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ABSTRACT Pinto beans, an underutilized legume, are abundant in protein content and contain a variety of beneficial phytonutrients. However, the commonly used protein extraction method, alkaline extraction, is associated with several drawbacks. These drawbacks include low extraction yield and purity as well as the production of large amounts of wastewater that can lead to environmental hazards. In this regard, membrane technology has gained considerable recognition as a superior method for extracting proteins. A combined processing scheme was developed, which included alkaline extraction at pH 10.5, ultrafiltration with a concentration factor of 5.5, diafiltration with a diavolume of 4, and isoelectric precipitation at pH 4.5 followed by freeze drying. The specific functional characteristics (nitrogen solubility index, water and oil holding capacity, and emulsifying and foaming properties) of the protein concentrates were assessed and compared with those of a commercially available soybean protein isolate. Based on pinto bean flour containing 23.9% protein, 85.5% of the protein was recovered in the products of this process: precipitated protein concentrate (PPC) with 86.4% protein, acid‐soluble protein concentrate (ASP‐C) with 56.3% protein, and meal residue with 6.1% protein. The mass yields were 17.3% in PPC, 3.9% in ASP‐C, and 54% in the meal residue. The precipitated protein showed higher emulsifying activity, and the acid‐soluble protein showed a high nitrogen solubility index (NSI) and oil‐holding capacity. Both proteins had comparable foaming properties to commercial soy protein isolate. The project demonstrated the feasibility of protein production from pinto beans and highlighted the proteins' useful food functionality and good potential for commercialization. [ABSTRACT FROM AUTHOR]

The pancreas gland (PG) is a secondary product of livestock processing; it contains a wide range of biologically active compounds. The purpose of this article is to analyze the efficiency of technological approaches for pancreas gland extraction with the help of trehalose and a glycine-proline mixture aimed for recovery and separation of the gland’s protein-peptide compounds. The extraction was conducted with 0.9% NaCl, 0.9% NaCl, with addition of 0.5 M trehalose (0.9% NaCl-0.5 M trehalose) and 0.9% NaCl with addition of 1% glycine and 0.1 M L-proline (0.9% NaCl-1% Gly-0.1M Pro), the ratio of pancreas gland to extractant was equal to 1:5. The concentration of the protein in the supernatants after their extraction was measured by the biuret reaction in a semi-automatic biochemical analyzer Biochem SA. The proteomic composition of the extracts and the native pancreas gland was assessed by one-dimensional Laemmli electrophoresis in a 12.5% polyacrylamide gel and by two-dimensional O’Farrell electrophoresis. When determining the intensity of the protein fractions, it was noted that the methodology of separation of protein-peptide mixtures extracted from the pigs pancreas gland with the extractant 0.9% NaCl-1% Gly-0.1M Pro, ensured the higher extraction of the proteins in comparison with the method of 0.9% NaCl-0.5 M trehalose. Notwithstanding the fact that application of amino acids (glycine and proline) mixture provided for a greater yield of proteins from the extract into the diafiltrate, the experiments in vitro showed that the diafiltrate obtained though trehalose featured higher activity. This may be explained by the fact that after dialysis removal of trehalose from the protein fraction with a molecular weight of less than 50 kDa, its residual quantities were still sufficient to prevent proteins aggregation and, as a consequence, the biological activity of the extracted proteins was preserved, while in the diafiltrate obtained through amino acids mixture where numerous protein aggregates were detected by 2-DE. This study allowed testing the biotechnological methodics on pig pancreatic tissues aimed to intensifying the extraction and separation of protein compounds. The results of the study are important for development of methodological approaches to obtaining the targeted substances for their further utilizing for various purposes.

The research was aimed at studying the combined effect of micro- and ultrafiltration technological parameters for justification of rational modes in the production of whey protein isolate. The process flow of whey protein isolate production was determined. It includes whey purification from casein dust and fat, pasteurization, ultrafiltration, microfiltration, ultrafiltration (combined with diafiltration), spray drying. Concentration was carried out from a dry matter mass fraction of 5.4-5.6% to 11.3-12.6% (protein concentration factor is 6.5-13). Microfiltration of the resulting retentate was used to maximize disposal of milk fat. The process was carried out using ceramic membranes (pore size from 0.14 pm to 1.4 pm). Their protein retention capacity was 0.2-0.4%, fat retention capacity was 64.6-76.2%. Rational microfiltration modes were selected. They are inlet pressure 0.15-0.2 MPa and temperature 10-15 °C. The microfiltration permeate was treated via repeated ultrafiltration combining it with diafiltration. It was possible to achieve the protein content in dry matter of the product not more than 87% using diafiltration with half the volume of water. However, it does not meet the requirements for the isolate. Increasing the amount of water for diafiltration caused a rise in the protein content in dry matter of the concentrate. The protein mass fraction in the whey protein isolate before drying was at least 17%. The whey protein isolate powder was characterized by the high protein content (93% in terms of SNF), quality and safety indicators met the requirements of regulatory documentation.

Bovine colostrum whey, despite being a source of bioactive peptides and prebiotic oligosaccharides with dietary and nutraceutical applications, presents significant processing challenges due to its high viscosity, making hollow fiber membranes, with their superior ability to control membrane fouling, well-suited for handling such fluids. The objective of this work was to determine optimal ultrafiltration conditions of a 10 kDa hollow fiber membrane to increase efficiency (i.e., permeate flux), selectively retain whey proteins, and facilitate the permeation of peptides (< 10,000 Da) and oligosaccharides (~600 Da) from bovine colostrum whey. The impact of feed flow rate (3, 6, 9 L min-1) and transmembrane pressure (1, 2, 3 bar) were evaluated on permeate flux, protein retention, and non-protein nitrogen permeation. Higher permeate fluxes were observed at higher feed flows and moderate transmembrane pressure, demonstrating the ability of turbulence to disrupt the concentration polarization layer at the membrane surface and decrease fouling, as exhibited in the resistance analyses. Higher feed flow significantly increased the cumulative permeate flux during colostrum whey concentration (64.6 ± 0.1 L h-1m-2 at 9 L min-1 vs. 12.5 ± 5.9 L h-1m-2 at 3 L min-1, at continuous diafiltration mode and 2 bar), with continuous diafiltration experiments yielding similar permeate fluxes to the ones achieved in discontinuous mode. Processing scale at 12.7 L (2 bar, 9 L min-1, and continuous diafiltration) enabled 97% protein retention and 95% peptide and oligosaccharide permeation, producing a permeate with 1.3 g L-1 peptide and 0.42 g L-1 oligosaccharide. The results of this study highlight the impact of key ultrafiltration conditions of bovine colostrum whey using a hollow fiber membrane for the effective recovery of peptides and oligosaccharides for subsequent evaluation of their biological properties and commercial applications. [ABSTRACT FROM AUTHOR]

Deep eutectic solvents (DES) are green alternatives for conventional solvents. They have gained attention for their potential to extract valuable compounds from biomass, such as seaweed. In this framework, a case study was developed to assess the feasibility of pressure-driven membrane processes as an efficient tool for the recovery of deep eutectic solvents and targeted biomolecules. For this purpose, a mixture composed of the DES choline chloride – ethylene glycol (ChCl-EG) 1:2, water and alginate was made to mimic a DES extraction from seaweed. An integrated separation process design was proposed where ultrafiltration-diafiltration-nanofiltration (UF-DF-NF) was coupled. UF and DF were found to be effective for the separation of alginate with an 85 % yield. DES was likewise recovered by 93 %, proving the membrane filtrations’ technical feasibility. The NF performance to separate the DES from the water, for its recycling, laid by a 45 %-50 % retention and a final concentrated DES solution of 18 %(v/v).

ABSTRACT: Processing temperature has a significant influence on the composition and functionality of the resulting streams following microfiltration (MF) of skim milk. In this study, MF and diafiltration (DF) were performed at 4 or 50°C to produce β-casein (β-CN)-depleted and nondepleted (i.e., native casein profile) micellar casein isolate retentates, respectively. Microfiltration combined with extensive DF resulted in a 40% depletion of β-CN at 4°C, whereas no β-CN depletion occurred at 50°C. Microfiltration at 4°C led to higher transmission of calcium into permeates, with retentate generated at 4°C containing less total calcium compared with retentate generated at 50°C, based on the volume of retentate remaining. Higher heat stability at 120°C was measured for retentates generated at 4°C compared with those at 50°C, across all pH values measured. Retentates generated at 4°C also had significantly lower ionic calcium values at each pH compared with those generated at 50°C. Higher apparent viscosities at 4°C were measured for retentates generated at 4°C compared with retentates generated at 50°C, likely due to increased voluminosity of β-CN-depleted casein micelles. The results of this study provide new information on how changing the composition of MF retentate, by appropriate control of processing temperature and DF, can alter physicochemical properties of casein micelles, with potential implications for ingredient functionality.

Self-driving laboratories and automated experiments can accelerate the design workflow and decrease errors associated with experiments that characterize membrane transport properties. Within this study, we use 3D printing to design a custom stirred cell that incorporates inline conductivity probes in the retentate and permeate streams. The probes provide a complete trajectory of the salt concentrations as they evolve over the course of an experiment. Here, automated diafiltration experiments are used to characterize the performance of commercial NF90 and NF270 polyamide membranes over a predetermined range of KCl concentrations from 1 to 100 mM. The measurements obtained by the inline conductivity probes are validated using offline post-experiment analyses. Compared to traditional filtration experiments, the probes decrease the amount of time required for an experimentalist to characterize membrane materials by more than 50× and increase the amount of information generated by 100×. Device design principles to address the physical constraints associated with making conductivity measurements in confined volumes are proposed. Overall, the device developed within this study provides a foundation to establish high-throughput, automated membrane characterization techniques.

Processing temperature has a significant influence on the composition and functionality of the resulting streams following microfiltration (MF) of skim milk. In this study, MF and diafiltration (DF) were performed at 4 or 50°C to produce β-casein (β-CN)-depleted and nondepleted (i.e., native casein profile) micellar casein isolate retentates, respectively. Microfiltration combined with extensive DF resulted in a 40% depletion of β-CN at 4°C, whereas no β-CN depletion occurred at 50°C. Microfiltration at 4°C led to higher transmission of calcium into permeates, with retentate generated at 4°C containing less total calcium compared with retentate generated at 50°C, based on the volume of retentate remaining. Higher heat stability at 120°C was measured for retentates generated at 4°C compared with those at 50°C, across all pH values measured. Retentates generated at 4°C also had significantly lower ionic calcium values at each pH compared with those generated at 50°C. Higher apparent viscosities at 4°C were measured for retentates generated at 4°C compared with retentates generated at 50°C, likely due to increased voluminosity of β-CN-depleted casein micelles. The results of this study provide new information on how changing the composition of MF retentate, by appropriate control of processing temperature and DF, can alter physicochemical properties of casein micelles, with potential implications for ingredient functionality. [ABSTRACT FROM AUTHOR]

In the production of biological therapeutics such as monoclonal antibodies (mAbs), ultrafiltration and diafiltration (UF/DF) are widely regarded as effective downstream processing steps capable of removing process equipment related leachables (PERLs) introduced upstream of the UF/DF step. However, clearance data available in the literature are limited to species with low partition coefficients (log P) such as buffer ions, hydrophilic organic compounds, and some metal ions. Additional data for a wide range of PERLs including hydrophobic compounds and elemental impurities are needed to establish meaningful, comprehensive safety risk assessments. Herein, we report the results from studies investigating the clearance of seven different organic PERLs representing a wide range of characteristics (i.e., log P (−0.3 to 18)), and four model elements with different chemical properties spiked into a mAb formulation at 10 ppm and analyzed during clearance using gas chromatography–mass spectrometry (GC–MS), liquid chromatography‐photodiode‐array‐mass spectrometry (LC‐PDA‐MS), and inductively coupled plasma mass spectrometry (ICP‐MS). The clearance data showed ideal clearance and sieving of spiked organic PERLs with log P < 4, partial clearance of PERLs with 4 < log P < 9, and poor clearance of highly hydrophobic PERLs (log P > 9) after nine diafiltration volumes (DVs). Supplemental clearance studies on seven additional PERLs present at much lower concentration levels (0.1–1.5 ppm) in the mAb formulation upstream of UF/DF and three PERLs associated with the tangential flow filtration (TFF) equipment also demonstrated the similar correlations between log P and % clearance. For model elements, the findings suggest that UF/DF in general provides ideal clearance for elements. Evidence showed that the UF/DF process does not only help mitigate leachables risk from PERLs introduced upstream of UF/DF, but also from the TFF operation itself as all three TFF‐related PERLs were effectively cleared. Overall, the UF/DF clearance presented in this work demonstrated whereas highly hydrophobic PERLs and elements that exist as charged species, particularly transition metal ions, may not be as effectively cleared and thus warrant further risk assessment; hydrophilic and some hydrophobic PERLs (log P < 4) are indeed well‐cleared and thus present a lower overall safety risk. [ABSTRACT FROM AUTHOR]

Diafiltration (DF) is a separation method used to separate and concentrate macromolecules, such as polysaccharides and proteins. To obtain high-purity target molecules by DF, appropriate conditions should be used. In this study, a mathematical model was developed to suggest appropriate ultra-filtration (UF) membrane modules for the separation of phycocyanin (PC) by multistep DF. PC is a protein produced by microalgae. The contribution of each UF membrane module to PC productivity and purity at each stage of the multistep DF process was quantified by the proposed model. The parameters required as model inputs (k, Fα1, and Fα2) were experimentally determined by permeating PC-containing solution through UF membrane modules (150, 30, and 10 kDa cutoffs). The resulting analytical solutions and those predicted by the model were in close agreement. The PC purity increased from 0.20 to 0.30 when a 10 kDa UF membrane module was used in two-step DF. An orthogonal table was used to determine the combination of UF membrane modules needed to achieve higher purity of PC. The model predicted that the 30 kDa UF membrane module would have the highest contribution to PC productivity and purity at any position in a three-step DF. The developed model can help identify appropriate conditions for separating macromolecules by DF. [ABSTRACT FROM AUTHOR]

Tangential flow filtration (TFF) is a central step in manufacturing of biopharmaceutics. Membrane clogging leads to decreased permeate flux, longer process time and potentially complete failure of the process. The effect of peristaltic pumping with tubings made of three different materials on protein particle formation during TFF was monitored via micro flow imaging, turbidity and photo documentation. At low protein concentrations, pumping with a membrane pump resulted in a stable flux with low protein particle concentration. Using a peristaltic pump led to markedly higher protein particle formation dependent on tubing type. With increasing protein particle formation propensity of the tubing, the permeate flux rate became lower and the process took longer. The protein particles formed in the pump were captured in the cassette and accumulated on the membrane leading to blocking. Using tubing with a hydrophilic copolymer modification counteracted membrane clogging and flux decrease by reducing protein particle formation. In ultrafiltration mode the permeate flux decrease was governed by the viscosity increase rather than by the protein aggregation; but using modified tubing is still beneficial due to a lower particle burden of the product. In summary, using tubing material for peristaltic pumping in TFF processes which leads a less protein particle formation, especially tubing material with hydrophilic modification, is highly beneficial for membrane flux and particle burden of the product. [ABSTRACT FROM AUTHOR]

In this study, ultrafiltration permeate obtained from acid whey (AWP) was applied as a diafiltration (DF) medium during skim milk microfiltration. The thereby induced acidification of differently concentrated retentates was assessed regarding suitability to generate gels from micellar casein concentrates further downstream. While aggregation was avoided during cold DF, subsequent heating induced controlled spontaneous formation of gels with concentration‐ and pH‐dependent firmness ranging from 26 to 1420 Pa. By employing its typical compounds, AWP as a rather problematic dairy side stream could thus be utilised for the production of calcium‐rich acidic casein gels providing sensory characteristics of cultured dairy products. [ABSTRACT FROM AUTHOR]

The enzyme β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, although for its effective application it is necessary to establish techniques for purification, concentration, or polishing, such as membrane separation processes, in particular ultrafiltration. The present study aimed to investigate ultrafiltration and diafiltration applied as initial steps for concentration and salt removal, respectively, in the β-galactosidase purification processes. Additionally, the influence levels of the pH (6.5, 7.7, or 7.5), membrane molecular weight cut-off (30, 50, 60, or 100 kDa), operating pressure (1.5, 2.0, or 2.5 kgf/cm2), and ionic strength of the ultrafiltration using NaCL or KCl (0.01–0.1 M) were evaluated considering the enzyme recovery, purification, retention, and concentration factors in relation to the proteins, volume, activity, and protein flux and yield of the processes. The ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm2 with a 50 kDa polyethersulfone membrane resulted in a volume concentration of the β-galactosidase extract up to 7.1-fold greater, a purification factor 1.2-fold greater, and an enzyme recovery rate of 108.9% by eliminating metabolites during the purification process. In addition, the lowest flux variation range (16.0 to 13.1 L/m2·h) was observed under these same conditions, thereby representing a decrease of 18.0%. An increase in the operating pressure and the addition of salts results in reduced enzyme recovery (up to 38% of the process yield (734.1 to 453.7 U/h) and up to 40% of the enzyme recovery rate (108.9 to 60.6%) during the ultrafiltration using NaCl, respectively). The operation in the diafiltration mode allowed salt removal after the purification of β-galactosidase (enzymatic recovery rates above 93.4%) via precipitation and ion-exchange chromatography elution and as part of an aqueous two-phase system using 6 diafiltration cycles, thereby revealing its application potential. [ABSTRACT FROM AUTHOR]

The aim of this study is extraction of potato protein via incorporation of ultrafiltration with different diafiltration times (90, 285 and 480 min), and evaluation of physicochemical and functional properties of extracted protein. So, in terms of functional characteristics (solubility, water and oil absorption capacity, emulsifying activity and emulsion stability) the concentrated permeate and retentate proteins, have been compared with control sample. In retentate and control samples the solubility increases significantly (P˂0.5) with raising the pH from 3 to 7, inverse it decreased between the 7 to 9. In addition, it was found that the solubility of retentate proteins were significantly (P˂0.5) more than control and permeated proteins. Also, the water absorption capacity of retentate proteins (18.85 g Water/ g protein Retentate 480 min) were more than the other samples (2.54 g Water/ g protein Permeate 90 min). Whereas, oil absorption capacity was significantly (P˂0.5) depended on diafiltration time and process. In a way that, by increasing the time this parameter dropped in all of the samples. It was also found that, the oil absorption of retentate samples (59.09 g oil/ g protein Retentate 90 min) were significantly (P˂0.5) more than the permeate ones (32.13 g oil/ g protein Permeate 90 min). Emulsifying activity and solubility of retentate samples were significantly grew by addition of diafiltration time, while these indexes reduced in permeate samples. The thermal analysis of retentate sample (with 480 diafiltration time) indicated that, it had higher water absorption capacity than control. [ABSTRACT FROM AUTHOR]

[Display omitted] The removal of organic solvents during the preparation of biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microparticles by an O/W- solvent extraction/evaporation process was investigated and controlled by diafiltration. Emulsification and steady replacement of the aqueous phase were performed in parallel in a single-vessel setup. During the process, the solidification of the dispersed phase (drug:PLGA:solvent droplets) into microparticles was monitored with video-microscopy and focused beam reflectance measurement (FBRM) and the residual solvent content was analyzed with headspace gas chromatography (organic solvent) and coulometric Karl-Fischer titration (water). Microparticles containing dexamethasone or risperidone were characterized with regard to particle size, morphology, encapsulation efficiency and in-vitro release. Diafiltration-accelerated solvent extraction shortened the process time by accelerating solidification of dispersed phase but reduced the residual dichloromethane content only in combination with increased temperature. Increasing the diafiltration rate increased particle size, porosity, and the encapsulation efficiency of risperidone. The latter effect was particularly evident with increasing lipophilicity of PLGA. A slower and more uniform solidification of end-capped and increased lactide content PLGA grade was identified as the reason for an increased drug leaching. Accelerated solvent extraction by diafiltration did not affect the in-vitro release of risperidone from different PLGA grades. The initial burst release of dexamethasone was increased by diafiltration when encapsulated in concentrations above the percolation threshold. Both porosity and burst release could be reduced by increasing the process temperature during diafiltration. Residual water content was established as an indicator for porosity and correlated with the burst release of dexamethasone. [ABSTRACT FROM AUTHOR]

Rare earth elements (REEs) are critical materials due to their utilization in high-flux magnets for wind turbines and electric motors. Purification and recycling of REEs are vital for maintaining their supply. This work explores and models nanofiltration (NF) followed by diafiltration for concentrating La3+ and polishing monovalent ions from the concentrated solution. Highly negative monovalent-ion rejections greatly enhance this polishing step, and proton concentrations decrease 3–4 orders of magnitude in 2.4 cell volumes of diafiltration. Studies with rotating membranes give a controlled, uniform boundary layer above the membrane to enable estimation of salt and ion permeances at various feed compositions. Insertion of these permeances in the solution-diffusion-electromigration model yields trends in ion rejections as a function of composition, solution flux, and boundary layer thickness. Simulated rejections of monovalent ions become much more negative with lower solution fluxes (up to a point) and higher ratios of La3+ to monovalent ions. La3+ rejections are much higher in the rotating membrane than in a stirred cell where the diffusion boundary layer is thicker and nonuniform. The combination of NF and diafiltration provides concentrated La3+ with a molar purity of 99 % when starting with equimolar mixtures of La3+ and Na+. Acid removal from La3+ solutions via diafiltration could prove useful for subsequent separations among REEs or other transition metals. [Display omitted] • Diafiltration efficiently polishes La3+ from Na+ or H+ due to negative rejection. • Uniform boundary layers on rotating membranes enable estimation of permeances. • The solution-diffusion-electromigration model gives trends in ion rejections. • Negative monovalent-ion rejections depend on flux, composition, and boundary layers. • Diafiltration yields 99 % La3+ from equimolar Na+ and La3+. [ABSTRACT FROM AUTHOR]

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing. [ABSTRACT FROM AUTHOR]

Chitosan (CS)-based polyplexes are produced by spontaneous electrostatic association with nucleic acids using CS in excess. Interactions of positively charged polyplexes, and the unbound CS, with negatively charged blood components limit the applicable dosage of such polymeric nanoparticles (NPs) and development of formulations with improved hemocompatibility and transfection efficiency is needed. Here, we introduce a strategy based on Tangential Flow Filtration (TFF) to remove unbound CS, concentrate polyplexes and subsequently coat with hyaluronic acid (HA) to improve hemocompatibility and bioactivity. Optimal TFF conditions were established. A library of HA with different molecular weights and degrees of sulfation was used at different carboxyl + sulfate to phosphate ratios for polyplex coating, bioactivity and hemocompatibility assessment. A systematic optimization of TFF conditions allowed for purification of polylpexes from excess unbound CS and subsequent coating with HA. Except for high molecular weight HA, for which macroscopic aggregation was observed, both sulfated and non-sulfated HAs resulted in small sized and homogenous coated complexes. However, sulfated HAs displayed higher stability during the second filtration process indicating their stronger binding affinity to polyplexes. Finally, we found that low molecular weight HA-coated polyplexes have equivalent silencing efficiency in vitro and improved hemocompatibility compared to uncoated polyplexes. [Display omitted] [ABSTRACT FROM AUTHOR]

We report the solvent-evaporation and ionic cross-linking mediated self-assembly of the shell cross-linked micelles of the amphiphilic triblock copolymer containing middle poly(methyl methacrylate) block (hydrophobic) and poly(2-dimethylamino)ethyl methacrylate end blocks (hydrophilic) on the membrane substrate to create molecular selective channels. The formation of selective channels on the substrate is attributed to the local increase of micelle concentration upon solvent evaporation, which leads to the core-core hydrophobic interaction. The post-ionic cross-linking of the shell part further reduces the intermicelle distance, thereby creating interstices for selective separation. The TUF-1:1 membrane prepared by the self-assembly of the cross-linked micelles (triblock copolymer:halide-terminated PEG-based = 1:1 w w -1 ) and by the post-ionic cross-linking shows molecular weight cutoff of 3000 g mol -1 and pure water permeance of 52 L m - 2 h -1 bar -1 . The membrane shows 99.5-99.9% rejection of Congo red and Direct red-80 in the presence or absence of salts and Na 2 SO 4 to dye separation factor of about 900. The added functionality (PEG) in the micelle structure provides good fouling-resistant properties toward dye and bovine serum albumin. This work provides the membrane formation mechanism and the advantages of the membrane for fractionation and resource recovery applications.

Evaluating the Gibbs-Donnan and volume exclusion effects during protein ultrafiltration and diafiltration (UF/DF) is crucial in biopharmaceutical process development to precisely control the concentration of the drug substance in the final formulation. Understanding the interactions between formulation excipients and proteins under these conditions requires a domain-specific knowledge of molecular-level phenomena. This study developed gradient boosted tree models to predict the Gibbs-Donnan and volume exclusion effects for amino acids and therapeutic monoclonal antibodies using simple molecular descriptors. The models' predictions were interpreted by information gain and Shapley additive explanation (SHAP) values to understand the modes of action of the antibodies and excipients and to validate the models. The results translated feature effects in machine learning to real-world molecular interactions, which were cross-referenced with existing scientific literature for verification. The models were validated in pilot-scale manufacturing runs of two antibody products requiring high levels of concentration. By only requiring a molecule's biophysicochemical descriptors and process conditions, the proposed models provide an in silico alternative to conventional UF/DF experiments to accelerate process development and boost process understanding of the underlying molecular mechanisms through rational interpretation of the models., (© 2024 Chugai Pharmaceutical Co., Ltd. Biotechnology Journal published by Wiley‐VCH GmbH.)

In this study, kinetic modeling of permeates flux and total hydraulic resistance of camel milk diafiltration in different conditions of pH (5.8, 6.3 and 6.8) and NaCl concentration (0, 75 and 150 mM) was performed by using 6 kinetic models. Finally, the homographic kinetic model has been selected for modeling permeates flux and exponential kinetic model has been selected for modeling total hydraulic resistance considering coefficient of determination (R2) and Root mean square deviation (RMSE). The results of ANOVA of homographic kinetic model illustrated that the linear effect of pH on all model parameters (initial flux (J0), steady-state flux (J∞), flux decline time constant (1/b) and flux decline extent (a)) and the linear effect of NaCl concentration at a 95% level on J0, 1/b and the interaction effects of pH-NaCl concentration at a 99% level on a were significant. The results of ANOVA of exponential kinetic model also showed that the linear effect of pH had a significant effect on steady-state hydraulic resistance (R∞) and the linear effect of NaCl concentration on initial hydraulic resistance (R0), R∞ and resistance increment rate (K) and the interaction effects of pH-NaCl concentration at a 95% level on k were significant. According to the results of sensitivity analysis for each 0.1 increase in pH about 1% R∞ decreased and for each 10 mM increase in NaCl concentration 2.75% R0 and 9% 1/b of the samples increased.

The study deals with nanofiltration for separating saccharides from phenolics and considers permeation of each individual sugar (mono- or disaccharide) from a plant hydrolysate compared to a model sugar solution. Experiments are carried out in a cross-flow flat-sheet cell (MaxiMem, Prozesstechnik GmbH) equipped with Nadir® NP030 P membrane. Nanofiltration in concentration and diafiltration mode has been investigated. It has been found that diafiltration mode ensures the appropriate conditions in the concentration polarization layer of the membrane that favors the sugar diffusion but prevents critical deposition and fouling. Due to the different mass transfer rates of sugars, a stepwise purification of phenolics in the retentate is obtained. Phenolics are first purified from galactose and mannose due to the negative rejections of these sugars at the beginning of diafiltration. Then purification from disaccharides increases with the increase in concentration polarization effect on diffusive transport of disaccharides through the membrane. At the same time purification from glucose also increases and becomes significant. Purification factors achieved in diafiltration mode vary from 3.9 to 10.2 and are up to 6.4-fold higher than in concentration mode. [Display omitted] • Diananofiltration versus concentration mode for separation of sugars and phenolics. • Higher sugar permeation and lower membrane fouling are achieved in diafiltration. • Concentration polarization leads to enhanced diffusive transport of dissacharides. • Smaller monosugars show negative rejections due to greater convective solute flux. • Due to different mass transfer rates of sugars, phenolics are purified stepwise. [ABSTRACT FROM AUTHOR]

Background and Objectives: Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell‐free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell‐free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. Materials and Methods: The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof‐of‐concept study, RBC units were expired at the time of washing (60–70 days old). Cell‐free Hb was quantified by UV–visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre‐ and post‐wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. Results: No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell‐free Hb reduction was observed within the first four diacycles with a starting cell‐free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Conclusion: Overall, this study investigated the proof‐of‐concept use of TFF for washing single RBC units with an emphasis on the removal of cell‐free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications. [ABSTRACT FROM AUTHOR]

A diafiltration-nanofiltration-reverse osmosis (DiaNF-RO) process is introduced to achieve solutes fractionation of divalent/monovalent ions, i.e., Mg/Na, to enable resource recovery in seawater desalination and brine management. The diafiltration process is applied at the NF stage (i.e., DiaNF) to enhance the Mg/Na fractionation performance of NF (i.e., SF1 Mg-Na), while the RO is incorporated to produce desalinated water as well as diluent required by the DiaNF. By initiating the ion fractionation at the pre-treatment rather than post-treatment (i.e., RO-NF), the DiaNF-RO mitigates challenges of high concentration in RO brine management, which includes limitation of maximum allowable operating pressure and high energy requirement. The semi-empirical model, with at least 77.4 % accuracy, is first applied to simulate the performance of DiaNF-RO at different operating conditions and configurations (i.e., multi-stage and recycling). The sensitivity analysis then suggests that dilution should only occur in the last NF stage and without any retentate recycling, while NF pressure and element number should be optimized. Using NF membrane with SF Mg-Na of 1.28 at 10 bar and feed seawater of 35 g/L TDS, the optimal 4-stage DiaNF-RO design achieves maximum SF1 Mg-Na of 12.19 at 5.72 kWh/m3, or a minimum energy consumption of 5.41 kWh/m3 at SF1 Mg-Na of 7.04. Schematic diagram of a DiaNF-RO process. [Display omitted] • DiaNF-RO initiates divalent-monovalent ion fractionation at pre-treatment stage. • DiaNF-RO amplifies separation factor of nanofiltration membrane by 12 times. • DiaNF-RO achieves 12.2 Mg-Na fractionation at a competitive 5.72 kWh/m3. • Practical simulations are validated to analyse the design in continuous mode. • Optimal operating conditions are identified for ion fractionation. [ABSTRACT FROM AUTHOR]

The diafiltration-nanofiltration-reverse osmosis (DiaNF-RO) is an innovative process that aims to achieve ion fractionation of divalent/monovalent ions, i.e., Mg/Na (SF1 Mg-Na), an important step towards resource recovery, while producing clean water in seawater desalination at a reasonable specific energy consumption (SEC). Firstly, the novelty of DiaNF-RO process is compared with the RO-DiaNF process where ion fractionation is performed at the pretreatment stage (i.e., seawater) in the former and post-treatment stage (i.e., SWRO brine) in the latter. DiaNF-RO outperforms RO-DiaNF in many aspects, including higher Mg recovery, higher SF1 Mg-Na , lower scaling risk, and more efficient cost utilization, which emphasizes the benefits of initiating ion fractionation at low salinity in brine management. Secondly, the process practicality is reiterated by showing how desirable performance is achievable at low number of stages with some modifications. One possible adjustment is to improve NF membrane performance in the priority order of higher water permeability, lower Na rejection, and higher Mg rejection, which enables the 2DiaNF-RO to achieve the desirable SF1 Mg-Na of 13.7 at 5.71 kWh/m3. Another possible adjustment is to integrate the 2nd RO stage for more product water recovery to achieve a competitive net SEC (4.01 kWh/m3). These results further solidify the industrial implementation possibility of the DiaNF-RO process. Comparison between DiaNF-RO and RO-DiaNF, and enhancement of the 2DiaNF-RO process with improved NF membrane performance or 2nd RO integration. [Display omitted] • Pre-treatment is more preferred than post-treatment for Mg/Na fractionation. • Pre-treatment has better cost utilization for ion fractionation. • Higher NF permeability triples the Mg/Na fractionation. • 2nd RO stage integration reduces energy by 30 % at lower water cost. • Desirable 2DiaNF-RO performance emphasizes process practicality. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop an extraction method to produce highly functional oat protein concentrates. We investigated the possibility of combining enzyme-aided slightly alkaline (pH 8.0) extraction with ultrafiltration and subsequent diafiltration for concentration of the extracted oat proteins. A further aim was to study how the deamidation of oat proteins with protein-glutaminase (PG) improves the solubility of proteins as a function of the following parameters: pH (6.0–9.0), enzyme dosage (4–20 U/g protein), and incubation time (1–4 h) with response surface methodology (RSM). Furthermore, we investigated selected functional properties, such as heat-induced gelation and solubility, of the oat protein concentrates. The chosen parameters for the enzymatic deamidation pre-treatment process by PG were as follows: pH 8.0, dosage 11.0 U/g protein, and an incubation time of 4 h (1 h at native pH and 3 h at pH 8.0). Two oat protein concentrates were produced, non-deamidated and ultrafiltered, and deamidated and ultrafiltered, with protein concentrations of 45.0 and 52.4%, respectively. The solubility of both oat protein concentrates was significantly improved at neutral and slightly alkaline pH compared to the solubility of proteins extracted from the starting material. Additionally, both oat protein concentrates produced equally strong heat-induced gel-like structures at a protein concentration of 10%. [ABSTRACT FROM AUTHOR]

Bottom-up approaches to producing aqueous crystal suspensions of active pharmaceutical ingredients (APIs), such as anti-solvent crystallisation, are gaining interest as they offer better control over surface properties compared to top-down approaches. However, one of the major challenges that needs to be addressed is the removal of organic solvents after the crystallisation step due to strict limitations regarding human exposure. Within this work, we investigated a process concept for the removal of solvent (i.e., ethanol) from the API crystal suspension using membrane-based diafiltration. A four-stage diafiltration process successfully reduced the ethanol concentration in the API (here, naproxen) crystal suspension below 0.5 wt% (the residual solvent limit as per ICH guidelines) with a water consumption of 1.5 g of added water per g of feed. The solvent exchange process had no negative influence on the stability of the crystals in suspension, as their size and polymorphic form remained unchanged. This work is a step towards the bottom-up production of API crystal suspension by applying solvent/anti-solvent crystallisation. It provides the proof of concept for establishing a process of organic solvent removal and offers an experimental framework to serve as the foundation for the design of experiments implementing a solvent exchange in API production processes.

The enzyme β-galactosidase catalyzes the hydrolysis of lactose into glucose and galactose, although for its effective application it is necessary to establish techniques for purification, concentration, or polishing, such as membrane separation processes, in particular ultrafiltration. The present study aimed to investigate ultrafiltration and diafiltration applied as initial steps for concentration and salt removal, respectively, in the β-galactosidase purification processes. Additionally, the influence levels of the pH (6.5, 7.7, or 7.5), membrane molecular weight cut-off (30, 50, 60, or 100 kDa), operating pressure (1.5, 2.0, or 2.5 kgf/cm2), and ionic strength of the ultrafiltration using NaCL or KCl (0.01–0.1 M) were evaluated considering the enzyme recovery, purification, retention, and concentration factors in relation to the proteins, volume, activity, and protein flux and yield of the processes. The ultrafiltration of the crude enzyme extract at pH 7.5 and 1.5 kgf/cm2 with a 50 kDa polyethersulfone membrane resulted in a volume concentration of the β-galactosidase extract up to 7.1-fold greater, a purification factor 1.2-fold greater, and an enzyme recovery rate of 108.9% by eliminating metabolites during the purification process. In addition, the lowest flux variation range (16.0 to 13.1 L/m2·h) was observed under these same conditions, thereby representing a decrease of 18.0%. An increase in the operating pressure and the addition of salts results in reduced enzyme recovery (up to 38% of the process yield (734.1 to 453.7 U/h) and up to 40% of the enzyme recovery rate (108.9 to 60.6%) during the ultrafiltration using NaCl, respectively). The operation in the diafiltration mode allowed salt removal after the purification of β-galactosidase (enzymatic recovery rates above 93.4%) via precipitation and ion-exchange chromatography elution and as part of an aqueous two-phase system using 6 diafiltration cycles, thereby revealing its application potential.

This research utilized a customized laboratory setup to compare the filtration performance and fouling buildup during microfiltration with polymeric membranes of skim milk using 2 diafiltration media: ultrafiltration permeate and ultrapure water. Two filtration modes were evaluated: in stage 1, the diafiltration media was added in a 1:1 ratio, with the collection of permeate continuing until the initial protein concentration was restored. In stage 2, retentates and permeates were recycled to simulate fouling accumulation in a steady-state without altering the retentate composition. Utilizing water as the diafiltration medium resulted in higher flux and lower resistance values compared with using ultrafiltration permeate, irrespective of the filtration mode. The concentration had a significant impact on membrane resistance, with no noticeable time-dependent effect on fouling layer development after 60 min of filtration when the retentate composition remained constant. The protein composition of the permeate and extracted foulants were comparable between the 2 media, with caseins predominating in the fouling layer., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Deep eutectic solvents (DES) are green alternatives for conventional solvents. They have gained attention for their potential to extract valuable compounds from biomass, such as seaweed. In this framework, a case study was developed to assess the feasibility of pressure-driven membrane processes as an efficient tool for the recovery of deep eutectic solvents and targeted biomolecules. For this purpose, a mixture composed of the DES choline chloride - ethylene glycol (ChCl-EG) 1:2, water and alginate was made to mimic a DES extraction from seaweed. An integrated separation process design was proposed where ultrafiltration-diafiltration-nanofiltration (UF-DF-NF) was coupled. UF and DF were found to be effective for the separation of alginate with an 85 % yield. DES was likewise recovered by 93 %, proving the membrane filtrations' technical feasibility. The NF performance to separate the DES from the water, for its recycling, laid by a 45 %-50 % retention and a final concentrated DES solution of 18 %(v/v)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Furfural, acetic acid, and sulfates are found in the hemicellulose (HMC) fraction of lignocellulosic biomass. Separation of furfural, acetic acid, and sulfates from monosaccharides by four nanofiltration (NF) membranes was evaluated with a model solution of glucose, xylose, furfural, acetic acid, and sulfates. Results showed that Alfa Laval NF99HF is the most promising membrane to purify monosaccharides, with the retentions of xylose (85%), glucose (95%), and with the minimum sulfate retention. pH has the highest impact on the retention of all solutes and there is no significant effect of temperature on the retentions of sulphates and acetic acid. Lower pH and temperature are favored to maximize the monosaccharide retention and to remove acetic acid while retaining more furfural with the monosaccharides. Moreover, fouling tendency is maximized at lower pH and higher temperatures. According to the statistical analysis, the retentions of glucose, xylose, furfural, sulfates, and acetic acid are 95%, 90%, 20%, 88%, and 0%, respectively at pH 3 and 25 °C. The presence of sulfates favors the separation of acetic acid and furfural from monosaccharides.

The milk protein concentrate (MPC) has been extensively studied; however, the MPC partial demineralization through the diafiltration (DF) and its effect on MPC ability to produce milk coagulate products has not been fully explored; therefore, it was considered studying the MPC demineralization process with DF and evaluate the effect of this treatment on the compositional and textural characteristics of enzymatically and acid-coagulated products. The MPC of ultrafiltration was diafiltered by two cycles, later this MPC was used to make a fresh cheese, a set yogurt and stirred yogurt. The application of a single DF cycle removed 22.2% of the ashes and 8.12% of the MPC calcium, but no statistically significant differences were present (P> 0.05) between the application of two DF cycles. The cheeses with MPC undergone to one cycle and two cycles of DF were less hard and presented less resistance to chewing, and the set yogurt showed lower springiness values due a total solids and calcium content, that was affected by DF. These phenomena increased the coagulation time and the formation of weaker gels. The DF achieved the maximum milk demineralization in a single cycle.

This study aimed to investigate the effect of pH levels (5.8, 6.3 and 6.8) and NaCl concentrations (0, 75 and 150 mM) on the camel milk diafiltration performance in terms of pseudo‐steady‐state permeate flux, total fouling resistance and compositions of retentate (protein, lactose, ash, total solids and 11 minerals). The results showed that the camel milk diafiltration performance can be enhanced by controlling the pH and NaCl concentrations. In this research, the optimum ultrafiltration performance conditions with the lowest fouling, lactose and ash, and the highest flux and protein were pH 6.6 and 110 mM NaCl. [ABSTRACT FROM AUTHOR]

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Streptokinase is a extracellular enzyme which is extracted from strains of beta Hemolytic streptococcus. The enzyme is a non-protease plasminogen activator that activates plasminogen to plasmin and degrades fibrin clot through its specific lysine binding site which is used in thrombolytic therapy. Purification of streptokinase produced from S.equisimilis in E.coli with N-terminal methionine was carried out in 3 Chromatography purification steps, 1) CM-Sepharose-FF at pH 4.2 followed by concentration and dialysis over night with Tris-HCl pH 8.0. Partially purified dialyzed enzyme sample was loaded on to 2) DEAE-Sepharose-FF column. The Purified fractions of DEAE column were pooled and applied on to Sephadex G-100 column. Enzyme purity was confirmed by SDS-PAGE and RP-HPLC.Its biological activity is determined by specific streptokinase assay and characterised the enzyme by Peptide mapping, MALDI-TOF, Isoelectric-focusing and RP-HPLC. The isoelectric point (pI) of streptokinase is around 4.98.The results of characterization shows that it contains two forms (Isomers) of streptokinase expressed in E. coli which was analyzed by RP-HPLC and chromogenic assay. The variation is formed by isomer-1 in which 85% of Streptokinase expressed without methionine (85000IU/mg) and Isomer-2 in which 15% of streptokinase expressed with methionine (nil activity) in E. coli. This phenomenon shows that the presence and absence of methionine in isomers of streptokinase varying the catalytic activity of the enzyme.

Partial removal of sugars in fruit juices without compromising their biofunctional properties represents a significant technological challenge. The current study was aimed at evaluating the separation of sugars from phenolic compounds in apple juice by using three different spiral-wound nanofiltration (NF) membranes with a molecular weight cut-off (MWCO) in the range of 200–500 Da. A combination of diafiltration and batch concentration processes was investigated to produce apple juice with reduced sugar content and improved health properties thanks to the preservation and concentration of phenolic compounds. For all selected membranes, permeate flux and recovery rate of glucose, fructose, and phenolic compounds, in both diafiltration and concentration processes, were evaluated. The concentration factor of target compounds as a function of the volume reduction factor (VRF) as well as the amount of adsorbed compound on the membrane surface from mass balance analysis were also evaluated. Among the investigated membranes a thin-film composite membrane with an MWCO of 200–300 Da provided the best results in terms of the preservation of phenolic compounds in the selected operating conditions. More than 70% of phenolic compounds were recovered in the retentate stream while the content of sugars was reduced by about 60%.

Ultrafiltration/diafiltration (UF/DF) is a typical step in protein drug manufacturing process to concentrate and exchange the protein solution into a desired formulation. However, significant offset of pH and composition from the target formulation have been frequently observed after UF/DF, posing challenges to the stability, performance, and consistency of the final drug product. Such shift can often be attributed to the Donnan and volume exclusion effects. In order to predict and compensate for those effects, a mechanistic model is developed based on the protein charge, mass and charge balances, as well as the equilibrium condition across the membrane. The integrated UF/DF model can be used to predict both the dynamic behavior and the final outcome of the process. Examples of the modeling results for the pH and composition variation during the UF/DF operations are presented for two monoclonal antibody proteins. The model predictions are in good agreement with a comprehensive experimental data set that covers different process steps, protein concentrations, solution matrices, and process scales. The results show that significant pH and excipient concentration shifts are more likely to occur for high protein concentration and low ionic strength matrices. As a special example, a self‐buffering protein formulation shows unique pH behavior during DF, which could also be captured with the dynamic model. The capability of the model in predicting the performance of UF/DF process as a function of protein characteristics and formulation conditions makes it a useful tool to improve process understanding and facilitate process development. [ABSTRACT FROM AUTHOR]

The design of effective ultrafiltration/diafiltration processes for protein formulation requires the use of membranes with very high protein retention. The objective of this study was to examine the effects of specific buffers on the retention of a model protein (bovine serum albumin) during ultrafiltration. Albumin retention at pH 4.8 was significantly reduced in phosphate buffer compared with that in acetate, citrate, and histidine. This behavior was consistent with a small change in the effective albumin hydrodynamic diameter as determined by dynamic light scattering. The underlying conformational changes leading to this change in diameter were explored using circular dichroism spectroscopy and differential scanning calorimetry. These results provide important insights into the factors controlling protein retention during ultrafiltration and diafiltration., (© 2024 The Author(s). Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

The high protein content of Arthrospira platensis makes this cyanobacterium a great component of food. Thus, the protein obtained should guarantee the nutrient characteristic, in this case, as bioactivity and techno-functional properties. Membrane separation processes (MSPs) have been highlighted by their capacity to separate proteins and keep their properties. Therefore, MSP was used to concentrate and separate the proteins from A. platensis, which were characterized by their physicochemical and techno-functional properties. Different ultrafiltration (UF) membranes and operational conditions (10- and 50-kDa pore size, conventional and tangential flow, hollow fiber type and pressures of 1.0, 1.5, and 2.0 bar) were tested and associated with diafiltration (DF). The maximized condition of UF/DF (hollow fiber, 50 kDa, tangential flow at 1.5 bar, associated with one DF) was up scaled (pilot scale) and allowed concentrate and permeate fractions with 81.75% and 61.20% of protein, respectively, to be obtained. A protein concentrate with 142 mg L−1 of phycocyanin, 90% of antioxidant activity, with proteins more soluble at high pH (50% soluble protein at pH 7.0) and molecular weight among 5–15 and 100 kDa, was obtained. Protein thermal and emulsifying properties were maintained by the MSP, and the concentrate fraction presented results similar as A. platensis biomass, which confirms the advantage of this method in guaranteeing the protein quality. [ABSTRACT FROM AUTHOR]

In this study, membrane-based methods were evaluated for the recovery of FucoPol, the fucose-rich exopolysaccharide (EPS) secreted by the bacterium Enterobacter A47, aiming at reducing the total water consumption and extraction time, while keeping a high product recovery, thus making the downstream procedure more sustainable and cost-effective. The optimized method involved ultrafiltration of the cell-free supernatant using a 30 kDa molecular weight cut-off (MWCO) membrane that allowed for a 37% reduction of the total water consumption and a 55% reduction of the extraction time, compared to the previously used method (diafiltration-ultrafiltration with a 100 kDa MWCO membrane). This change in the downstream procedure improved the product’s recovery (around 10% increase) and its purity, evidenced by the lower protein (8.2 wt%) and inorganic salts (4.0 wt%) contents of the samples (compared to 9.3 and 8.6 wt%, respectively, for the previously used method), without impacting FucoPol’s sugar and acyl groups composition, molecular mass distribution or thermal degradation profile. The biopolymer’s emulsion-forming and stabilizing capacity was also not affected (emulsification activity (EA) with olive oil, at a 2:3 ratio, of 98 ± 0% for all samples), while the rheological properties were improved (the zero-shear viscosity increased from 8.89 ± 0.62 Pa·s to 17.40 ± 0.04 Pa·s), which can be assigned to the higher purity degree of the extracted samples. These findings demonstrate a significant improvement in the downstream procedure raising FucoPol’s recovery, while reducing water consumption and operation time, key criteria in terms of process economic and environmental sustainability. Moreover, those changes improved the biopolymer’s rheological properties, known to significantly impact FucoPol’s utilization in cosmetic, pharmaceutical or food products.

The purification of phycocyanin (PC) from Spirulina generally involves a combination of different techniques. Here, we report the results on PC yields from a combined aqueous extraction-ultrafiltration (UF) process of a strain of Arthrospira maxima cultivated in a farm devoted to producing PC with food-grade purity. Samples optimized from different biomass/solvent ratios were purified by using a polyethersulphone (PES) membrane with a molecular weight cut-off (MWCO) of 20 kDa. The UF system was operated at 2.0 ± 0.1 bar and at 24 ± 2 °C up to a volume concentration factor (VCF) of 5. A diafiltration (DF) process was conducted after UF in order to increase the PC recovery in the retentate. Samples were collected during both UF and DF processes in order to evaluate membrane productivity and PC purity. The average permeate fluxes of about 14.4 L/m2h were measured in the selected operating conditions and more than 96% of PC was rejected by the UF membrane independently ofthe extraction yields and times. The concentration of PC in the final retentate was 1.17 mg/mL; this confirmed the observed rejection and the final VCF of the process (about 5-fold when compared to the concentration of PC in the crude extract). In addition, the combination of UF and diafiltration allowed the removal of about 91.7% of the DNA from the crude extract, thereby improving the purity of the phycocyanin in the retentate fraction.

Membrane technology has been successfully applied for the purification of bromelain, a protease enzyme from pineapple. However, the current system operates less optimally in terms of flux and separation primarily due to properties of the feed, such as viscosity. Hence, in this study, enzymatic pretreatment and diafiltration operation were employed in a two-stage ultrafiltration (UF) system to enhance the performance of the purification and concentration process of bromelain enzyme from an extract of pineapple crude waste mixture (CWM). Pretreatment of the CWM extract using either pectinase or cellulase, or the combination of both, was applied and compared regarding the apparent viscosity reduction. Diafiltration step was introduced in UF stage 2 and observations on the flux performance, enzyme recovery and enzyme purity were made. A 12 % apparent viscosity reduction was achieved when the CWM extract was pretreated with pectinase which led to 37-38 % improvement in the flux performance of both UF stages, as well as higher enzyme recovery in UF stage 1. The introduction of diafiltration mode in UF stage 2 managed to sustain high flux values while yielding 4.4-fold enzyme purity (higher than a 2.5-fold purity achieved in our previous work); however, high diluent consumption was needed. The outcomes of this study showed that the flux performance and bromelain separation can be enhanced by reducing the viscosity with the employment of enzymatic pretreatment and diafiltration operation. Thus, both techniques can be potentially applied in a large-scale membrane-based process for bromelain production.

The aim of this study was to develop an extraction method to produce highly functional oat protein concentrates. We investigated the possibility of combining enzyme-aided slightly alkaline (pH 8.0) extraction with ultrafiltration and subsequent diafiltration for concentration of the extracted oat proteins. A further aim was to study how the deamidation of oat proteins with protein-glutaminase (PG) improves the solubility of proteins as a function of the following parameters: pH (6.0–9.0), enzyme dosage (4–20 U/g protein), and incubation time (1–4 h) with response surface methodology (RSM). Furthermore, we investigated selected functional properties, such as heat-induced gelation and solubility, of the oat protein concentrates. The chosen parameters for the enzymatic deamidation pre-treatment process by PG were as follows: pH 8.0, dosage 11.0 U/g protein, and an incubation time of 4 h (1 h at native pH and 3 h at pH 8.0). Two oat protein concentrates were produced, non-deamidated and ultrafiltered, and deamidated and ultrafiltered, with protein concentrations of 45.0 and 52.4%, respectively. The solubility of both oat protein concentrates was significantly improved at neutral and slightly alkaline pH compared to the solubility of proteins extracted from the starting material. Additionally, both oat protein concentrates produced equally strong heat-induced gel-like structures at a protein concentration of 10%.

Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low‐buffered media due to the change in local H+ concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration. The model involves no adjustable parameters, with the protein charge evaluated directly from the protonation/deprotonation of the ionizable amino acids accounting for the electrostatic interactions between the charged mAb and the H+ ions. Model predictions are in excellent agreement with experimental data for the pH and ion concentrations during diafiltration of a mAb and fusion protein with different isoelectric points and different formulation conditions. Model simulations are then used to obtain fundamental insights into the factors controlling the diafiltration behavior as well as guidelines for development of diafiltration processes to achieve target bufferless formulation conditions. [ABSTRACT FROM AUTHOR]

Cheese made from microfiltration (MF) retentate may suffer from textural defects due to a high Ca concentration. The reduction of colloidal minerals by the acidification of milk before MF at pH below 6.0 has been well documented in the literature. This process, however, creates less valuable side streams to the MF process and induces changes in the casein micelles that negatively affect their coagulation properties. The objective of this study was to determine whether a minor reduction in pH by using different acidifiers in the diafiltration (DF) water could induce changes in composition and renneting properties of the MF retentate. A 2-stage filtration process was used, with the first designed to increase the casein concentration to 8% and the second to slightly reduce the casein concentrate by 0.1 pH unit by DF, without influencing the total protein concentration. Four acidifying agents were tested during DF: lactic acid, hydrochloric acid, citric acid, and carbon dioxide. Diafiltration with water was used as a reference. At the start of DF, the retentates of acid DF had a slightly reduced pH, with an average of 0.09, whereas the pH of the reference retentate increased by an average of 0.07 unit. The reference retentate regained its starting pH by the end of DF. The carbonated retentate gradually increased in pH during processing, whereas the pH of the lactic, hydrochloric, and citric acid retentates remained constant. The permeate from the lactic acid and carbonated treatments had a reduced whey protein content compared with the reference. The total P and inorganic phosphate were lowered in the retentate by using carbonation. The total amount of Mg and Na were lowered in the retentate by using citric acid. The ionic Ca content in the retentate increased with use of lactic or hydrochloric acid. The type of acidifier used reduced the rennet clotting time. Combined acidified diafiltration with a slight reduction affects the permeate composition and improves the retentate clotting time despite the minimal mineral modification. [ABSTRACT FROM AUTHOR]

A membrane process for the recovery of = 90 % of the soluble peptides (with antioxidant activity) found in concentrated lecithin-free egg yolk hydrolysates (LFEYH) was established. Hydrolysis of concentrated free lecithin egg yolk protein dispersions (10 and 20 %) was conducted with Alcalase 2.4 L, an endoprotease from Bacillus Licheniformis. The obtained hydrolysates were fractionated by ultrabltration (UF), where the effect of the membrane material and molecular weight cut-off (MWCO) was studied. 'I/No flat sheet regenerated cellulose membranes and three tubular ceramic membranes with different MWCOs (50, 10 and 5 kDa) were tested. Different hydrodynamic operating conditions were established in all membranes to work under sustainable flux during UF. The 50 kDa ceramic membrane was found to be the most efficient in terms of flux (78.45 and 47.84 kgm-2 h-1 for 10 and 20 % protein dispersions, respectively) and showed lower fouling. The use of diafiltration (DF), allowed recovering more than 90 % of soluble and heat-stable peptides, which also showed high antioxidant activity, from hydrolysates produced in both concentrated (10 and 20 %) egg yolk protein dispersions. [ABSTRACT FROM AUTHOR]