Extraction, characterization and quantification of milk proteins

Following the selection of the protein standard, protein extracts were quantified.

Results are present in Figure 18:

Figure 18 – Quantification of proteins extracts. M stands for mean value

The extraction of milk proteins from skimmed milk followed by the desalting step resulted in a protein extract with a protein content of 96.17%, while the protein content of the powdered milk samples was about 82.02%. Nonetheless, the protein content of samples in both groups varied greatly. This can be due to the different content of casein isoforms skimmed milk and powdered, as the quantification of proteins in dependent on the binding of dyes to specific amino acids. As powdered milk is subject to high temperatures during its manufacturing process, protein structure might me altered and differ greatly from the casein standard used for the calibration curve. As such, the bind of dyes to these proteins might be significantly different compared to non-treated casein.

1.2. Characterization of milk proteins

Both milk protein extracts were loaded into SDS-Page gel (12%), along with different protein standards and the commercial mixtures of milk proteins. The resulting gel is presented in Figure 19

Figure 19 – SDS-gel resulting from the skimmed milk and powdered milk extracts: L1;

L2 and L3 – skimmed milk extract; LP1; LP2 and LP3 – powdered milk extract; Cas – casein standard. The protein molecular weight standard (first lane) used was Nzytech’s Protein Colour Marker II.

The bands present in the lane of the protein extract obtained from powdered milk are similar to the bands in the lane in powder milk. This indicates that major milk proteins were successfully extracted with the described method. The same can be said for the extracted obtained from skimmed milk, as the bands present in the lane where it was loaded are similar to the bands in the lane where untreated skimmed milk was loaded.

As such, it is possible to conclude that the extraction and purification processes were successful in extracting the major milk proteins present in the different dairy products.

The polls of both skim milk and powder milk proteins extracts were then loaded into SDS-Page gels to follow up the protein profile. Samples of skimmed milk and powdered milk were also loaded into to gel (Figure20).

Figure 20 – SDS-gel resulting from the skimmed milk and powdered milk extracts: LA –α-lactalbumin standard; LG – β-lactoglobulin standard; WP – whey protein commercial mixture; Cas – casein standard; PM – powdered milk; PME – powdered milk extract; SM – skimmed milk; SME – skimmed milk extract; WPH – hydrolyzed whey milk. The protein standard used was Nzytech’s Protein Marker.

As in the previous gel (Figure 19), the predominant proteins in both samples were caseins. The isoform αs1 and αs2 were again the main isoforms of milk powder extract.

αs2 was again present in smaller quantities in skim milk extract, while β-casein and κ-casein were present in similar quantities in both samples. In the lane were skimmed milk and skimmed milk protein extract were loaded, low molecular weight bands (14 and 18 kDa) were again visible. These bands had similar weights as the bands present in the lanes were α-lactalbumin (14kDa) and β-lactoglobulin (18kDa) were loaded. This thus confirms the presence of whey proteins both in skimmed milk and the skimmed milk protein extract. Whey protein hydrolysate, composed of peptides resulting from the hydrolysis of whey proteins, was also loaded into the gels. The resulting peptides have low molecular weights and has such no bands were visible in the lane where the hydrolysate was loaded. These results thus confirm the success of the scaled-up extraction process.

The major protein both in the skim milk and the powder milk extract was casein. Four isoforms of casein were present in the lanes where skimmed milk and the powdered milk extract were loaded, similarly to the lane where the commercial standard of casein was loaded: αs1 (23.6kDa), αs2 (25.2kDa), β-casein (24.0kDa) and κ-casein (19.0kDa) (Cabana, 2017). However, the concentration of each isoform of casein varied in each

sample. The isoform αs1 and αs2 were the main isoforms of milk powder extract, while only αs1 was present in the skim milk extract. β-casein and κ-casein were present in similar quantities in both samples. Bands of with lower molecular weights (14kDa and 18kDa) and high molecular weights (66kdA) were also present in the skim milk extract but were only present in vestigial amounts in the powder milk extract. This bands likely correspond to whey proteins and BSA, respectively. These results thus demonstrate the different protein composition of the two distinct types of milk products. These differences could be explained by the manufacturing process used in powdered milk production.

Powdered milk is currently manufactured using primarily dry heating processes, like spray drying and drum drying.

In the spray drying method, milk products like skimmed milk or whole milk are firstly concentrated in an evaporator until they reach approximately 50 percent milk solids. After this, the concentrated product is sprayed into a heated chamber where water is almost instantly evaporated, resulting in a fine powder of milk solids.

In the drum drying method, milk products are applied as a thin film to the surface of a heated drum. The resulting milk solids are then scraped off.

The dry heating used in the industrial processes used to produce powdered milk productions enhances chemical modifications of amino acids, like oxidation and glycation. The heating of proteins can also lead to their denaturation.

Alternately, milk products can be freeze dried. Although the costs of these processes being about five times as much as the cost of conventional drying methods, freeze-drying of milk proteins significantly reduces the modification. Despite that, freeze-drying also leads to proteins denaturation, although to a lesser extent.

Differences in the protein structure due to processing of dairy products can significantly alter proteins digestibility and amino acid availability. This can, in turn, alter the immunogenic properties of these proteins, as their differential digestion can result in the formation of different peptides during digestion. As such, the substitution of non-proceed milk proteins for processed milk products can constitute an effective alternative in the prevention of IgE-mediated food allergies.

The skim milk protein extract was thus select for use in the bioaccessibility and bioavailability experiments, has the percentage of protein in the extracted was superior to the protein content of the extracted obtained from powered milk. Besides that, the

protein content of this extract into that of commercial casein standards, as opposed to the powered milk extract.