The proteins coming from the milk whey have numerous functional properties. Among the proteins with high bioactivity, α-lactoalbumin (α-La) and β-lactoglobulin (β-Lg) are present in large quantities in the milk whey. In the separation process of proteins, it is important to choose techniques which besides ensuring purity and high yield will not affect the molecule biological activity. The aqueous two-phase systems (ATS) have been utilized with success in the partition of these proteins, however, the studies were performed using protein in its pure form. Studies using milk whey in-nature and goat milk whey have not been found yet. In this context, the objective of this study was to evaluate the liquid liquid equilibrium of aqueous two-phase systems (ATS) in the partition of α-La and β-Lg from goat milk whey in-nature. Equilibrium data were performed considering ATS comprised of polyethylene glycol, potassium phosphate and water at 25°C and pH 7.0. The influence of the polymer molecular weight and amount of goat milk whey in-nature on the partition coefficient of these proteins were assessed. The partition coefficient, selectivity, process yield and purity of α-lactoalbumin and β-lactoglobulin proteins were determined. The results showed that the separation technique by aqueous biphasic systems is applicable indicating high efficiency in the whey proteins separation process.
The whey consists of the aqueous fraction obtained as result of the milk coagulation during cheese production or in casein production; it retains about 55% of milk nutrient [
Most protein purification processes involves several steps and solvent consumption. Aqueous two-phase systems (ATS) have been applied with success in the recovery of biomolecules from natural products [
An aqueous biphasic system can be formed by different ways, mixing two hydrophilic polymers or a polymer and salt organic or inorganic [
Due to the high water content presented in the aqueous biphasic systems, around 80% to 90%, and the low interfacial tension, it is possible to separate bio-molecules under mild conditions in a suitable environment, without affecting its biological activity [
The partition of α-La e β-Lg using ATSs has been presented in literature by applying of different systems, such as Polyethylene glycol (PEG) + sodium citrate + water and PEG + potassium phosphate + water [
In this sense, the aim of this study was to evaluate the liquid liquid equilibrium of aqueous two-phase systems applied to the partition of α-La and β-Lg from goat milk whey in-nature. The aqueous two-phase systems were formed by polyethylene glycol (PEG), potassium phosphate (KPi) and water. All systems were maintained at 298 K and pH 7.0. The partition coefficient, selectivity, process yield and purity of α-La e β-Lg proteins were determined. The amount of the goat milk whey in-nature necessary for the separation and the influence of the polymer molecular weight on the partition of these proteins were also evaluated.
The goat milk whey was provided by Association of Small Ranchers of the Angicos wilderness (Associação dos Pequenos Agropecuaristas do Sertão de Angicos-APASA). The polymers utilized were PEG of molecular weight 1500 Daltons (Impex-Lot 35263-D), 4000 (Synth-Lot 152264), 8000 (Sigma-Lot 120M0004V). The salt utilized was potassium phosphate (KPi), monobasic (Vetec-Lot 1200572, Purity 99%) and dibasic (Isofar-Lot 111027, Purity 98%). In all systems, deionized water was used. The samples were filtered, filtered through cellulose acetate membrane with a porosity of 0.45 µm using acetonitrile (Sigma, Lot SHBD1824V, Purity 99.9%) and trifluoroacetic acid (Sigma-Lot BCBM0756V, Purity 99%).
The aqueous biphasic systems were formed according to the methodology described by Coimbra et al. [
The quantification of α-La and β-Lg protein was performed by high performance liquid chromatography (Shimadzu, Prominence, Kyoto, Japan series) containing a ternary pumping system (LC-20AT), autosampler (SIL-20AHT), column oven (CTO-20A), detector per diode array (SPD-M20A) and interface (CBM-20A). LC solution data were evaluated using an acquisition software, version 1.25 and Rigaku data treatment. The chromatographic separation occurred in reversed phase, the column and parameters utilized were the same as presented in Buffoni [
The separation process was evaluated in terms of partition coefficient, selectivity, yield and purity, which were determined through as described below.
a) Partition Coefficient (K): it was determined considering the ratio between the protein concentration in the upper phase and the concentration of the same protein in the lower phase:
where K is the partition coefficient, CUP and CLP are the protein concentrations in the upper and lower phase, respectively.
b) Selectivity: it is defined by the ratio between the partition coefficients of α-La and β-Lg in the two balance phases:
where S represents selectivity of α-La compared to β-Lg, KαLa and KβLg are the partition coefficients of α-La and β-Lg, respectively.
c) Yield and Purity: These two values were calculated according to equations defined by Chen [
where YαLa,UP and KβLg,LP correspond to α-La yield in the upper phase and β-Lg yield in the lower phase, respectively; KαLa and KβLg are the partition coefficients of α-La and β-Lg, respectively; Vr correspond to the volume ratio between the phases.
And for the α-La and β-Lg proteins purity:
where PαLa,UP and PβLg,LP correspond to the purity percentage of α-La proteins in the upper phase and β-Lg in the lower phase, respectively.
According to
To determine tie-line, different methodologies can be used. Chumpitaz [
Good adjust of the tie-line can be observed for the tie-line obtained in this work (s.d = 0.01). However, the tie-line obtained by Cumpitaz [
From
According to
The results also demonstrated the efficiency of aqueous two-phase systems in the separation of these proteins, since 85% of β-Lg was recovery in the lower phase, and 92% of α-La in the upper phase.
It is important to emphasize that the partition coefficient of these proteins were coherent with studies of literature that also applied polyethylene glycol, potassium phosphate and water at the same operational conditions, however using pure α-La e β-Lg proteins [
After the determination of the best amount of goat milk whey to be used, it was evaluated the partition of the proteins in the ATSs formed by PEG (150, 4000, 8000) + KPi + water.
According to
Goat milk whey amount (mL) | Kα-La | Kβ-Lg | Selectivity | Yield (%) | Purity (%) | ||
---|---|---|---|---|---|---|---|
α-Laup | β-Lglp | α-Laup | β-Lglp | ||||
1 | 5.78 | 0.09 | 63.71 | 78.61 | 94.54 | 77.08 | 94.99 |
5 | 2.25 | 0.05 | 45.57 | 58.91 | 96.95 | 81.84 | 91.00 |
10 | - | - | - | - | - | - | - |
*up = upper phase, lp = lower phase.
PEG | Kα-La | Kβ-Lg | Selectivity | Yield (%) | Purity (%) | Separation Time (s) | ||
---|---|---|---|---|---|---|---|---|
α-Laup | β-Lglp | α-Laup | β-Lglp | |||||
1500 | 5.78 | 0.091 | 63.71 | 78.61 | 94.54 | 77.1 | 94.99 | 144 |
4000 | 0.35 | - | - | 41.02 | 100 | 100 | 87.90 | 117 |
8000 | - | - | - | - | - | - | - | 74 |
*up = upper phase, lp = lower phase.
mer promotes decreasing of the partition coefficient [
The results demonstrated good efficiency in the separation of proteins for the system consisted by PEG 1500 + KPi + water, where: 78.61% of α-La was concentrated in the upper phase, and 95% of β-Lg was concentrated in the lower phase.
The results demonstrate that the phase diagram provided information about the condition to be applied in the recovery of whey protein in the two-phases. The amount of goat milk whey in-nature needs to be considered in order to promote the best separation of the proteins. The increase of the molecular weight of the polymer caused an increase in the biphasic region. On the other hand, increasing the polymer molecular weight caused a decrease in partition coefficient of the protein as well as a decrease in time separation of the phases the aqueous systems. The system consists of PEG 1500 + KPi + water showed the best results in terms of partition coefficient, yield and purity of the α-La e β-Lg proteins from goat milk whey in-nature. The results showed that the aqueous two-phase systems are applicable in the recovery of proteins from real systems.
The authors are grateful to CNPq for its financial support and DEQ/PPGEQ by encouraging and supporting the research.
Freire, L.A.C. and Pereira, C.G. (2016) Aqueous Two-Phase Systems Applied to Partition Proteins from Goat Milk Whey In-Nature. Advances in Bioscience and Biotechnology, 7, 350-359. http://dx.doi.org/10.4236/abb.2016.79034