The current study focuses on the fouling of membranes at each step of this ED and EDBM process. Despite a reduction in the membrane conductivities and some changes in the mineral composition of the membranes, no scaling was evident after three runs of the process with the same membranes. However, it appeared that the main fouling was due to the presence of isoflavones, the main polyphenols in tofu whey. Indeed, a higher concentration was observed on the AEMs, giving them a yellow coloration, while small amounts were found in the CEMs, and there were no traces on the BPMs. The glycosylated forms of isoflavones were present in higher concentrations than the aglycone forms, probably due to their high amounts of hydroxyl groups, which can interact with the membrane matrices. In addition, the higher concentration of isoflavones on the AEMs seems to be due to a combination of electrostatic interactions, hydrogen bonding, and π-π stacking, whereas only π-π stacking and hydrogen bonds were possible with the CEMs. To the best of our knowledge, this is the first study to investigate the potential fouling of BPMs by polyphenols, report the fouling of IEMs by isoflavones and propose potential interactions.

Evaluating nano-metal oxide mixed matrix membranes for whey protein separation using hybrid intelligent optimization learning

Ultrafiltration (UF) membranes offer cost-efficient and sustainable techniques for the extraction of whey protein in cheese whey wastewater. This strategy substantially aids in waste minimization and fosters the progression of a circular economy paradigm within the dairy industry. The emerging discipline of machine learning has benefited UF by enhancing the separation performance and deepening the filtration mechanism. In this study, adaptive neuro-fuzzy inference systems (ANFIS) coupled chemometric learning was implemented for the performance evaluation of whey protein recovery in nano metal oxide embedded mixed matrix membranes (MMMs) UF from whey wastewater. The selected input variables are nano metal oxides concentration (NC), pore radius (S), and contact angle (CA). The targeted responses for the parametric dependency analysis are individual whey protein permeability (lysozyme (PLys), lactalbumin (PLA) and bovine serum albumin (PBSA)) and protein rejection (%) (lysozyme (RLys), lactalbumin (RLA) and bovine serum albumin (RBSA)). The Jarque-Bera indicated that the input variables did not deviate from normality, and S and CA have significantly influenced the whey protein rejection and permeability. ANFIS models demonstrate superior predictive capabilities with minimal error for flux under various transmembrane pressure (TMP) in MMMs due to their inherent gradient descent optimization. Among the proteins, a Pearson correlation coefficient approaching unity denotes a significant positive relationship between lysozyme flux (FLys). The ANFIS models exhibited excellent predictive performance in real-time cheese whey effluent flux and rejection data, achieving a mean square error of 0.0003 for experimental flux. This study provides valuable insights for the development of models tailored to small datasets, offering a foundation for predicting and enhancing membrane performance.

Nitrosative Stress in Astronaut Skeletal Muscle in Spaceflight.

Long-duration mission (LDM) astronauts from the International Space Station (ISS) (>180 ISS days) revealed a close-to-normal sarcolemmal nitric oxide synthase type-1 (NOS1) immunoexpression in myofibers together with biochemical and quantitative qPCR changes in deep calf soleus muscle. Nitro-DIGE analyses identified functional proteins (structural, metabolic, mitochondrial) that were over-nitrosylated post- vs. preflight. In a short-duration mission (SDM) astronaut (9 ISS days), s-nitrosylation of a nodal protein of the glycolytic flux, specific proteins in tricarboxylic acid (TCA) cycle, respiratory chain, and over-nitrosylation of creatine kinase M-types as signs of impaired ATP production and muscle contraction proteins were seen. S-nitrosylation of serotransferrin (TF) or carbonic anhydrase 3 (CA3b and 3c) represented signs of acute response microgravity muscle maladaptation. LDM nitrosoprofiles reflected recovery of mitochondrial activity, contraction proteins, and iron transporter TF as signs of muscle adaptation to microgravity. Nitrosated antioxidant proteins, alcohol dehydrogenase 5/S-nitrosoglutathione reductase (ADH5/GSNOR), and selenoprotein thioredoxin reductase 1 (TXNRD1) levels indicated signs of altered redox homeostasis and reduced protection from nitrosative stress in spaceflight. This work presents a novel spaceflight-generated dataset on s-nitrosylated muscle protein signatures from astronauts that helps both to better understand the structural and molecular networks associated to muscular nitrosative stress and to design countermeasures to dysfunction and impaired performance control in human spaceflight missions.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2–3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Therapeutic benefit of oregano oil in the acute idiosyncratic hepatotoxicity induced by carbon tetrachloride in rats: Adverse effects of high dose of oreganum.

Toxicity caused by carbon tetrachloride (CCl4) can lead to serious liver injury. The aim of the study is to investigate the protective effects of oregano oil (Origanum minutiflorum extract oil) against CCl4-induced liver injury. Two doses of oregano oil were used in the experiment: a low dose (LD; 20 mg/kg) and a high dose (HD; 60 mg/kg) during 2 weeks. CCl4 caused severe liver damage, nucleolus destruction in hepatocytes and cytogenetic changes in the nucleus. Indirectly, CCl4 causes decreased protein synthesis and significantly high creatinine and urea values. Hematological disorders have been recorded, such as decreased RBC and hemoglobin concentration, increased WBC and deformability of the erythrocyte membrane. Both doses of oregano oil had protective effects. Improved protein synthesis and high globulins level, creatinine and urea were found in both groups. Cytogenetic changes in the nucleus of hepatocytes were reduced. A high dose of oregano oil had maximal protective effects for RBC, but a very weak effect on hemoglobin synthesis. Also, WBC and lymphocyte values were low. Origanum stimulates protein synthesis and recovery of hepatocytes after liver injury, reduces the deformability of the erythrocyte membrane. High doses of oregano oil decreased WBC and lymphocytes which may lead to a weakening of the immune response. However, high doses are more effective against severe platelet aggregation than low doses, suggesting an effective treatment against thrombocytosis.

Production of techno-functional proteins and plant biostimulants from Nannochloropsis gaditana

Nannochloropsis gaditana is produced mainly for its lipid content and fatty acid profile. However, its protein content is comparable to or even higher than that of most conventional protein sources. In this work, the microalga N. gaditana BEA 1202B purchased from the Spanish Bank of Algae (BEA) was produced using 3.4 m3 tubular photobioreactors achieving a biomass productivity of 0.1 g L−1·day−1 and a protein content of 29.2 ± 4.5 % (d.w.). The biomass produced was used as a source of proteins; the protein recovery was optimised following a response surface methodology. Under optimal conditions, which were solubilisation at pH = 10.2 and precipitation at pH = 3.1, the protein recovery yield was 17.5 g·100 g−1. The extract exhibited a water-holding and an oil-holding capacity of 2.1 and 3.0 g g−1, respectively. The leftovers from protein extraction were assessed as plant biostimulants and demonstrated a positive effect on root development, weight gain, and chlorophyll retention. The process described in this work presents a sustainable and environmentally friendly approach to harness the potential of the microalga N. gaditana in producing high-value food proteins and plant biostimulants. The integration of this microalgal-based biorefinery concept could contribute to more sustainable agriculture and address the growing global demand for protein-rich products and plant promoters.