Continuous fractionation of whey protein isolates by using supercritical carbon dioxide
Graphical abstract
Introduction
Whey proteins have high biological value and can be marketed in the form of concentrates (WPC) or isolates (WPI). WPI exhibits high protein content (over 90%) and is considered as a food supplement [1]. Whey protein is a source of peptides, which can result in health benefits to the consumers, as it has antihypertensive, antimicrobial, antioxidant, and opioid activities, and it has applications in the food industry [2,3]. Whey proteins include α-Lactalbumin (α-La) and β-Lactoglobulin (β-Lg), which represent the majority of the total whey proteins (20% α-La and 40% β-Lg) [4,5].
α-La is found in human milk and also in bovine serum [4,6]. It helps in the absorption of minerals and has antibacterial, immunomodulatory, and antitumor activities [7]. Furthermore, it can be used in anticancer drugs and in the prevention of type-2 diabetes [8]. A food enriched in α-La can complement the diet of infants and the elderly [9]. β-Lg represents the major protein in bovine serum. The addition of β-Lg into food products enhances some technological and functional properties, such as emulsion stability, viscosity, and gelling capacity. Foods enriched with β-Lg protein are used in sports nutrition [9]. However, it is the most allergenic protein among whey proteins and is absent in human milk [8,10]. Glycomacropeptide (GMP) is also an important component present in WPI. It has the ability to bind cholera and Escherichia coli enterotoxins, to promote the growth of bifidobacteria, to suppress gastric secretion and to inhibit viral or bacterial adhesion to intestinal epithelia cells [11].
Each whey protein has an individual application, therefore, there is a demand for technologies to fractionate these proteins efficiently [12,13]. Selective separation of α-La and β-Lg proteins by membrane filtrations is compromised due to the similarity of their molar masses (α-La = 14,186 Da and β-Lg = 18,281 Da) [14,15]. Other methods that have been already used to separate whey proteins include acid, isoelectric, and salting out precipitation; adsorption; liquid chromatography as ion exchange or affinity chromatography; dialysis; gel electrophoresis; reverse osmosis membranes; nanofiltration; ultrafiltration and microfiltration [7,[16], [17], [18], [19], [20], [21], [22]].
In a recent work, protein solution models containing α-La, β-Lg and casein (1:1:1), with a final concentration of the three proteins in water equal to 0.75%, were treated with high hydrostatic pressure (HHP) and acidified to pH 4.6 with HCl [23]. This treatment resulted in an α-La recovery up to 77% and 86% purity. Marciniak et al. [23] used a model dairy solution (no presence of minor proteins and GMP), extreme pressures up to 600 MPa, solvent (HCl), and a batch operation mode.
Toro et al. [24] developed a α-La fractionation using many steps including thermal precipitation of α-La, separation of native β-Lg from the precipitate via microfiltration and ultrafiltration, purification of β-Lg, resuspension of the precipitate, and purification of α-La, resulting in protein fractions with a purity of 91.3% for α-La and 97.2% for β-Lg. These results are outstanding due to the high purity fractionation achieved. Differential scanning calorimetry (DSC) analysis was performed by Toro et al. [24] to determine if the protein is attached to an apo or holo structure. However, DSC analysis does not revel if the degree of evolution of the protein is in the native state, although if the fractionated protein shows the same heat flux as the standard protein, there is a strong indication that does not have denaturation.
The multistep process of ultrafiltration followed by tryptic hydrolysis and a second ultrafiltration resulted in 15% α-La recovery [25], and the ion exchange and affinity chromatography methods reached a recovery of 36% and 48%, respectively [26].
Selective hydrolysis of β-Lg from WPI was also performed in a previous study by using α-chymotrypsin to obtain purified α-La, resulting in 74% of α-La without the presence of β-Lg [27]. Lisak et al. [27] reported that trypsin has enzymatic activity which digests β-Lg whereas α-La remains more or less in its native state. However, no structural analysis of the proteins was conducted.
The use of carbon dioxide (CO2) under supercritical conditions (above 31.1 °C and 7.39 MPa) and in batch mode has been studied for the precipitation and separation of whey proteins [1,28]. The result is a rich phase of α-La (precipitate) and another phase rich in β-Lg (in solution). The main advantage of this method is that the pH of the system returns to neutral on depressurizing, which dispenses with the need for toxic or organic solvents and avoids the presence of additional contaminants [1]. Supercritical CO2 (scCO2) exhibits desirable characteristics for application in the pharmaceutical and food industry and is widely used. The properties of CO2, like viscosity, diffusivity, and density, depend on the temperature and pressure used. CO2 presents low critical temperature and moderate critical pressure, which allows its application for the proteins, with no degradation [29,30].
CO2 dissolves partially in solution with a high-water content to form carbonic acid (H2CO3), which dissociates into bicarbonate (HCO3−), carbonate (CO32−) and hydrogen (H+) ions. It is worth mentioning that the solubility of carbon dioxide in water is only around 1 mole % in CO2 at 25 MPa and 40 °C and decreases with the decrease in pressure and/or increases in temperature [31]. This mechanism (Eqs. (1), (2), (3), (4)) is responsible for the pH decrease of the solution [1,32].CO2(g) ↔ CO2(aq)CO2(aq) + H2O ↔ H2CO3H2CO3 ↔ HCO3− + H+HCO3− ↔ CO32− + H+
The isoelectric point of the α-La protein, which was reported to be between 4.2 and 4.5 [7], can be achieved by adjusting the pressure and temperature conditions of the reaction, which promotes its precipitation. Under these conditions, β-Lg protein remains soluble because its isoelectric point is different from the isoelectric point of α-La (between 5.35 and 5.49 [7]). This results in a fraction that is rich in precipitated α-La protein and has a low β-Lg content, and another fraction, rich in soluble β-Lg and low in α-La content.
ScCO2 solvent was applied for the fractionation of β-Lg and α-La from a WPI solution by Bonnaillie and Tomasula [1]. The authors used a batch reactor with a capacity of 1 L (laboratory scale), with the pressure varying between 5.5 and 34 MPa and temperature ranging between 60 and 65 °C. They reported that the α-La was enriched up to seven-fold (60 °C and 31 MPa) and the β-Lg up to five-fold (65 °C and 11.7 MPa). Tomasula et al. [33] studied α-La precipitation from whey protein concentrate containing 75% protein (WPC75), also in a batch reactor. The maximum precipitation of α-La (55.4% α-La in the precipitated fraction) was obtained by using a solution of 7% WPC75 with the experimental conditions of 64 °C and 4.1 MPa. In a later study, Yver et al. [28] modeled and estimated the cost of application of scCO2 in the fractionation of α-La and β-Lg proteins from WPI, by using the same experimental apparatus as Tomasula et al. [33] with modification. Under conditions of T = 60 °C, C = 5% WPI, P = 8.3 MPa, the production cost was determined to be $8.65 per kilogram of WPI treated, resulting in the highest α-La purity, 61%, with 80% α-La recovery in the solid fraction. The residence time used was 2 h in a batch reactor.
Furthermore, some techniques have been used in the past for GMP purification. Silva et al. [11] partitioned GMP by using the poly(ethylene glycol) (PEG)-sodium citrate system and a protein recovery of higher than 85% was obtained. Xu et al. [34] used the anion exchange resin amberlite IRA 93 to absorb GMP selectively from cheddar cheese whey.
The use of a continuous reactor results in higher safety, higher speed, and improved efficiency, and better quality than with a batch reactor [35]. Thus, in the present study, we have applied the key methodological difference of the use of a continuous-flow reactor with scCO2 as a solvent in an aqueous medium to obtain two fractions enriched with α-La and β-Lg proteins from WPI. The recycling of CO2 is possible with this configuration, resulting in an environmentally more sustainable process. Also, the final product obtained is free of organic or toxic solvents, which will avoid later steps to remove them. Previous studies used a batch method for fractionating whey protein isolates [1,28,33], therefore, there is demand to perform the fractionation in continuous mode.
The α-La protein causes a buffering effect of the solution [36,37] and its precipitation leads to slight pH changes in the medium. This effect results in fewer α-La molecules achieving the isoelectric point, loss of the fractionation capacity, and, consequently, lower yield. So, a fine adjustment in pH and temperature can compensate these losses and improve the yield. Continuous removal of the α-La from the reaction medium minimizes the buffering effect, which can be achieved by the continuous-mode reaction. In addition, the continuous mode leads to lower cost, mainly due to the fewer pauses in the fractionation process.
The processing of WPI with scCO2 could result in alterations in the structure and properties of the proteins, such as formation of a gel with higher strength [38] and increased turbidity and particle size [39]. As far as we know, there is no study that has evaluated the effect of processing of WPI with scCO2 in continuous mode on the proteins.
Therefore, the main objective of this study was to evaluate the best process conditions (temperature and pressure) to fractionate α-La and β-Lg proteins from WPI by using a continuous-flow reactor with scCO2 as a solvent in an aqueous medium. Circular dichroism was used to examine the structure of α-La and β-Lg proteins before and after the treatment with scCO2 using a continuous reactor.
Section snippets
Materials
Commercial spray-dried WPI from cheese whey was purchased from Medicinal Pharmacy and Handling Ltda (Maringa, PR, Brazil). Bovine milk protein α-La and β-Lg were purchased from Sigma–Aldrich (purity > 85% and > 90%, respectively, Merck KGaA, Darmstadt, Germany). Glycomacropeptide (purity > 80%) was kindly gifted from Davisco Foods International (Eden Prairie, MN, USA). Trifluoracetic acid (TFA, analytical grade, Merck KGaA, Darmstadt, Germany) and acetonitrile (HPLC grade, Merck KGaA,
WPI initial composition
The compositional analysis was conducted with WPI with no supercritical treatment and the results are given in Table 1. The protein content in the starting WPI was 89.6%, of which 16%, 43%, and 17.8% corresponded to α-La, β-Lg, and GMP, respectively. The other components (12.8%) were assigned to the minor proteins, immunoglobulins, bovine serum albumin, lactoferrin, and casein fragments [6].
Protein quantification in α-La and β-Lg enriched fractions
The phase behavior of CO2 and water mixtures was previously studied [47]. Throughout the ranges of
Conclusion
The selected pressure and temperature operating conditions allowed the fractionation of α-La and β-Lg proteins from WPI by using scCO2 as a solvent in a continuous-flow reactor. For the lowest temperature and pressure (8 MPa and 55 °C), the α/β ratio increased for the α-La-rich fraction (40.5% α-La and 9.9% β-Lg) with 20.9% protein precipitation, which was shown to be the best conditions to obtain α-La. The α/β ratio under these conditions was nine times higher than the initial ratio in WPI. On
Acknowledgment
The authors thank the CAPES: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CNPq: Conselho Nacional de Desenvolvimento Científico e Tecnológico, FAPEMIG: Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Fundação Araucária and COMCAP (UEM): Complexo de Centrais de Apoio à Pesquisa da Universidade Estadual de Maringá for financial support and scholarships.
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