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Background: This study assesses the feasibility of producing hyperimmune anti-COVID-19 intravenously administrable immunoglobulin (C-IVIG) from pooled convalescent plasma (PCP) to provide a safe and effective passive immunization treatment option for COVID-19. Materials & methods: PCP was fractionated by modified caprylic acid precipitation followed by ultrafiltration/diafiltration to produce hyperimmune C-IVIG. Results: In C-IVIG, the mean SARS-CoV-2 antibody level was found to be threefold (104 ± 30 cut-off index) that of the PCP (36 ± 8.5 cut-off index) and mean protein concentration was found to be 46 ± 3.7 g/l, comprised of 89.5% immunoglobulins. Conclusion: The current method of producing C-IVIG is feasible as it uses locally available PCP and simpler technology and yields a high titer of SARS-CoV-2 antibody. The safety and efficacy of C-IVIG will be evaluated in a registered clinical trial (NCT 04521309).

In the framework of modern bioprocessing continuous ultrafiltration/diafiltration (UF/DF) is getting increasingly popular. However, while continuous UF can be easily implemented using a so-called single pass tangential flow filtration (SPTFF) module, continuous DF requires a more complicated setup including several SPTFF modules and intermittent dilution steps. Recently, we introduced a novel module design for continuous DF allowing simultaneous delivery of fresh buffer while withdrawing the permeate, thus achieving high degrees of buffer exchange within a single unit. In addition, the system allows to cyclically switch the flow direction of DF buffer through the membranes. Those uncommon features, however, also make it more difficult to determine an operation optimum experimentally by means of trial and error. Therefore, here a detailed finite element model of the physical processes within the module is presented, predicting key figures such as the obtained diafiltration efficiency and the resulting pressures. Because within the module all flow channels are filled by a 3D-printed porous grid supporting the membranes from both sides, the modified Brinkman equation was used to simulate the hydrodynamics, while common mass balance differential equations including accumulation, convection, and an anisotropic dispersion term were used for the simulation of concentration profiles of dissolved species. The predicted key figures are in good agreement with experimental results, obtained for feed solutions including up to 50 g/L of protein and being operated with and without switching the flow direction of the diafiltration buffer. A thorough parameter study reveals that the module shows the best performance for unidirectional flow of the diafiltration buffer, reaching diafiltration efficiencies independence to the applied diavolumes which are comparable to the ones of a conventional multi-stage setup using three SPTFF modules. Therefore, the simulation-based evaluation of optimum operation conditions reveals that the new module design has the potential to realize truly continuous diafiltration setups with high efficiency, requiring only one unit and no extra external piping for returning diafiltration in counterflow. Such simplified setups should be especially useful in small, flexible processing plants as they are increasingly demanded in the biopharmaceutical industry.

This paper presents the results of preparation of highly purified acid protease eluates from the native solution of Aspergillus oryzae strain 55. The analysis of the component composition of the native solution of the Aspergillus oryzae was carried out by the elution gel chromatography method. In addition, the molecular weight of the acid protease of Aspergillus oryzae was determined. The study of the native solution by the method of electrophoresis confirmed the obtained data. Isolation and purification of acid protease from ballast impurities was performed by sorption on various cation exchangers and by ultrafiltration. Optimal conditions for sorption of acid protease from the native solution of Aspergillus oryzae on cation exchangers KU-23, С-106, KB-2 were chosen, and equilibrium parameters of sorption of acid protease on these sorbents were studied. Sorption isotherms were constructed and the distribution coefficient Kd was calculated. Isotherms of protease sorption on carboxyl cation exchangers (KB-2 and C-106) are characterized by the presence of a maximum. Sorption of the protease on the sulfate cationite KU-23 corresponds to the Langmuir model. The maximum selectivity and sorption capacity were obtained on the KU-23 and C-106 sorbents, the values are comparable and are m=5800 U/g, Kd=36.2 сm3/g for KU-23 and m=5600 U/g, Kd=35.0 сm3/g forC-106.A kinetic-dynamic analysis of the sorption of acid protease on ionites C-106 and KU-23 with speed of process 0.4-0.9 ml/min was carried out. The maximum yield of the desired product was obtained on a KU-23 sulfate cationite at a process rate of ω=0.8 сm3/ min. It is shown that the maximum yield of the targetproduct is achieved on the sulfocationite KU-23, and is 66% at the sorption stage and 76% at the desorptionstage. Purification of acid protease eluate using ultra - and diafiltration can increase the specific activity by 4.5 times.

There is little information concerning the fractionation by microfiltration (MF) of casein micelles and immunoglobulins plus other minor whey proteins with ceramic gradient membranes. The order of transmission was α-lactalbumin (α-La, 2.3 nm)> β-lactoglobulin (β-Lg; 4.2 nm)>IgG (10.7 nm)> lactoperoxidase (8.2 nm)> IgA (18.1 nm)> IgM (23.8 nm)> lactoferrine > blood serum albumin (7.8 nm), irrespective of the applied transmembrane pressure (0.6–3 bar) and equal to 55% > 50% > 47% > 41%>39% > 32% > 22% > 19%. Including preconcentration, it was possible to obtain 90% of the initial IgG, IgA and IgM within 85, 119, and 160 min, based on 1 m2 of membrane area and 50 L of skim milk volume. The long-term process exposure at 50 °C did not affect α-La and IgG but β-Lg (3–5% denaturation), which, however, was selectively retained by the MF. In conclusion, MF is not only suitable for fractionation of the major whey proteins and caseins, but also for the minor and far bigger immunoglobulins.

For producing demineralized whey concentrates (dry matter (DM) contents: 12–24%) with high whey protein nativity, the suitability of a gentle two-step preservation process (extended shelf life (ESL) process) combining microfiltration and subsequent thermal treatment was investigated. Concentration and demineralization of whey was achieved by using solely nanofiltration (NF) or NF combined with subsequent diafiltration. The influence of the degree of demineralization (number of diafiltration steps: 0; 0.75; 1.5) and DM content on each process step, whey protein denaturation and shelf life of whey concentrates was studied. Applying the ESL process, a strong bacterial reduction of 5.3–7.8 log cycles was achieved depending on the initial bacterial count. For the heat sensitive whey protein β-lactoglobulin, low thermal denaturation of 10–28% was observed, which was independent of DM content and degree of demineralization. However, the shelf life of NF whey concentrates was strongly dependent on their degree of demineralization and ranged between 2 and 16 weeks.

Plants as a platform for recombinant protein expression are now economically comparable to well-established systems, such as microbes and mammalian cells, thanks to advantages such as scalability and product safety. However, downstream processing accounts for the majority of the final product costs because plant extracts contain large quantities of host cell proteins (HCPs) that must be removed using elaborate purification strategies. Heat precipitation in planta (blanching) can remove ∼80% of HCPs and thus simplify further purification steps, but this is only possible if the target protein is thermostable. Here we describe a combination of blanching and chromatography to purify the thermostable transmission-blocking malaria vaccine candidate FQS, which was transiently expressed in Nicotiana benthamiana leaves. If the blanching temperature exceeded a critical threshold of ∼75 °C, FQS was no longer recognized by the malaria transmission-blocking monoclonal antibody 4B7. A design-of-experiments approach revealed that reducing the blanching temperature from 80 °C to 70 °C restored antibody binding while still precipitating most HCPs. We also found that blanching inhibited the degradation of FQS in plant extracts, probably due to the thermal inactivation of proteases. We screened hydrophobic interaction chromatography materials using miniature columns and a liquid-handling station. Octyl Sepharose achieved the highest FQS purity during the primary capture step and led to a final purity of ∼72% with 60% recovery via step elution. We found that 30–75% FQS was lost during ultrafiltration/diafiltration, giving a final yield of 9 mg kg−1 plant material after purification based on an initial yield of ∼49 mg kg−1 biomass after blanching.

Looking at current trends within downstream processing (DSP) of high value bioproducts, it shows that there are ongoing efforts in replacing batch processes by continuous variants. However, a unit procedure which still lacks a simple and compact continuous variant is diafiltration. Here, we present such a single piece of diafiltration equipment achieving continuous buffer exchange of up to 99.90%. The device is composed of a 3D-printed single pass diafiltration (SPDF) module containing two commercial ultrafiltration membranes. While the retentate is flowing through a narrow channel between the two membranes, the channels above and below can supply diafiltration buffer or remove permeate solution. The obtained results illustrate systematically the vulnerability of the device to the effect of concentration polarization at the membrane surface, and that this problem can be strongly reduced using an alternating direction of diafiltration buffer perfusion through the membranes as process inherent backflush. By this, a quasi-stationary operation could be obtained during continuous diafiltration, making the device an interesting option for in-process buffer exchange.

In this study we investigated the effect of different aqueous fractionation processes on the suitability of pea protein isolates (PPI) to substitute whey protein isolate (WPI) in heat-set gels. We found that a milder fractionation process based on diafiltration was successful in substituting WPI, yielding similar gel strength (i.e. elastic modulus) at a range of concentrations. Three different pea protein isolates were analysed, one obtained using diafiltration (PPId), another obtained using isoelectric precipitation (PPIp), and a commercial one (PPIc) as a reference. The isolates PPIp and PPId contained mainly native proteins, whereas the proteins in PPIc were denatured. PPId had a protein solubility almost similar to that of WPI at pH 7, while PPIp and PPIc were less soluble. PPIp and PPIc had better thickening capacities, larger aggregate/particle sizes and higher viscosities compared to PPId. After heat-induced gelation all PPI's showed similar or higher gel strength than WPI between a 7–13 wt % protein concentration. Between 13 and 15 wt % PPId showed a similar gel strength compared to WPI. Above 15 wt % WPI formed the strongest gels. It was concluded that PPId can fully replace WPI up to protein concentrations of 15 wt %. For mixtures of WPI with the other PPI's, it turned out that up to half of the WPI could be replaced by any of the PPI's without compromising on gel strength. This makes us conclude that PPI is a suitable substitute for WPI in heat-set gels.

This work gives novel information about the antimicrobial effect and volatiles of melanoidins isolated from Maria biscuit, common and soft bread. Melanoidins were isolated from scraped and sieved crusts (1 mm), after gluten digestion, 10 kDa ultrafiltration, and diafiltration. Finally, they were freeze-dried. Headspace solid-phase dynamic extraction coupled with a gas chromatograph with a mass spectrometer was used to determine the volatile profiles. The antimicrobial effect was evaluated against isolated strains of the most relevant food spoilage and pathogen microorganisms, together with some molds and yeasts. Melanoidins from common bread exhibited the most extensive antimicrobial activities and showed the most composite volatile profile. No undesirable compounds, such as furfural and 5-hydroxy-methyl-furfural, were found in any of the melanoidins studied. The obtained data pointed out that bakery melanoidins can exert effective food technological properties as natural antimicrobials that can improve shelf-life and security of foodstuffs, together with a possible contribution to food aroma., Government of Autonomous Community of Castile-Leon and FEDER funds [JCyL/FEDER, BU243P18].

Background: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

The Chilean granado bean (Phaseolus vulgaris L.) contains nutritionally valuable proteins, and there was indication that the proteins can help in the prevention of diabetes. To further explore tis potential purified samples of the bean proteins is required. A membrane-based process was developed for the isolation of proteins from granado beans, adapted from methods reported earlier for mustard protein processing. The optimised process consists of alkaline protein extraction from granado bean flour at pH 10, ultrafiltration at concentration factor 4 and diafiltration with diavolume 4 followed by isoelectric protein precipitation at pH 4. The process starting with granado beans containing 28% protein, recovered 60.1% of the protein as precipitated protein isolate (PPI) and 7.2% as acid soluble protein isolates (SPI). The losses in the process system were approximately 26% of mass and 18.8% of nitrogen due to removal of non-protein nitrogen and small molecular weight components, likely carbohydrates. The protein contents of the PPI and SPI were ~92 % and ~ 62% on a moisture-free basis; the protein content of the SPI produced is considerably lower than typical isolates. This may be due to the co-recovery of high molecular weight carbohydrates. The water absorption capacity and nitrogen solubility index, of the PPI and SPI were measured and compared to other oilseed isolates. The PPI showed high water absorption (

Optimizing protein intake for very low birth weight (1,500 g) infants is fundamental to prevent faltering postnatal growth with the potential association of impaired neurodevelopment. The protein content of human milk is not sufficient to support the growth of very low birth weight infants. To meet their elevated protein requirements, human milk is currently fortified using typically bovine milk-based protein isolates (85% on a dry basis). However, these products have several limitations for use in this vulnerable population. To overcome the shortcomings of bovine milk-based protein supplement, a human milk protein concentrate (HMPC) was developed. In preliminary attempts using 10 kDa ultrafiltration (UF) membranes, it was not possible to reach the protein content of commercial protein isolates, presumably due to the retention of human milk oligosaccharides (HMO). Consequently, it was hypothesized that the use of a UF membrane with a higher molecular weight cut-off (50 kDa rather than 10 kDa) could improve the transmission of carbohydrates, including HMO, in the permeate, thus increasing the protein purity of the subsequent HMPC. The results showed that permeate fluxes during the concentration step were similar to either UF molecular weight cut-off, but the 50-kDa membrane had a higher permeate flux during the diafiltration sequence. However, it was not sufficient to increase the protein purity of the human milk retentate, as both membranes generated HMPC with similar protein contents of 48.8% (10 kDa) and 50% (50 kDa) on a dry basis. This result was related to the high retention of HMO, mainly during the concentration step, although the diafiltration step was efficient to decrease their content in the HMPC. As the major bioactive proteins (lactoferrin, lysozyme, bile salt stimulated lipase, and α1-antitrypsin) in human milk were detected in both HMPC, the 50-kDa membrane seems the most appropriate to the preparation of HMPC in terms of permeation flux values. However, improving the separation of HMO from proteins is essential to increase the protein purity of HMPC.

Whey protein is a mixture of globular proteins with high nutritional value and excellent functional properties availability. Purified individual whey proteins exhibit better functionality than in their original mixture, thus there is an increasing interest in developing more efficient methods to recover individual whey protein fractions, especially membrane technology. However, whey protein fractionation from the protein mixture is still a huge challenge for membrane separation. Herein, a dual-gating pHresponsive membrane with the heterogeneous structure was developed for whey protein fractionation. The heterogeneous structure of the membranes was obtained by grafting the dimethylaminoethyl methacrylate (DMAEMA) and 4-vinylpyridine (4VP) near the topside and inside the pores of the ethylene vinyl alcohol copolymer (EVAL) membranes based on selective modification of grafting sites. The PDMAEMA and P4VP gates responded to pH individually and provided a multi-stage regulation of the surface charge and sieving property of the membranes. The major whey protein fractionation from the mixture could be achieved by single dual-gating pH-responsive membrane via the sequential stepwise pH gradient from 3.0 to 8.0. The α-Lactalbumin (α-La), β-Lactoglobulin (β-Lg), lactoferrin (LF) and bovine serum albumin (BSA) were separated and enriched in the permeate at pH 3.0, 4.2, 6.4 and 8.0 by the diafiltration process, respectively. This work not only proposed a novel concept of pHresponsive membrane, but also offered a sustainable solution to fractionate the whey protein from the complicated mixture.

Sugarcane molasses distillery wastewater (DWW) was fractionated by ultrafiltration (UF) using 100 kDa and 10 kDa membranes. The isolated fractions were characterized for antioxidant activity and content of melanoidins, total phenols, proteins and carbohydrates were measured. Chemical composition of the fractions was determined using GC–MS analysis and antimicrobial property was tested against pure microbial cultures. DWW has high antioxidant activity with the melanoidins content around 2 × 103 times the total phenols content. Diafiltration leads to loss in antioxidant activity. Removing the bound melanoidins compounds from the pure melanoidins core does not affect the antioxidant activity. Among all fractions, the highest antioxidant activity is shown by the >100 kDa fraction without diafiltration. Ethanol fractionation of >100 kDa fraction further separates phenols and melanoidins rich fractions from its structure. Ethanol soluble fraction shows high phenols content while the precipitate or aqueous fraction has high melanoidins and carbohydrate content. Antimicrobial potency is mainly observed in arginine rich >100 kDa fraction against Bacillus licheniformis.

Whey is a low calorie milk serum remaining after casein removal in cheese manufacturing. Composition and properties of whey depend on the type of cheese production process, type and the quality of the used milk. Whey mostly consists of lactose, proteins, vitamins, minerals, enzymes, hormones and growth factors. Functional and nutritional roles of whey components meet the requirements for dietary food. Whey proteins are liquid fractions of 20% milk proteins, which remain in whey after casein removal. The increase in the temperature of milk processing leads to denaturation of proteins and a decrease in their content. The most common whey proteins are albumin and globulin. Separation and fractionation of whey proteins may be carried out as precipitation, combined with acid treatment, high temperature treatment or centrifugation (centry-whey process), and as a membrane process (usually ultra- or microfiltration followed by a diafiltration and spray drying). Whey proteins can also be separated based on their molecular mass, charge and hydrophobicity. It is a well-known fact that whey proteins possess admirable nutritional and functional components, such as essential amino acids, bioactive peptides, antioxidants and immunopotentiators. Research of bioactive compounds and nutrients pushed whey protein to the forefront of the functional food sector, therefore, the implementation of these proteins in the diet and supplements has the potential to address many metabolic imbalance-caused diseases, and it seems imperative to harness their potential.

A combination of nanofiltration (NF) and spray drying was selected for this study to remove low molecular weight sugars and consequently to avoid the use of carrier agents and to increase the purity of fructans from Agave salmiana juice. NF experiments were performed in a stirred-cell unit using hydrophilic cellulose membrane with MWCO of 1000 Da, operated in concentration and diafiltration mode with volume reduction factor (VRF) = 2.7, 4 and volume dilution factor (VDF) = 1, 2.7 and 4, respectively. Retentates were collected and dried in a mini spray drier B-290 at inlet temperatures between 160 and 180 °C. A rotatable central composite design with five center points was used and powder yield (PY), moisture content (MC), purity value (PV) and glass transition temperature (Tg) of powders were measured as dependent variables. Response surface plots (p

Processes for the production of protein isolates from camerlina Camelina sativa were developed by modifying the procedure used for other seeds from the Brassicaceae family, such as rapeseed and mustard. The procedure consisted of defatting the seed followed by alkaline extraction at pH 11, ultra- and diafiltration using a 5-kDa membrane, isolelectric precipitation of the proteins at pH 5 and recovery of the acid soluble protein isolates (SPI) after further filtration and drying. Protein yields as precipitated protein isolate and SPI were 10.7 and 11.4%, containing 67 and 42%, respectively. Due to the high concentration of mucilage in camelina, a water-to-seed ratio of 30 had to be used for protein extraction from the hexane-defatted seed and most of the mucilage had to be removed prior to the defatting process. A rapid mucilage extraction process using water at 55 °C was developed. Camelina mucilage absorbs water and oil. Viscosity measurements of dried and redissolved mucilage showed the highest values at natural pH, and the viscosity increased rapidly above 1% solids concentration. It may be a useful product in the food and pharmaceutical industries.

Corn stover samples were subjected to hydrothermal treatment in order to solubilize the hemicellulosic fraction, obtaining hemicellulose-derived saccharides with up to 11.7 g/L of xylooligosaccharides (XOS). Oligomers-containing liquors were nanofiltered by two-step membrane-based separation (discontinuous diafiltration and concentration) showing an increase in XOS concentration up to 21.94 g/L, purified by ion-exchange processing and freeze-dried, obtaining final products with up to 89% of purity. The presence of both substituted (with 4- O -methyl-glucuronic acid or acetyl substituents) and unsubstituted oligosaccharides, mainly made up of xylose and with a degree of polymerization in the range of 5–29, was observed in the purified products. The effects of the various XOS substrates on the metabolic activity and on the dynamics of the microbial populations were studied in media inoculated with human fecal sample. Short chain fatty acids generation and changes in the three selected bacterial groups ( Bifidobacterium genus, Lactobacillus−Enterococcus and Bacteroides−Prevotella group) confirm the suitability of this work to obtain XOS fractions with potential functional properties for human health.

BACKGROUND Once peroxidase purification can improve substrate degradation capacity, peroxidase from rice bran was purified by expanded bed adsorption (EBA) coupled to diafiltration/ultrafiltration (DF/UF) and applied for deoxynivalenol (DON) reduction. RESULTS The DF/UF step was optimized according to membrane molecular weight cutoff (MWCO), DF cycles number, operation temperature and CaCl2 addition to the DF buffer. DON reduction tests were carried out with both clarified and purified peroxidase. The use of MWCO of 10 kDa, 10 °C, six DF cycles and adding 4 mmol L−1 CaCl2 to DF buffer promoted enzyme purification of 4.4-fold and enzymatic recovery of 79.4%. The global purification process combining two techniques resulted in enzyme purification of 75.1-fold with an enzymatic recovery of 22.8%. Peroxidase purified by EBA combined with DF/UF resulted in DON reduction of 81.7% in a model system. CONCLUSION Peroxidase purification was achieved by scale-up integrated strategies. The use of purified peroxidase from agricultural by-products could be a promising alternative for enzymatic DON levels reduction. © 2017 Society of Chemical Industry

Huauzontle (Chenopodium berlandieri subsp. Nuttalliae) is a species native to Mexico. Over the centuries, important nutritional and medical properties have been attributed to this species in ancient Mexican tradition. In contrast, quinoa (Chenopodium quinoaWilld.), an Andean crop genetically related to huauzontle, has been widely studied and acknowledged for its outstanding potential as a high quality protein source. Considering this, the main objective of this study was to obtain and purify the protein fractions of huauzontle seeds using alkaline extraction, isoelectric precipitation, and ultrafiltration membrane technology. Different pH settings were tested for the extraction and precipitation operations and pH 11 and 3.5 were selected, respectively. The protein mass balance for both extraction and precipitation, accounted for 96.6% and 98.7% of the proteins respectively. Using a 10 kDa ultrafiltration membrane, the protein was concentrated and purified by ultrafiltration and diafiltration at 45-50 °C with a concentration factor of 4 and a diafiltration volume of 4. A precipitated protein isolate was obtained with 78.96% purity. In this study, a process that combines alkaline extraction, isoelectric precipitation and ultrafiltration membrane technology was successfully employed to separate and purify huauzontle protein fractions to a level comparable to those reported in the literature for other proteinaceous seeds. In addition, this work will help to establish huauzontle, an ancient native Mexican crop, as an important source of protein, which can also be used as a protein supplement for staple foods.

Sargassum muticum was processed by hydrothermal extraction under previously optimized non-isothermal conditions (up to 187 &deg, C). The alginate free crude hydrolysate was further concentrated by ultrafiltration, operating in diafiltration mode to produce an extract (SmE) enriched in the fucoidan and the phlorotannin fractions and with low mineral content and antiradical capacity equivalent to that of Trolox. In order to explore the potential of this concentrated product for food or feed additive, the in vivo antioxidant potential was assessed. Male Sprague&ndash, Dawley rats were fed SmE dissolved in distilled water at doses of 0.5, 1.0 or 2.0 g kg&minus, 1, administered via an intragastric tube daily for three weeks. The weight and organ gain was not significantly affected in the different groups in relation to the control group fed a standard diet. Serum glucose was significantly lowered in the groups receiving the higher SmE doses, liver GPx levels were reduced and liver TBARS levels decreased in rats administered the extract, but no effect on SOD activity in either liver or erythrocytes was observed.

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column’s full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone’s action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone’s travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3–6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5–fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2–9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two ‘protein-free’ systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the ‘state-of-the-art’ technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.

The Paralytic shellfish neurotoxins (PST) are of increasing interest for biomedical applications. The chemical synthesis is often complex and expensive that’s why the purification by membrane filtration of PST from Alexandrium minutum dinoflagellate was investigated. Disrupted micro-alga cells by ultrasonic treatment were diafiltred to let pass toxins through an ultrafiltration membrane. Then, the mean permeate was concentrated and diafiltrated by nanofiltration. Mean permeate fluxes equal to 187, 135 and 135 L h−1 m−2 were obtained during the first diafiltration, the concentration step and the final diafiltration respectively. Up to 57% (mol/mol) and 78% (mol/mol) of organic matters and salts were removed respectively. Divalent ions were sparsely removed contrary to monovalent ones. C1 and C2 toxins were successfully purified since more than 75% (mol/mol) were recovered. However, only 27 to 50% (mol/mol) of GTX2, GTX3 and STX were recovered.

A 3D printed ultrafiltration/diafiltration (UF/DF) module is presented allowing the continuous, simultaneous concentration of retained (bio-)molecules and reduction or exchange of the salt buffer. Differing from the single-pass UF concepts known from the literature, DF operation does not require the application of several steps or units with intermediating dilution. In contrast, the developed module uses two membranes confining the section in which the molecules are concentrated while the sample is passing. Simultaneously to this concentration process, the two membranes allow a perpendicular in and outflow of DF buffer reducing the salt content in this section. The module showed the continuous concentration of a dissolved protein up to a factor of 4.6 while reducing the salt concentration down to 47% of the initial concentration along a flow path length of only 5 cm. Due to single-pass operation the module shows concentration polarization effects reducing the effective permeability of the applied membrane in case of higher concentration factors. However, because of its simple design and the capability to simultaneously run UF and DF processes in a single module, the development could be economically beneficial for small scale UF/DF applications.

We present here a novel method for recovering ß-galactosidase (ß-gal) from the yeast Kluyveromyces lactis (NRRL 1118) by means of sarkosyl permeabilization. The yeast was permeabilized with 0.2% (w/v) sarkosyl in 0.1 M potassium phosphate buffer for 30 min at 25 °C. After centrifuging and washing, the permeabilized cells were incubated in buffer at pH 7.2, 35 °C for 6–7 h resulting in a nearly soluble extract containing 80% of the ß-galactosidase and about 40% of the cell protein. Transmission electron microscopy showed that permeabilized cells underwent gross structural changes during enzyme release but did not exhibit significant physical breakage. An extract obtained from a 40 mg mL–1 suspension of permeabilized cells was partially purified and concentrated 10-fold through diafiltration and ultrafiltration. The concentrated enzyme solution mixed with 50% (v/v) glycerol proved to be stable for at least 10 months at 5 °C. The kinetic properties of the ß-galactosidase preparation including the hydrolysis of milk, were similar to those exhibited by a commonly used commercial ß-galactosidase derived from the same yeast species. Sarkosyl is a biodegradable detergent with extremely low toxicity, thus making this compound an attractive permeabilizing agent for the downstream -processing of enzymes used in the food industry. Fil: Voget, Claudio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina

Bovine whey protein was hydrolysed using cardosins A and B purified from dried flowers of Cynara cardunculus by combining diafiltration, anion-exchange chromatography and ultrafiltration. The proteolysis experiments were performed using different whey protein concentrations and enzyme/substrate (E/S) ratios. Complete hydrolysis of the main whey proteins, β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La), was achieved after 4 h, at E/S ratios of 1/150 U/mg, regardless the initial protein concentration. In previous reports, the authors suggested that cardosins could not hydrolyse β-lactoblogulin. However, our promising results open up new possibilities to further explore the action of cardosins on whey proteins for the production of bioactive peptides.

Utilisation of cheese whey for production of healthier food has been the main task of this study. Cheese whey, which is often considered as a waste material, was treated by a series of processes including fermentation, membrane filtrations, mixing with barley flour, drying, and/or extrusion to produce barley sourdough. In the first series of experiments, the effect of membrane ultrafiltration (UF) and nanofiltration (NF) on separation of whey components has been studied. Non-fermented sweet cheese whey was treated by pilot-plant membrane ultrafiltration (50 kDa tubular ceramic membrane, TAMI Industries, France), followed by three step diafiltration to minimise losses of lactose, glucose, galactose, and organic acids (namely lactic acid) in retentate. The UF permeates were used for the subsequent nanofiltration and diafiltrations (spiral wound membrane NF 270-2540, Filmtec, Dow Chemicals, USA). The results showed high recovery of proteins (81%) during UF and reduction of lactose and propionate losses by using diafiltration. Rejections of components on the NF membrane were: 93% lactose, 77% galactose, and 76% lactic acid. However, the diafiltration on the NF membrane reduced the relative recovery both for carbohydrates and organic acids. In the second series of experiments, cheese whey was concentrated by reverse osmosis (RO) and the obtained retentate was fermented with Lactobacillus plantarum , Lactobacillus sanfranciscensis and Propionibacterium freudenreichii subsp . freudenreichii. Part of the fermented whey was mixed with barley flour and dried by cold air fluid drying to produce sourdough pellets. The other part of fermented whey was filtered by nanofiltration and both permeate and retentate were sprayed on the surface of sourdough pellets in several layers and dried again. The extrusion of a mixture containing barley flour and fermented whey was tested for pellet production as well. The main aims were to obtain sourdough rich in natural preservatives, and maintain the present microorganisms active. That is why all the technological processes, including drying, were carried out under low temperatures. The content of organic acids (lactic, acetic and propionic) was analysed in final sourdough samples.

Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

The combination of nanoparticles with proteins to form functional hybrid systems is receiving undiminished attention because of its many biotechnological and medical applications. The separation of these hybrid materials from unbound free biomolecules has posed difficult challenges to fractionation and purification. Here, a model study has been carried out by removing proteins (bovine serum albumin (BSA) or lysozyme (LYS)) from the dispersion mixtures with silica nanoparticles (nominal size 20 nm) using ultrafiltration (UF) membranes. Regenerated cellulose (RC) and polyethersulfone (PES) membranes with nominal molecular weight cut-off (NMWCO) of 100 kDa, and a PES UF membrane (NMWCO 300 kDa) functionalized with UV-grafted amphoteric polymer hydrogel layer consisting of N -[3-(dimethylamino)propyl]-acrylamide (DMAPAA) and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and having an experimentally determined cut-off of 180 kDa (identical with the experimental data for PES 100 kDa) were studied. Membrane properties and filtration conditions, in particular pH value and flux, were selected or adapted based on data for single component feeds to achieve maximum protein transmission, complete silica retention and, hence, maximum silica/protein selectivity. Batch dead-end and continuous diafiltration processes were used for fractionation and purification. Overall, the performance of PES UF membranes was inferior compared to the other membranes because of too strong fouling. With membrane RC 100, the transmission data of LYS and BSA from the mixture with silica were 80% and 30%, respectively. With the hydrogel-functionalized PES membrane, the respective transmissions from the mixture were ∼35% and ∼15% for LYS and BSA, respectively. In both cases, quantitative rejection of silica could be achieved. Using continuous diafiltration, membrane RC 100 had better purification efficiency, removing a total of 91% of LYS using 6 diavolumes (DV) in 2.4 h and 84% of BSA using 10 DV in 5.5 h. With the hydrogel-functionalized PES membrane, 82% of LYS and 74% of BSA were removed using 6 and 10 DV within larger time, i.e. 4.0 and 6.8 h, respectively. Importantly, the retained silica nanoparticles remained stable in the dispersion, without any indication of aggregation. Overall, this study will add valuable knowledge to the most efficient use of ultrafiltration sieving properties for the removal or purification of proteins from systems comprising other colloidal particles having a size which is larger by a factor of only 3–10.

Lactoferrin is an important protein found in sweet whey that has several biological activities, such as nutritional, antimicrobial, antiviral, anti-inflammatory, antioxidant, anticarcinogenic and immunomodulation. However, it is found in very low concentration in whey and its capture and purification is a challenge. This study proposes a simple strategy in which two operations for the separation and purification of lactoferrin are integrated. Sweet whey containing 0.12 mg/mL of lactoferrin was subjected to microfiltration for clarification, followed by ultrafiltration and diafiltration in order to reduce in 10 times the total volume with simultaneous concentration and pre-purification of lactoferrin to 1.1 mg/mL. In the second operation, the protein was purified by a preparative chromatography based on cationic exchange expanded bed chromatography (EBC). Adsorption isotherms were determined which allowed establishing the conditions for the chromatographic step. Hydrodynamic studies of the expanded bed and its dynamic binding capacity were also accomplished. The final concentration of the purified lactoferrin was 17.4 mg/mL, resulting in a purity of 92.7% and recovery of 87.0%.

International audience; Exopolysaccharides (EPS) from cell-free Porphyridiwn cruentum media were concentrated then purified (diafiltration) on a 0.14 ism ceramic membrane. The influence of cross-flow velocities on filtration performances was investigated. Mean permeate fluxes equal to 49.8, 68.9 and 81.9 L h(-1). m(-2) were obtained during the concentration at 4 bars for respectively cross-flow velocities inside the membrane lumen equal to 2.5, 3.3 and 4.2 m s(-1); 49.7 Lm(-1). M-2 for the diafiltration at 3.3 m s(-1). Permeate fluxes were correctly predicted from polysaccharide concentrations (10% deviation). Volume reduction factors higher than 7.8 were reached. Rejection rates of polysaccharides and proteins varied according to the cross-flow velocities. Thus, the EPS recovery rate or time of filtration could be modulated following the cross-flow velocity. Polysaccharides were concentrated 6.3 to 10.4 times in such a way that the final sugars concentration reached 1.74-2.26 g L-1. Rheological behavior of filtered solutions changed following the concentration progress. More than 80% (w/w) of polysaccharides were recovered while 49% and 99% of proteins and salts were removed respectively. The filtrations allowed reaching a final monosaccharide content of dry matter equal to 48.9% against 0.6% (w/w) initially.

Stevioside, a white crystalline compound derived from a small perennial shrub, stevia, has been used for centuries as a safe bio-sweetener for patients suffering from diabetics, obesity and other medical complaints. However, its high cost of production renders it inaccessible to the poor and underprivileged. The potential of membrane-based pressure driven processes, such as microfiltration (MF), ultrafiltration (UF) and nanofiltration (NF), has been investigated for the separation and purification of the stevioside from crude stevia leaf extract to make it affordable to the common populace, which constitutes the main objective of this work. After dewaxing with hexane at 60°C, stevia was extracted using hot water at 120°C. The aqueous extract was pre-filtered by a filter press to obtain a turbid stevia solution (20 L), which was subjected to MF using a polypropylene micron cartridge (5 µm) and ceramic tubular module (0.2 µm) for removing suspended solids at a trans-membrane pressure of 0–2 kg/cm2 and temperature of 29°C. Permeate from MF was subjected to UF system having polyethersulfone membrane of 10 kDa MWCO to reject high molecular weight impurities and permeate stevia. UF reject containing remnants of stevia extract was subjected to a series of five diafiltration (DF) steps using deionized water to extract the maximum amount of stevia with each DF step, continuing until 90–95% of permeate containing stevia was recovered. The permeate coming from each DF step was combined and fed to a NF membrane of 150 Da MWCO at a feed pressure of 21 kg/cm2 until a permeate recovery of 93% was achieved. During the NF step, the high M.Wt. (829) stevia was concentrated while low molecular weight impurities, like salts, passed through into permeate. The resultant NF reject, enriched in stevia, was subjected to evaporation and crystallization to yield high purity stevia product.

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis, which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli. The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS–PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis. Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis.

Two virus propagation methods (in broth and on double agar overlay) and three purification procedures (PEG precipitation, centrifugal diafiltration and CsCl density gradient centrifugation) were comparatively evaluated using MS2 and P22 bacteriophages as model viruses. The prepared stocks were characterized in terms of electrophoretic mobility as a function of pH, particle size distribution, surface tension components and the overall hydrophobicity of the virus, as well as the percentage of infectious and total virus recovered. The obtained data were used to rank the purification methods according to six criteria of likely practical relevance. Regardless of the purification method applied, virus propagation in broth media resulted in higher purity virus stocks as the growth on double agar overlay introduced difficult-to-remove residual agar. CsCl density gradient centrifugation gave the highest quality bacteriophage suspensions, recovered infectious P22 at least as efficiently as the other two purification methods and selected for intact P22 virions over damaged ones. The impurities remaining in the virus suspension after PEG precipitation and centrifugal diafiltration broadened the size distribution and interfered with electrophoretic mobility measurements. The residual impurities had a major impact on the free energy of virus-virus interfacial interaction (the quantitative measure of virus hydrophobicity/hydrophilicity) leading to an incorrect determination of P22 bacteriophage as hydrophilic. The trends in measured physicochemical properties can be rationalized by considering impurity-coated virions as permeable soft particles.

Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50-60 nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials.

In this paper, the production of powder enriched in lactoferrin (Lf) and immunoglobulin G (IgG) from untreated feta cheese whey is studied. More specifically, the influence of transmembrane pressure (Δp) and temperature on flux and separation ability during ultrafiltration combined with continuous diafiltration is investigated. Two different types of membranes were used, a spiral polyvinylidene fluoride (PVDF) (molecular weight cut-off [MWCO ∼ 75 kDa) and a set of 18 cylindrical PVDF membranes (MWCO ∼ 100 kDa). For the production of the whey powder, two drying methods were compared: spray and freeze drying. All combinations lead to powder with high total protein content and with a notable content in these two bioactive proteins. However, cylindrical membranes (at a temperature of 20°C and a transmembrane pressure of 4 bar) in combination with freeze drying resulted in the highest yield from whey into Lf and IgG and excellent sensory characteristics. Practical Applications Whey powder enriched in the multifunctional proteins lactoferrin and immunoglobulin G have very large potential both as nutrition additives and for pharmaceutical purposes. The systematic study of the parameters affecting all unit operations involved leads to the most efficient and cheapest production. In order to achieve this, the methodology was kept as simple and low cost as possible. This way, a strong tool could be created for the utilization of the cheese-making by-product whey, which still causes large environmental problems.

The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).

Polyelectrolyte (PE) wrapping of colloidal nanoparticles (NPs) is a standard method to control NP surface chemistry and charge. Because excess polyelectrolytes are usually employed in the surface modification process, it is critical to evaluate different purification strategies to obtain a clean final product and thus avoid ambiguities in the source of effects on biological systems. In this work, 4 nm diameter gold nanoparticles (AuNPs) were wrapped with 15 kDa poly(allylamine hydrochloride) (PAH), and three purification strategies were applied: (a) diafiltration or either (b) one round or (c) two rounds of centrifugation. The bacterial toxicity of each of these three PAH-AuNP samples was evaluated for the bacterium Shewanella oneidensis MR-1 and is quantitatively correlated with the amount of unbound PAH molecules in the AuNP suspensions, as judged by X-ray photoelectron spectroscopy, nuclear magnetic resonance experiments and quantification using fluorescent assay. Dialysis experiments show that, for a 15 kDa polyelectrolyte, a 50 kDa dialysis membrane is not sufficient to remove all PAH polymers. Together, these data showcase the importance of choosing a proper postsynthesis purification method for polyelectrolyte-wrapped NPs and reveal that apparent toxicity results may be due to unintended free wrapping agents such as polyelectrolytes.

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.

Keratinases are proteases that have several important applications in the daily life. Although they have attracted the attention of researchers in the field of biotechnology, their use in industrial processes requires efficient purification techniques allowing scaling-up. Among these techniques, the aqueous two-phase system and ultrafiltration stand out. This study reports the purification of keratinase from Bacillus sp. P45 by aqueous two-phase system two-stage process integrated to ultrafiltration. In the aqueous two-phase system, the addition of sodium chloride in polyethylene glycol-potassium phosphate system favored the partitioning of keratinase. The keratinase purified by an aqueous two-phase system two-stage process integrated to ultrafiltration in diafiltration mode presented purification factor of 6.1-fold and enzymatic recovery of 56.3%, and the enzyme was separated from polyethylene glycol. The purified keratinase was capable of hydrolyzing both soluble and insoluble protein substrates more efficiently than the crude enzyme, evidencing the beneficial effect of enzyme purification on its catalytic activity.

Vi capsular polysaccharide is the major component of Vi polysaccharide typhoid vaccines. Vi is synthesized during growth of Salmonella enterica subspecies enterica serovar Typhi and is released into the fermentation broth in large quantities. Along with the Vi considerable amounts of impurities consisting of bacterial protein, nucleic acid and lipopolysaccharide (LPS) as well as media components contaminate the fermentation broth. A purification method based on selective precipitation of Vi using the cationic detergent cetavlon was developed to separate impurities from Vi. A novel method for handling the Vi precipitate using 0.2 μm sterilizing grade filters to trap and wash the Vi and then, after re-solubilization, allow the Vi to pass through the filter was developed. Cetavlon selectively precipitates Vi and is the major purification step in the process, however, the conditions must be carefully controlled otherwise LPS will co-precipitate in large quantities. Various diafiltration steps help to remove contaminating protein, nucleic acid and fermentation media components as well as chemicals added during the process to induce precipitation of either Vi or contaminants. The final yield of purified Vi was approximately 45% and the bulk concentrate complied with the specifications defined in the WHO recommendations for Vi polysaccharide vaccine. Analysis of the Vi by size exclusion chromatography revealed a uniform peak with a narrow size distribution. The Nuclear Magnetic Resonance spectrum was similar to Vi produced by other methods. The method developed produces large quantities of Vi using low cost production methods translating into Vi based vaccines that can be produced at affordable prices for use in developing countries.

In the present investigation, the purification of β-galactosidase synthesized from Bacillus safensis (JUCHE 1), a new strain isolated from casein whey, has been considered. Broth harvested after 28 h (100.8 × 103 s) was first centrifuged, and the sedimented cell pellet was sonicated to isolate intracellular β-galactosidase. After sonication, the intracellular fluid was subjected to microfiltration (MF) (0.2 × 10–6 m) and ultrafiltration (UF) (300 kg mol–1) in series using a flat-sheet poly(ether sulfone) (PES) membrane in a novel rotating-disk membrane module. In the present investigation, four-stage discontinuous diafiltration (DD) at a constant volume concentration factor (VCF = 2) was carried out. Hydrodynamic studies were conducted to determine the appropriate stirrer speed, transmembrane pressure (TMP), and membrane rotation speed. Finally, β-galactosidase was purified based on the principle of salting-out upon addition of 40% (w/v) ammonium sulfate, followed by dialysis using a 1 kg mol–1 membrane. ...

Soluble and natural mixed pea proteins (PP) were extracted from defatted pea seeds according to acidic precipitation (PPP) or ultrafiltration/diafiltration (PPDF) procedures. The isolates contained proteins with a low level of denaturation. Mixed pea globulins isolates presented quite similar solubility and thermal profiles, also a similar polypeptide composition. Vicilin/convicilin 7S (Vic) and legumin 11S (Leg) fractions were obtained by batch chromatography using a salt gradient for the elution. Several incompatible systems were built by mixing the pea proteins with an anionic polysaccharide (sodium alginate, SA), when biopolymers were both negatively charged. Most of mixtures exhibited a phase separation phenomenon. From phase diagrams, experimental binodal curves obtained with either mixed globulins or legumin fraction were apparently very close. However phase boundary was better-defined with the Leg fraction. No macroscopic phase separation was evidenced for mixtures with the vicilin fraction. Microstructure of the PP-SA mixtures was investigated by confocal microscopy (CLSM) according to PP composition and biopolymer initial composition. The Leg-SA and most of PPP-SA mixtures exhibited a droplet-like structure, while structure of PPDF-SA mixtures was aggregated-like. With mixed PP, an alginate entrapment within the PP-enriched phase would disturb phase separation. Also density and shape of the protein-enriched microdomains influenced kinetics of demixing. Polydispersity within the PP-SA mixtures, in terms of wide range molecular weights distribution and charge heterogeneity would explain such differences.

Wild type ethanologenic microorganisms display very low ethanol yields on xylose in lignocellulosic hydrolysate due to low tolerance to ethanol and other metabolic inhibitors present in hydrolysates. The major thrust of research on xylose conversion has been towards the development of recombinant microorganisms capable of simultaneous uptake of glucose and xylose. These genetically modified ethanologenic strains also suffer from low yields, low productivities and genetic instability. Nanofiltration and membrane assisted cell recycle are process engineering strategies which are being described in the current study for the first time to increase ethanol yield and productivity from lignocellulosic hydrolysates using wild type strains. Various nanofiltration membranes have been evaluated for high pentose-hexose separation and high permeate flux. Operating conditions viz. temperature and transmembrane pressure have been optimized for achieving a maximal xylose to glucose separation factor of 3.4. The hydrolysate achieved by enzymatic saccharification of chemically pretreated biomass is subjected to nanofiltration and thereby split into two streams. The retentate stream is enriched in glucose while the permeate stream is enriched in xylose and depleted in metabolic inhibitors. The retentate and permeate steams are fermented by Saccharomyces cerevisae and Pichia stipitis respectively to obtain maximum yields. The retentate stream can be subjected to diafiltration by the fermented permeate from the first cycle to further increase the recovery of xylose and thus the overall ethanol yield from xylose. The permeate stream can be subjected to an additional nanofiltration based concentration step to further increase the ethanol titer. Overall yields between 0.39 and 0.43 have been achieved on validation of the process with hydrolysate of sorghum bagasse.

Agaricus bisporus is a safe and rich source of tyrosinase and lectin. To address the increasing demand for these proteins, a facile method was developed for their simultaneous preparation from A. bisporus. The method includes demelanization of the raw extract followed by stepwise precipitation of the proteins from the aqueous extract. Employing the right organic solvent in demelanization and precipitation steps reduces the intermolecular interactions between these proteins. This enhances the efficacy of gel-permeation chromatography and results in preparation of partially purified lectin and tyrosinase suitable for many applications in biotechnology and bio-research. The method can be performed without diafiltration.

Measles virus and mumps virus (MeV and MuV) are enveloped RNA viruses used for production of live attenuated vaccines for prophylaxis of measles and mumps disease, respectively. For biotechnological production of and basic research on these viruses, the preparation of highly purified and infectious viruses is a prerequisite, and to meet that aim, knowledge of their stability and biophysical properties is crucial. Our goal was to carry out a detailed investigation of the stability of MeV and MuV under various pH, temperature, shear stress, filtration and storage conditions, as well as to evaluate two commonly used purification techniques, ultracentrifugation and diafiltration, with regard to their efficiency and effect on virus properties. Virus titers were estimated by CCID50 assay, particle size and concentration were measured by Nanoparticle tracking analysis (NTA) measurements, and the host cell protein content was determined by ELISA. The results demonstrated the stability of MuV and MeV at pH9 and above pH 4 and 5, respectively, and aggregation was observed at pH9. Storage without stabilizer did not result in structural changes, but the reduction in infectivity after 24 hours was significant at +37 °C. Vortexing of the viruses resulted in significant particle degradation, leading to lower virus titers, whereas pipetting had much less impact on virus viability. Diafiltration resulted in higher recovery of both total and infectious virus particles than ultracentrifugation. These results provide important data for research on all upstream and downstream processes on these two viruses regarding biotechnological production and basic research.

International audience; IN order to improve bioethanol production by yeast fermentation of lignocellulosic hydrolysates, sugar/ inhibitor separation by nanofiltration was studied on a bench-scale unit equipped with a spiral-wound membrane. Therefore, a model solution containing 3 sugars and 4 inhibitors was treated with two previously selected membranes (NF270 from DOW Filmtec and DK from GE Osmonics). Both membranes led to high sugar rejection, especially at high permeate flux (>90% for glucose and arabinose and >85% for xylose). Although its water permeability was smaller, DK membrane was preferred for its higher transmission of the inhibitors, especially for the largest ones (vanillin and 5-hydroxymethyl furfural), ensuring a better detoxification level. Diafiltration was applied to improve sugar purity of the treated hydrolysate. With a diavolume equivalent to 1.25 times that of the feed, acetic acid concentration wasdivided by 5 and brought back to concentrations lower than 1 g L-1. A simulation model was proposed to predict the diavolume to apply, depending on the initial concentrations. Finally, processed hydrolysates were tested for the fermentation ability with a Pichia stipitis species. Fermentation tests showed that diafiltration followed by concentration led to retentates as fermentable as an equivalent pure sugars solution.

8 páginas, 3 figuras, 3 tablas, The goal of the present work was to optimise the different environmental friendly processes involved in the extraction and purification of chondroitin sulphate (CS) from Prionace glauca head wastes. The experimental development was based on second order rotatable designs and evaluated by response surface methodology combined with a previous kinetic approach. The sequential stages optimised were: (1) the enzymatic hydrolysis of head cartilage catalysed by alcalase (55.7 °C/pH 8.2); (2) the chemical treatment of enzyme hydrolysates by means of alkaline-hydroalcoholic saline solutions (NaOH: 0.54 M, EtOH: 1.17 v, NaCl: 2.5%) to end the protein hydrolysis and to precipitate and selectively redissolve CS versus the peptidic material and (3) the selective purification and concentration of CS and the concomitant protein permeation of extracts which were obtained from previous treatment using ultrafiltration and diafiltration (UF–DF) technologies at two different cut-offs, Financial support from projects MARMED 2011-1/164 (Atlantic Area Programme, EU), 0687_NOVOMAR_1_P (POCTEP Programme, EU) and iSEAS LIFE13 ENV/ES/000131 (LIFE+ Programme, EU) is acknowledged

Lipase was produced by Penicillium camembertii KCCM 11268 under solid state fermentation (SSF), and the production process was optimized by using statistical experimental designs. The initial moisture content, cultivation time, inoculum size and concentration of basal medium were considered as the factors of optimum conditions for SSF. P. Camembertii KCCM 11268 was cultivated in SSF using wheat bran as the substrate for lipase production. Under the optimized condition, lipase activity was reached around 7.8 U/ml after eight days fermentation. To partially purify the lipase, ammonium sulfate (80% saturation) was added to the crude lipase solution and concentrated using a diafiltration (VIVAFLOW 50). The concentrated lipase solution from P. Camembertii KCCM 11268 (PCL) was immobilized on silica gel by cross-linking method. Also, PCL was mixed with a commercial lipase solution from Candida rugosa (CRL), and this mixture was co-immobilized on silica gel. The immobilized and co-immobilized lipase activities were 1150.1 and 7924.8 U/g matrix, respectively. Palm oil and methanol were used as substrates and 1mmol of methanol was added every 1.5 h and 2 times during biodiesel production. The reaction was carried out at temperatures of 30, 40, 50, 60 and 70 °C. The maximum biodiesel conversion by co-immobilized lipase was 99% after 5 h at 50 °C.

The yellow color of Cheddar cheese whey arises from a residual amount of annatto that partitions into the whey during Cheddar cheese manufacture. Bleaching of the color using hydrogen peroxide or benzoyl peroxide is often a prerequisite to produce an acceptable neutral-colored whey protein concentrate and isolate. However, the use of these strong oxidizing agents often generates off-flavors as a result of lipid oxidation and results in loss of nutritive value due to protein oxidation. The objective of this study was to determine the extent of partitioning of annatto between protein, milk fat globule membrane (MFGM), and aqueous (serum) phases of cheese whey so that a simple method can be developed to remove annatto from cheese whey. The MFGM was separated from Cheddar cheese whey using a recently developed novel method. Quantitative analysis of the distribution of annatto in the fat-free whey protein isolate (WPI), the MFGM fractions, and the serum phase revealed that annatto was not bound to the protein fraction but was mostly distributed between the serum phase and the MFGM fraction. The results showed that a colorless WPI or whey protein concentrate could be produced from Cheddar cheese whey by separation of MFGM from the whey, followed by diafiltration. This approach will negate the need for using bleaching agents.