Immunoreactivity of Pressurized Milk Samples

The results of HHP induced changes in the immunoreactivity of milk protein fractions can be best demonstrated on the 2D-PAGE separations.

Control and pressurized (600 MPa, 5 mins) milk samples were first separated by 2D-PAGE in 12-20% gradient gel then blotted. Again, for immunoblotting milk positive human blood serum was used, and the conjugate was horseradish peroxidase-labeled anti-human IgE.

The most promising results were found for mare’s and goat milk, and the least changes in immunoreactivity appeared in bovine milk.

Although the 2D-PAGE of control and pressure treated mare’s milk samples were very much alike, indicating, that HHP didn’t cause any changes in the protein fractions (not shown), the difference between the immunoblots was great. While α-La and β-Lg of control mare’s milk gave definite immune responses, no antigen-antibody complex could be detected in pressurized samples (Fig. 37.).

Control mare’s milk Pressurized mare’s milk

Figure 37. A. Immunoblot of control mare’s

milk following 2D-PAGE Figure 37. B. Immunoblot of pressurized mare’s milk following 2D-PAGE β-lg α-la

2D-PAGE of goat milk didn’t show many differences in the intensity of milk protein fractions of control and HHP treated samples (not shown). But again, after immunoblotting, the decrease in the immunoreactivity, primarily in the casein fraction was significant. The intensive line, indicating the casein fraction in control milk, disappeared, only a few spots remained showing immunoreactivity (Fig. 38.). At the same time the intensity of the immune response observed in the position corresponding to β-Lg became slightly weaker. Using the present method, the immunoreactivity of α-La hasn’t shown any change.

Control goat’s milk Pressurized goat’s milk

Figure 38. A. Immunoblot of control goat milk following 2D-PAGE

Figure 38. B. Immunoblot of pressurized goat milk following 2D-PAGE

Casein β-Lg α-La

Changes in the immunoreactivity of protein fractions in bovine milk were found as well.

Casein in the pressurized sample gave weaker immune responses than in the control sample.

Intensity of the immune reaction caused by β-Lg decreased as a result of pressure treatment. No immunoreactivity of α-La could be found after 5 minutes treatment at 600 MPa (Fig. 39.).

Control bovine milk Pressurized bovine milk

Figure 39. A. Immunoblot of control bovine

milk following 2D-PAGE Figure 39. B. Immunoblot of pressurized bovine milk following 2D-PAGE Casein β-Lg α-La

In another test of immunoreactivity of milk proteins, bovine milk was treated applying 300, 400, 600, and 800 MPa each for 5 mins. Antigen-antibody complexes were investigated by using anti-β-lactoglobulin antibody IgG developed in rabbit, and human sera for IgE, respectively.

No differences were detected between the immunoreactivity of casein and α-La fractions neither in control nor in pressurized samples in the measurements with anti-β-lactoglobulin antibody IgG developed in rabbit. But interesting changes occurred in the immunoreactivity of β-Lg. An enlarged section of the densitogram demonstrates it well (Fig. 40.).

OD

Rf

Figure 40. Densitogram section of the immunoblot by anti-β-lactoglobulin antibody IgG developed in rabbit

Skim milk control Skim milk 300 MPa, 5 min Skim milk 600 MPa, 5 min The densitogram demonstrates clearly that decrease in immunoreactivity of β-Lg corresponded to the decrease in the intensity of this protein. Three hundred MPa treatment affected β-Lg B (first band from left) in a different way than A (second band from left). At this pressure the intensity of β-Lg B was about half of the original intensity but β-Lg A showed only a very slight decrease. This affirms the finding of Botelho et al. (2000) who reported that β-Lg B was significantly more sensitive to pressure denaturation than β-Lg A. At 600 MPa the intensity of both β-Lg isoforms showed similar values.

Decrease in immunoreactivity could be noticed only in skim milk but not in whole milk according to the applied conditions of the experiment.

When immunochemical reactions with milk positive human serum were studied, casein fractions gave definite responses. High pressure decreased the immunoreactivity of these fractions, but the rate of decrease reached its maximum at 400 MPa treatment. No further reduction was obtained at higher pressures. According to the densitogram (not shown), the ODu value of immunoblotted casein bands changed from 0.060 (control) to 0.037 ODu, an approximate decrease of 40%. The other protein fractions didn’t show immunochemical reactions, most likely because the human serum originated from a patient who was sensitive only to casein.

Summing up the results, HHP seemed to decrease the immunoreactivity of certain protein fractions in the different milk types, but the extent of the decrease was not significant, except for mare’s milk, according to the applied separation and immunoblotting methods. Thus HHP treatment alone did not prove to be useful in efforts to produce hypoallergenic milk or milk

products. However, in combination with other methods, HHP treatment was effective in decreasing or even cancelling the immunochemical reactivity of milk proteins. Bonomi et al.

(2000; 2003) hydrolysed pressurized β-Lg with proteolytic enzymes. The authors found that the immunoreactivity of the whole hydrolysates was related to their content of residual intact β-Lg, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolisis under pressure at 600 MPa. The results indicated that chymotripsin effectively hydrolised hydrophobic regions of β-Lg that had been temporarily exposed during the pressure treatments, and that were not accessible in the native protein or in the protein that had been previously pressure treated.