Paula Jauregi

Recovery of lactoferrin and lactoperoxidase from sweet whey using colloidal gas aphrons (CGAs) generated from an anionic surfactant, AOT

The recovery of lactoferrin and lactoperoxidase from sweet whey was studied using colloidal gas aphrons (CGAs), which are surfactant‐stabilized microbubbles (10–100 μm). CGAs are generated by intense stirring (8000 rpm for 10 min) of the anionic surfactant AOT (sodium bis‐2‐ethylhexyl sulfosuccinate). A volume of CGAs (10–30 mL) is mixed with a given volume of whey (1–10 mL), and the mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Each of the phases is analyzed by SDS‐PAGE and surfactant colorimetric assay. A statistical experimental design has been developed to assess the effect of different process parameters including pH, ionic strength, the concentration of surfactant in the CGAs generating solution, the volume of CGAs and the volume of whey on separation efficiency. As expected pH, ionic strength and the volume of whey (i.e. the amount of total protein in the starting material) are the main factors influencing the partitioning of the Lf·Lp fraction into the aphron phase. Moreover, it has been demonstrated that best separation performance was achieved at pH = 4 and ionic strength = 0.1 mol/L i.e., with conditions favoring electrostatic interactions between target proteins and CGAs (recovery was 90% and the concentration of lactoferrin and lactoperoxidase in the aphron phase was 25 times higher than that in the liquid phase), whereas conditions favoring hydrophobic interactions (pH close to pI and high ionic strength) led to lower performance. However, under these conditions, as confirmed by zeta potential measurements, the adsorption of both target proteins and contaminant proteins is favored. Thus, low selectivity is achieved at all of the studied conditions. These results confirm the initial hypothesis that CGAs act as ion exchangers and that the selectivity of the process can be manipulated by changing main operating parameters such as type of surfactant, pH and ionic strength.

Characterisation of colloidal gas aphrons for subsequent use for protein recovery

Abstract Colloidal gas aphrons (CGAs) were first reported by Sebba (J. Colloid Interface Sci., 35 (4) (1971) 643) as micro bubbles (10-100 μm), composed of a gaseous inner core surrounded by a thin surfactant film, which are created by intense stirring of a surfactant solution. Since then, these colloidal dispersions have been used for diverse applications (clarification of suspensions, removal of sulphur crystals, separation of organic dyes from wastewater, etc.). However, there have been no reports, as yet, of their direct application for protein recovery. In this study, CGAs are created from an anionic surfactant (AOT) and are characterised in terms of stability and gas hold-up for a range of process parameters relevant to their proposed use for protein recovery, at a later stage. A statistical experimental design was developed in order to study the effect of different factors (surfactant concentration, salt concentration, pH, time of stirring and temperature) on the stability and gas hold-up of CGAs. The analysis of results from the experimental design provides predictive statistical models. Stability was found to depend mainly on salt and surfactant concentration. Several interactions are shown to be significant including the time-temperature interaction. Gas hold-up was found to depend mainly on salt and surfactant concentration and time of stirring. Also, results from power measurements are presented and the minimum energy for the formation of CGAs, for one set of solution properties, is determined.

Protein recovery using gas-liquid dispersions

Two separation techniques, foam separation and colloidal gas aphrons (CGAs), both of which are based on gas-liquid dispersions, are compared as potential applications for protein recovery in downstream processing. The potential advantages of each method are described and the concentration and selectivity achieved with each method, for a range of proteins is discussed. The physical basis of foam separation is the preferential adsorption of surface active species at a gas-liquid interface, with surface inactive species remaining in bulk solution. When a solution containing surface active species is sparged with gas, a foam is produced at the surface: this foam can be collected, and upon collapse contains surface active species in a concentrated form. CGAs are microbubble dispersions (bubble diameters 10-100 microm) with high gas hold ups (>50%) and relatively high stability, which are formed by stirring a surfactant solution at speeds above a critical value (typically around 5000 rpm). It is expected that when proteins are brought into contact with aphrons, protein adsorbs to the surfactant through electrostatic and/or hydrophobic forces. The aphron phase can be separated easily from the bulk solution due to its buoyancy, thus allowing separation of protein in a concentrated form.

Colloidal gas aphrons: A novel approach to protein recovery

Sebba (1987) defined colloidal gas aphrons (CGA) as microbubbles stabilized by surfactant layers, which are created by stirring surfactant solutions at speeds greater than a critical value. A high shear impeller is used for stirring and critical values for the impeller speed must be exceeded to create these stable gas liquid dispersions (typically >5000 rpm). Although there have been no previous reports of direct protein recovery using CGA, it is likely that, with appropriate choice of surfactant, proteins should adsorb to these surfactant bubbles by means of electrostatic and/or hydrophobic interactions. This is the basis of this study, in which the use of CGA for protein recovery from aqueous solution is considered. A surfactant which has been characterized previously for generation of CGA was chosen (Jauregi et al., 1997), i.e., the anionic surfactant sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT). Lysozyme, a well-characterized protein, was chosen as the protein to be recovered. Lysozyme was recovered successfully from aqueous solution using CGA generated from AOT. At optimum conditions, lysozyme recovery, enrichment ratio, and separation ratio were 95%, 19 and 302 respectively, with enzyme activity maintained. These results indicate the exciting potential of this technique. A wide range of process conditions including initial concentration of protein and surfactant, surfactant/protein molar ratio, pH, and ionic strength were considered. High recoveries and enrichments were generally obtained at protein concentrations </=0.41 mg/mL, and surfactant concentrations >0.11 mg/mL. However, at high ionic strength (0.29M) poor separation and recoveries were obtained at low protein concentrations (counter-ions diminishing electrostatic interactions between protein and aphrons at this condition). In general, (ns/np)a was determined to be between 10 and 16 for experiments in which high levels of recovery/separation parameters were found. For most conditions, protein precipitation was observed; however, this precipitate could be resolubilized without loss of enzyme activity.

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5.

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC50 = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC50 = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.

Antioxidant, antibacterial and ACE-inhibitory activity of four monofloral honeys in relation to their chemical composition

Different monofloral honeys from Castilla-La Mancha (Spain) have been studied in order to determine their main functional and biological properties. Thyme honey and chestnut honey possess the highest antioxidant capacity, which is due to their high vitamin C (in thyme honey) and total polyphenolic content (in chestnut honey). On the other hand, chestnut honey showed high antimicrobial activity against Staphylococcus aureus and Escherichia coli, whilst others had no activity against S. aureus and showed very small activity against E. coli. Moreover it was found that the antimicrobial activity measured in chestnut honey was partly due to its lysozyme content. In addition the angiotensin I-converting enzyme (ACE) inhibitory activity was measured, and the ACE inhibition is one mechanism by which antihypertensive activity is exerted in vivo. All the types of honey showed some activity but chestnut honey had the highest ACE inhibitory activity.

Recovery of gallic acid with colloidal gas aphrons (CGA)

In the present paper the potential application of colloidal gas aphrons (CGA) to the recovery of antioxidants from wine-making waste extracts is investigated. CGA were generated by stirring a buffered solution (400 ml) of a cationic surfactant(cetyltrimethylammonium bromide, CTAB) at 8000 rpm for 10 minutes. Trials were carried out on standard solutions (2 ml) of gallic acid (GA) 200 mg/l with varying volumes of colloidal gas aphrons (20-60 ml) generated with varying concentrations of CTAB (2 and 4 mM). Influence of pH, solvent (buffered aqueous solution and ethanol), CTAB to GA molar ratio on recovery were studied. Best recovery (63%) was achieved from an aqueous solution of GA and at a CTAB to GA molar ratio of 16. Separation is mainly driven by electrostatic interactions but pH conditions are to be optimised to preserve the GA antioxidant power.

Production of angiotensin-I-converting enzyme inhibitory peptides from β-lactoglobulin- and casein-derived peptides: An integrative approach

Angiotensin I‐converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β‐lactoglobulin‐ and casein‐derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N “Amano” in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein‐derived peptides with IC50 = 285 μg/mL. In this hydrolysate we identified the well‐known potent ACE‐inhibitor and antihypertensive tripeptide Ile‐Pro‐Pro (IPP) and another novel octapeptide Gln‐Asp‐Lys‐Thr‐Glu‐Ile‐Pro‐Thr (QDKTEIPT). The second hydrolysate was mainly composed of β‐lactoglobulin derived peptides with IC50 =128 μg/mL. This hydrolysate contained a tetrapeptide (Ile‐Ile‐Ala‐Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Development of an Integrative Process for the Production of Bioactive Peptides from Whey by Proteolytic Commercial Mixtures

The aim of this work is to develop an integrative process based on the use of an ion exchange resin for the enzymatic hydrolysis of β-lactoglobulin to produce peptides with angiotensin converting enzyme (ACE) inhibitory activity using whey as a feed stock. Unlike the conventional methods, the main advantage of this approach is that by integrating the selective separation of β-lactoglobulin from whey and its hydrolysis less complex mixtures of peptides are produced. Furthermore, peptides of similar charge as β-lactoglobulin remain adsorbed achieving further purification. In this work, the enzyme protease N Amano at an enzyme to substrate ratio of 1/100 (wt/wt) was added directly to the adsorbed proteins in a thermostatically controlled membrane reactor operated in batch mode. Separation of the smaller peptides from the enzyme and larger peptides was achieved with a 1 kDa molecular weight cut-off ultrafiltration membrane. Also, this step enables the recycling of non-hydrolyzed substrates, large peptides, and enzyme. The adsorbed protein was re-solubilised in a 10 mM potassium phosphate buffer (pH 7 and 45°C). The different fractions were assayed for their bioactivity in terms of angiotensin converting enzyme inhibition percentage (ACEi%) and IC50 which is the concentration of peptides that can inhibit the ACE activity by 50%. Results show that permeates of 2 and 6 hrs hydrolysis have the highest bioactivity with IC50 = 67 and 98 µg/ml respectively.

Astringency reduction in red wine by whey proteins

Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and β-lactoglobulin (β-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but β-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein-tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.