Removing proteins from an aerated yeast fermentation by pulsing carbon dioxide

Salting-out is a common technique used for precipitating proteins and other materials from fermentation and tissue culture processes. It leaves a salt residue in the system. Foam fractionation can also be used to remove proteins by protein precipitation from a dilute solution. In doing so, there is usually a trade-off between enrichment and recovery. An increase in the airflow rate will increase the recovery, but only at the expense of the enrichment. A new method for increasing the recovery in foam fractionations and in yeast fermentations is to add a burst of CO₂ to the process and then restore the air. This CO₂ acts like a temporary salt, but it does not leave behind a residue. The recovery increases as a result of the joint use of these gases, perhaps by more than 10-fold, without sacrificing the enrichment. Chicken egg albumin in a foam fractionation column can serve as a simple, experimental model for the proposed recovery process in lieu of the fermentation process.     

Modeling a protein foam fractionation process

A simple staged model for the protein foam fractionation process is proposed in this article. This simplified model does not detail the complex foam structure and gas-liquid hydrodynamics in the foam phase but, rather, is built on the conventional theoretical stage concept considering upward bubbles with entrained liquid and downward liquid (drainage) as counter-current flows. To simulate the protein concentration distribution in the liquid along the column by the model, the bubble size and liquid hold-up with respect to the position must be known, as well as the adsorption isotherm of the protein being considered. The model is evaluated for one stage by data from the semibatch foam fractionation of egg albumin and data from the continuous foam fractionation of bovine serum albumin. The effect of two significant variables (superficial gas velocity and feed protein concentration) on enrichment is well predicted by the model, especially for continuous operation and semibatch operation when initial concentration is high.     

Batch foam fractionation of kudzu (Pueraria lobata) vine retting solution

The aqueous protein solution from kudzu(Pueraria lobata) vine retting broth, without the addition of other surfactants, was foam-fractionated in a vertical tubular column with multiple sampling ports. Time-varying trajectories of the total protein levels were determined to describe the protein behavior at six positions along the 1-m column. The lowest two trajectories of this batch process represented a loss of proteins from the bulk liquid and tended to merge and decay together in time; the other trajectories displayed a gain in proteins in the foam phase. These upper column port protein concentration trajectories generally increased in time up to 45 min, followed by a decrease, reflecting the removal of proteins from the column ports. The foam became dryer as it passed up the column to the top port. The protein concentration was about 5–8×higher in the top port foam than in the initial bulk solution, mainly as a result of liquid drainage from the foam along the column axis. This concentration increase in the collected foam was dependent on the initial pH of the bulk solution. The mol-wt profile of the proteins in the concentrated foam effluent was determined by one-dimensional gel electrophoresis. An analysis of the gel electropherograms indicated that the most abundant proteins could be cellulases and pectinases.     

Construction of a comprehensive beer proteome map using sequential filter‐aided sample preparation coupled with liquid chromatography tandem mass spectrometry

The quality traits of beer, which include flavor, texture, foam stability, gushing, and haze formation, rely on contributions from beer proteins and peptides. Large‐scale proteomic analysis of beer is gaining importance, not only with respect to authenticity of raw material in beer but also to improve quality control during beer production. In this work, foam proteins were first isolated from beer by virtue of their high hydrophobicity. Then sequential filter‐aided sample preparation coupled with liquid chromatography and tandem mass spectrometry was used to analyze both beer protein and foam protein. Finally, 4692 proteins were identified as beer proteins, and 3906 proteins were identified as foam proteins. In total, 7113 proteins were identified in the beer sample. Several proteins contributing to beer quality traits, including lipid transfer protein, serpin, hordein, gliadin, and glutenin, were detected in our proteins list. This work constructed a comprehensive beer proteome map that may help to evaluate potential health risks related to beer consumption in celiac patients.     

Measurement of bubble size distribution in protein foam fractionation column using capillary probe with photoelectric sensors

Bubble size is a key variable for predicting the ability to separate and concentrate proteins in a foam fraction ation process. It is used to characterize not only the bubble-specific interfacial a rea but also coalescence of bubbles in the foam phase. This article describes the development of a photoelectric method for measuring the bubble size distribution in both bubble and foam columns for concentrating proteins. The method uses a vacuum to withdraw a stream of gas-liquid dispersion from the bubble or foam column through a capillary tube with a funnel-shaped inlet. The resulting sample bubble cylinders are detected, and their lengths are calculated by using two pairs of infrared photoelectric sensors that are connected with a high-speed data acquisition system controlled by a microcomputer. The bubble size distributions in the bubble column 12 and 1 cm below the interface and in the foam phase 1 cm above the interface are obtained in a continuous foam fractionation process for concentrating ovalbumin. The effects of certain operating conditions such as the feed protein concentration, superficial gas velocity, liquid flow rate, and solution pH are investigated. The results may prove to be helpful in understanding the mechanisms controlling the foam fractionation of proteins.     

A multi point conductivity measurement system for characterisation of protein foams

Protein foams play an important role in both food and biotechnological processes. A sound understanding of foaming properties of proteins relevant to such processes is useful e.g. to allow adequate control of unwanted foams and appropriate choice of protein-physical system when foams of certain characteristics are required. In general, measurements of changes in foam volume (volumetric method) are used for foam characterisation. However, recently there has been increased interest in the use of measurement methods based on conductivity and capacitance. Simple relative techniques based on electrical conductivity measurements provide information on both foamability and foam stability. A multi point conductivity measurement system has been designed and used for characterisation of model protein foams (0.1 and 1.0 mg ml⁻¹ Bovine serum albumin, BSA). The solution of BSA was sparged with nitrogen or carbon dioxide gas at constant flow rate (90 cm³ min⁻¹) via a stainless steel sinter (0.5 or 2.0 μm in pore size). A comparison of foaming properties determined by volumetric and conductimetric techniques is provided. Both methods show that more stable foams are obtained for solutions at higher BSA concentrations. At all BSA concentrations, higher foamability and stability are achieved with a smaller sinter pore size. When nitrogen rather than carbon dioxide is used as a dispersed phase, higher foamability and foam stability are obtained. The conductivity measurements indicate that foamability is dependent on gas type, whereas, volumetric measurements do not show such differences.     

A study of the effect of proteins and endogenous cations on a lipophilic beta-cyclodextrin-based potentiometric lidocaine sensor using discrete solution and flow-injection analysis

2,6 Didodecyl beta cyclodextrin (2,6ddbetaCD) modified ion-selective electrodes (ISEs) were used in discrete solutions and in a flow injection analysis manifold for monitoring the local anaesthetic lidocaine hydrochloride (lignocaine) in the presence of endogenous cations and proteins. Membrane matrices comprised of either high molecular weight poly (vinyl chloride) (PVC) or polyurethane (Tecoflex SG 80) were compared. The behaviour of these electrodes in the presence of bovine serum albumin (BSA), alpha(1) acid glycoprotein (AAG) and human serum (HS) indicate that the Tecoflex-based membrane matrices are preferable to PVC as the drift induced by proteins on baseline lines and peak height reproducibility was considerably reduced in the former. Interference from 'serum' levels of sodium, potassium and calcium (145 mmol dm(-3) Na(+), 1.26 mmol dm(-3) Ca(2+), 4.30 mmol dm(-3) K(+)) was negligible (-Log K(ij)(POT) (overall) >/=4.0). The major organic interferents were molecules of similar size and structure, which caused reduced slope and drift in baseline potentials, at equimolar concentration levels. A reduction in interferent concentration by a factor of 10 negated these effects.     

Emulsifying and foaming properties of amaranth seed protein isolates

The emulsifying and foaming properties of amaranth seed protein isolates prepared by wet extraction methods, such as isoelectric precipitation and dialysis, were investigated. The various isolates differ from each other in many ways. The isolate prepared by isoelectric precipitation mainly contains the globulin but not the albumin fraction and a considerable amount of polysaccharides, while the other isolate prepared by the dialysis method contains all the globulin and albumin fractions. The protein-polysaccharide complexes enhance emulsion stability due to steric repulsion effects. Measurements of the emulsion stability show that the studied protein isolates act as effective stabilizing agents. Foam expansion is dominated by the surface activity and availability of protein in the solution, while foam stability is determined by the properties of the interfacial layer. The results show that amaranth protein isolates act as an effective foaming agent. Both foaming properties intensified from the presence of protein-polysaccharide complexes.     

羽毛蛋白溶解度、起泡力及保水力的特性研究

本文探讨了羽毛蛋白的某些重要功能性质,并与常用标准卵清蛋白比较。结果表明,羽毛蛋白具有较高的起泡力、水可溶性、保水力及较低的保泡力,是具有较为优良功能性质的蛋白质。通过扫描电镜观察,较好地揭示了羽毛蛋白微观结构和功能性质之间的内在联系。     

Foaming and Structural Studies on the Acidic Subunit of Amaranth 11S Globulin Modified with Antihypertensive Peptides as a Function of pH and Ionic Strength

Some studies aimed at revealing the relationship between protein structure and their functional properties. However, the majority of these reports have been carried out using protein isolates. There are limited reports on the possible relationship between the functional properties and the structure of a purified protein. In this work the amaranth 11S globulin acidic subunit (AAC) and five mutations of the same protein that were modified in their variable regions with antihypertensive peptides (VYVYVYVY and RIPP), were analyzed at two ionic strength (2.9 and 17.6 g/L NaCl) and pH (3.0–7.0). Results revealed better solubility for the proteins mutated at the terminal ends (AACM.1 and AACM.4) and lower solubility for the protein inserted with RIPP peptide. Spectroscopy studies revealed an increase of β-sheet structure at high salt concentration for all proteins. It was also observed that salt concentration acted as a modulator, which allowed a better foam features for all modified proteins limiting movement of side chains and reducing red-shifted displacement of λmax. All proteins showed foam capacity ranging from 76 to 93% although foam stability was twofold better for modified proteins than for AAC at high salt concentration. This study allowed better understanding about the structural changes that influence the foaming properties of engineered proteins.     

Effect of environmental humidity on static foam stability

The quality of foaming products (such as beer and shampoo) and the performance of industrial processes that harness foam (such as the froth flotation of minerals or the foam fractionation of proteins) depend upon foam stability. In this study, experiments are performed to study the effect of environmental humidity on the collapse of static foams. The dependency of the rate at which a foam collapses upon humidity is demonstrated, and we propose a hypothesis for bubble bursting due to Marangoni instability induced by nonuniform evaporation to help explain the dependency. This hypothesis is supported by direct experimental observations of the bursting process of isolated bubbles by high speed video recording and the thinning of isolated foam films under different values of humidity and temperature by microinterferometric methods.     

Responsive Aqueous Foams

Remarkable properties have emerged recently for aqueous foams, including ultrastability and responsiveness. Responsive aqueous foams refer to foams for which the stability can be switched between stable and unstable states with a change in environment or with external stimuli. Responsive foams have been obtained from various foam stabilizers, such as surfactants, proteins, polymers, and particles, and with various stimuli. Different strategies have been developed to design this type of soft material. We briefly review the two main approaches used to obtain responsive foams. The first approach is based on the responsiveness of the interfacial layer surrounding the gas bubbles, which leads to responsive foams. The second approach is based on modifications that occur in the aqueous phase inside the foam liquid channels to tune the foam stability. We will highlight the most sophisticated approaches, which use light, temperature, and magnetic fields and lead to switchable foam stability.     

Affinity foam fractionation of Trichoderma cellulase

Cellulase could not be selectively collected from fermentation broth by simple foam fractionation, because of the presence of other more surface-active compounds. A new approach of affinity foam fractionation was investigated for improvement. A hardwood hydrolysate (containing cellulose oligomers, substrates to cellulase) and two substrate analogs, i.e., carboxymethyl cellulose (CMC) and xylan hydrolysate, were added before the foaming process. The substrates and substrate analogs were indeed found to bind the cellulase selectively and form more hydrophobic complexes that partition more readily onto bubble surfaces. In this study, the effects of the type and concentration of substrate/analog as well as the presence of cells at different growth stages were examined. The foam fractionation properties evaluated included foaming speed, foam stability, foamate volume, and enrichment of filter paper unit (FPU) and individual cellulase components (i.e., endoglucanases, exoglucanases, and β-glucosidases). Depending on the broth and substrate/analog employed, the foamate FPU could be more than fourfold higher than the starting broth FPU. Addition of substrate/analog also deterred the enrichment of other extracellular proteins, resulting in the desired cellulase purification in the foamate. The value of E/P (enzyme activity-FPU/g/L of proteins) in the foamate reached as high as 18, from a lactose-based fermentation broth with original E/P of 5.6. Among cellulase components, exoglucanases were enriched the most and β-glucosidases the least. The study with CMC of different molecular weights (MW) and degrees of substitution (DS) indicated that the CMC with low DS and high MW performed better in cellulase foam fractionation.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Effect of protein denaturation on void fraction in foam separation column

Foam fractionation is a cost-effective process that uses air to extract protein from a liquid (in this case “crude” dilute egg-albumin solution). This article deals with how the void fraction (fraction of air in the aerated solution) of foam is affected by heat denaturation of the protein. A 2-mm glass tube was used to sample the foam-liquid interface fluid in a 35-mm-diameter column in order to detect small changes in void fraction and foam production, which are not easily detected directly from the bulk foam. The main control variablein this study was the protein solution preheating time. As the preheating time increased, the initial void fraction in the column decreased. The initial void fraction of the undenatured solution ranged from about 0.73 to 0.80, and the void fraction for significant preheating times of 5 min ranged from approx 0.68 to 0.72. Furthermore, the period of foam production increased from 5 to 7 min for undenatured proteins in solution to as long 15 min for 5-min preheated solutions. Side-port sampling through a small capillary tube has the potential to be used as a rapid and inexpensive way to determine the level of protein denaturation by directly determining the void fraction and then estimating the effect of denaturation from a protein denaturation calibration curve of the void fraction.     

Restoration of protein foam stability through electrostatic propylene glycol alginate-mediated protein–protein interactions

The action of propylene glycol alginate in the enhancement of foam stability of a destabilised Tween 20/bovine serum albumin mixed system was evaluated. A significant increase in the foam stability was observed in the presence of low concentrations of propylene glycol alginate. A pseudo-plateau level of foam stability was obtained in the presence of approximately 0.8 μg/ml propylene glycol alginate in the solution used to form the foam. Foam stability enhancement due to bulk viscosity changes and surface effects were elucidated. The increase in foam stability was investigated by reference to the properties of thin liquid films and the macroscopic interface of test solutions. Propylene glycol alginate was found to slow the rate of thin film drainage, increase the equilibrium thickness of the films, slow the lateral diffusion of a fluorescent probe molecule located in the adsorbed layer and increase the elasticity of the interface. Data are consistent with propylene glycol alginate-induced crosslinking of protein in the adsorbed layer. This polysaccharide presents a means for controlling protein foam stability.     

Effect of protein aggregates on foaming properties of β-lactoglobulin

Our paper aims at determining the respective part of protein aggregates and non-aggregated proteins in the foam formation and stability of β-lactoglobulin. We report results on fractal aggregates formed at neutral pH and strong ionic strength (aggregates size from 30 to 190 nm). Pure aggregates and mixtures of non-aggregated/aggregated proteins at varying ratios were used. The capacity of aggregates to form and stabilize foams has been studied in relation with their ability to absorb at air/water interfaces. Our results show that protein aggregates are not able by themselves to improve the foaming properties but participate to a better foam stabilization in the presence of non-aggregated proteins. Non-aggregated proteins appear to be necessary to produce stable foams. We have shown that the amount and the size of aggregates had an influence on the drainage rate.     

A proposed mechanism for detergent-assisted foam fractionation of lysozyme and cellulase restored with β-cyclodextrin

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as beta-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to beta-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.     

Hydrophobicity Enhances the Formation of Protein-Stabilized Foams

Screening proteins for their potential use in foam applications is very laborious and time consuming. It would be beneficial if the foam properties could be predicted based on their molecular properties, but this is currently not possible. For protein-stabilized emulsions, a model was recently introduced to predict the emulsion properties from the protein molecular properties. Since the fundamental mechanisms for foam and emulsion formation are very similar, it is of interest to determine whether the link to molecular properties defined in that model is also applicable to foams. This study aims to link the exposed hydrophobicity with the foam ability and foam stability, using lysozyme variants with altered hydrophobicity, obtained from controlled heat treatment (77 °C for 0–120 min). To establish this link, the molecular characteristics, interfacial properties, and foam ability and stability (at different concentrations) were analysed. The increasing hydrophobicity resulted in an increased adsorption rate constant, and for concentrations in the protein-poor regime, the increasing hydrophobicity enhanced foam ability (i.e., interfacial area created). At higher relative exposed hydrophobicity (i.e., ~2–5 times higher than native lysozyme), the adsorption rate constant and foam ability became independent of hydrophobicity. The foam stability (i.e., foam collapse) was affected by the initial foam structure. In the protein-rich regime—with nearly identical foam structure—the hydrophobicity did not affect the foam stability. The link between exposed hydrophobicity and foam ability confirms the similarity between protein-stabilized foams and emulsions, and thereby indicates that the model proposed for emulsions can be used to predict foam properties in the future.