kDa Molecular Weight Cutoff Research Articles

Fractionation of whey protein hydrolysates using charged UF/NF membranes

The possibility to separate peptides from a tryptic hydrolysates of whey proteins with charged UF/NF membranes has been investigated. A total hydrolysate (TH) was prepared by tryptic hydrolysis of a commercial whey protein isolate followed by UF-treatment using a 10 kDa MWCO in order to remove the enzyme and non-hydrolyzed material from the reaction mixture. Firstly, five different membrane materials were evaluated for the fractionation of TH and the membrane SG13 with a molecular weight cut-off (MWCO) between 1000 and 5000 g mol −1 showed the best separation properties accompanied with high fluxes. The separation behavior of the TH by SG13 membranes at pH 5 and 9, with or without 0.5 M NaCl additions was further investigated. It was observed that pH increase from 5 to 9 not only raised the flux and nitrogen transmission values but also decreased the tendency to fouling. However, NaCl additions increased permeability but also increased the fouling. A detailed examination of the peptide separation revealed that negatively charged peptides were in lower proportion in the permeates whereas the opposite trend was observed for neutral and positively charged peptides. The results are explained by charge effects related to the rising of a Donnan potential at the membrane surface during the fractionation of hydrolysates.

Microplitis croceipes teratocytes: in vitro culture and biological activity of teratocyte secreted protein

Teratocytes originate from the dissociation of the extraembryonic serosal membrane in some Braconidae and Scelionidae. Methods used to culture teratocytes in vitro are described and the yield of teratocyte secreted proteins (TSP) was measured. Although 90% are viable after 6 days, in vitro teratocytes reached only half the diameter (32 μm) of the same age teratocytes obtained in vivo. Teratocytes cultured in vitro secrete as much as 0.7 μg of protein per day per larval equivalent (≈900 cells). Presence of parasitoid larvae enhanced teratocyte viability while periodic exchange of medium did not. However, medium exchange significantly increased the total amount of protein secreted. Size and viability were improved with the addition of 10% FBS to the Ex-cell 400 culture medium. Non-denaturing PAGE showed at least 15 proteins with molecular sizes estimated to be between 24 to 347 kDa in medium containing teratocytes. An in vitro fat body assay was developed to measure the effect of TSP on protein synthesis and juvenile hormone esterase (JHE) activity. Crude TSP inhibited in vitro incorporation of [ 35S]-methionine into protein synthesized by the fat body. The amount of JHE released from in vitro fat body treated with crude TSP was significantly less than controls, most likely caused by the inhibition of general protein synthesis. The active fraction of TSP passed through a 30 kDa molecular weight cutoff filter but was retained by a 3 kDa filter. SDS-PAGE revealed four proteins with molecular weights between 8 and 20 kDa not present in control medium incubated without teratocytes.

Modulation Of Calcium Fluxes In Human Platelets By Human Milk in vitro † 21

The effect of adding human milk (HM) to washed fura-2 loaded human platelets was investigated in vitro. The effect of thrombin (0.01 U/ml) to increase intracellular free calcium ([Ca2+]i), when calcium was present in the medium, was inhibited approx. 60% by a brief (10 second) treatment with HM (6% produced the maximum inhibition). Similar inhibition of the ability of vasopressin to increase ([Ca2+]i) was also observed. The effect of thrombin to mobilize intracellular calcium in platelets (5 mM EGTA in the medium) was inhibited approx. 70% by HM (4% produced the maximum inhibition). This result suggests the HM contains a factor or factors that prevent thrombin from increasing([Ca2+]i) in platelets, perhaps by either stimulating calcium efflux from the platelet, inhibiting intracellular calcium release or stimulating calcium uptake into the intracellular stores. When calcium influx into platelets was measured using barium ions as a calcium surrogate, HM blocked the influx by approx. 70% (4% produced the maximum inhibition). This result also implies that HM also contains a factor that inhibits calcium influx across the plasma membrane. Significant effects could be observed with 1% HM on all three parameters. The inhibitory activity did not pass through a 10 KDa molecular weight cut off filter. The inhibitory effect of HM was substantially reduced by heating the milk for 15 min. at 100 C. Treating HM with 0.3 M HCl or 0.5M NaOH at room temperature for 10 min. had no effect on activity. It is concluded that HM contains a factors or factors that reduce the ability of thrombin to increase [Ca2+]i in platelets by both blocking calcium influx and preventing mobilization from internal calcium stores, alternatively the plasma membrane calcium ATPase pump may be activated. The factor is stable to mild acid and base treatment is heat labile and has a molecular weight in excess of 10 KDa. Because platelets participate in propagation of the acute inflammatory response, human milk factors that modify intracellular signalling in platelets might contribute to the anti-inflammatory character of human milk.

Fractionation of BSA and lysozyme using ultrafiltration: effect of pH and membrane pretreatment

Selective transmission of a solute through membranes proves to be a challenge in ultrafiltration processes. This is because the transport of a solute through an ultrafiltration membrane does not depend on size alone, but on several other factors such as solute-solute and solute-membrane interactions. By manipulating physicochemical parameters and process variables (eg. pH, ionic strength, concentration of solute, etc.) and by membrane modification, it is possible to enhance the transmission of a particular solute and thus enhance fractionation of solutes. In this paper, the effect of pH on fractionation of BSA and lysozyme by ultrafiltration through 50 kDa MWCO (molecular weight cut off) polysulfone membrane has been examined. It was found that the selectivity of solute separation for dilute mixtures of BSA and lysozyme was very much pH dependent and varied from 3.3 at pH 5.2 to 220.0 at pH 8.8. However, at a higher feed concentration, the transmission of lysozyme through polysulfone membrane decreases quite dramatically resulting in lower throughput of product. An attempt has been made to enhance the transmission of lysozyme through the polysulfone ultrafiltration membrane by pretreating the surface of the membrane by adsorption of another protein, myoglobin. An increase in lysozyme transmission of up to 63% with respect to native membrane was observed. The stability of this pretreatment and its effect on permeate flux have been examined. The pretreated membrane was used to fractionate BSA/lysozyme mixtures. Even at higher feed concentration, enhanced fractionation with respect to native membrane was observed due to highly enhanced transmission of lysozyme through the pretreated membrane.

Screening tool for hollow-fiber bioreactor process development

Fundamental research of factors affecting cell growth in hollow-fiber bioreactors is hindered by the lack of an efficient screening tool. To address this issue, a hollow-fiber micro-bioreactor has been developed. Hollow fibers with 10 kDa molecular weight cutoffs are housed within a piece of silicone tubing. Cells are inoculated within the hollow fibers which provides a 0.2-mL culture volume. The space between the fibers and silicone tubing (5 or 16 mL) is used as a medium reservoir sufficient to feed the cells for at least 24 h. Oxygenation is provided directly through the silicone tubing so that a pump for medium recirculation is not required. As a result, many conditions can be tested simultaneously in a single incubator. Three days after inoculation at 5 x 10(6) cells/mL in the micro-bioreactor, the rho 1D4 murine hybridoma cell line reached 2.8 x 10(7) cells/mL with an antibody concentration of 0.17 mg/mL. When inoculated at 5 x 10(7) cells/mL, the cell concentration reached 1.8 x 10(8)/mL after 3 days with an antibody concentration of 1.0 mg/mL. Results from a series of experiments with the micro-bioreactor suggested that the initial growth phase of this cell line in a hollow-fiber system is dependent on the serum concentration in the medium reservoir. This prediction was tested by simultaneously inoculating two production-scale hollow-fiber bioreactor systems. The cell side of the membrane for each bioreactor contained 10% serum, but serum was added to the reservoir side of only one of the bioreactors. The cells with only basal medium in the reservoir died after a few days, while the cells with 10% serum in the medium reservoir grew rapidly. These results demonstrate that the micro-bioreactor developed here can support good cell growth and that it can be used as a research tool to predict the performance of large-scale hollow-fiber systems.

Development and testing of a vaccine for the prevention of infectious bovine keratoconjunctivitis

We have purified, concentrated and stabilized the protein cytotoxin from Moraxella bovis. The toxin is concentrated on a filter with a 500,000 kDa molecular weight cut off. Small proteins are removed by extensive diafiltration on the same filter. The cytotoxin is concentrated approximately 500 fold, and remains active after 5 months of freezing. A vaccine trial for toxicity and efficacy were performed.

Effect of nonaqueous solvents on the flux behavior of ultrafiltration membranes

Flux experiments were conducted to determine the effect of aqueous methanol, resistance behavior of 10 and 30 kDa molecular weight cutoff cellulose and polysulfone membranes. The resistance of the 10 kDa cellulose membrane increased as the solubility parameter of the aqueous solvent solution ( δ S) approached that of the membrane ( δ P). The opposite effect was observed with the 30 kDa cellulose membrane: a resistance minimum was observed when δ S ≅ δ P. Differences in flux behavior are believed to be a consequence of the 10 kDa membrane having a much more anisotropic structure as compared to the 30 kDa membrane. Basically the same flux maxima and minima trends were observed with the flow of solvent solutions throgh 10 and 30 kDa polysulfone membranes. However, acetonitrile, because of its low hydrogen bonding capabilities, dramatically reduces the glass transition temperature of polysulfone. As a result, the presence of this solvent at high concentrations disrupted the integrity of the 10 kDa polysulfone membrane, leading to an over ten fold decrease in flow resistance.