Cow’s milk allergen β-lactoglobulin immunoreactivity affected by pulsed light treatment Janire Orcajo, Dr. Iñigo Martínez de Marañón, Dr. María Lavilla (
[email protected]). AZTI-Tecnalia. Astondo bidea, Edif. 609. Derio (48160) Bizkaia, Spain www.azti.es
Food Research Division
Introduction
Results
Results (cont.)
Conclusions
Pulsed Light (PL) is a technology that consists of a successive repetition of short duration and high power flashes of broadband emission light (200-1000 nm) that has been shown to be effective in inactivating a wide range of microorganisms. Proteins are among the major targets for photo-induced modifications due to the abundance of endogenous chromophores within their structure. Despite these findings, the effect of PL on the allergenicity of proteins is still a matter of speculation.
Changes in β-lg Immunoreactivity to IgE
Native-PAGE
A gradual increase of the I% with the fluence applied was found (Fig 1). The highest inhibition (43%) was reached at a fluence of 16 J cm-2 for phosphate buffer, where the β-lg is presented as dimers; only a 29% was found for acidic medium when octamers of β-lg prevail in the solution buffer.
Native-PAGE confirms a molecular alteration of β-lg structure when PL treatments are applied. Nontreated β-lg can be seen in Fig 3 as a band of 18.4 kDa at pH 4.6 and 6.6 (L1 and L2, respectively). When β-lg is exposed to a fluence of 8 (L3 and L4) and 16 J cm-2 (L5 and L6) some bands of higher molecular weight appear due to protein aggregation by disulfide bonds (Elmnasser et al., 2008). No differences due to pH is detected by Native-PAGE.
1.- Pulsed light technology inhibits the affinity between the β-lg and the IgE (implied in Type I hypersensitivity reactions). This means that the selected monoclonal IgE does not recognize the βlg when the protein is exposed to PL as accurately as in its native state. A conformational change in the protein structure caused by the pulsed light treatment may affect the union antigen-antibody.
Inhibition (%)
Thus, the aim of this study was to determine how such treatment affects the immunoreactivity of the β-lactoglobulin (βlg), the main whey protein fraction in bovine milk and the major allergen causing cow's milk allergy (CMA) encountered by a newborn child.
50 40 30
10 0 0
Samples preparation: 2 mg/ml β-lg lyophilized powder in phosphate buffer 0.1 M, pH 6.6 (~ milk pH), and acetate buffer 0.1M, pH 4.6 (~ acid whey pH).
ELISAs: • A competitive ELISA was developed for IgE immunoreactivity (recombinant IgE). Polyclonal goat antihuman Kappa-HRP was used as conjugate. Changes in IgE binding were calculated as percentage inhibition:
• A Sandwich ELISA Proteon β-lg detection kit (ZEUInmunotec) was used for IgG immunoreactivity measurement. Native-PAGE: Performed with a PhastSystem™ electrophoresis system in a PhastGel gradient 8-25% and PhastGel Native buffer strips following the manufacturer Separation Technique File #120.
8 12 Fluence (J cm-2)
16
Changes in β-lg Immunoreactivity to IgG IgG affinity to β-lg decreases gradually with the fluence applied as can be seen in Fig 2. The major effect is observed in phosphate buffer meanwhile the aggregation in octamers in acetate buffer seems to protect the protein from PL treatment.
𝑶𝑫 𝟒𝟓𝟎𝒔 − 𝑶𝑫 𝟒𝟓𝟎𝒄 ∗ 𝟏𝟎𝟎 𝑶𝑫 𝟒𝟓𝟎𝒂 − 𝑶𝑫 𝟒𝟓𝟎𝒄
OD 450 s: Abs of samples (PL treated) OD 450c: Abs of control (not PL treated) OD 450a: Abs of neg. control (no β-lg)
4
Fig 1. Inhibition percentage (I%) of the β-lg and IgE union by pulsed light treatment
120 100 Detection (%)
𝐈% =
Acetate buffer, pH 4.6
20
Materials and Methods
Pulsed-Light treatments: Performed at room temperature. Total fluence : 4, 8, 12 and 16 J cm-2.
Phosphate buffer, pH 6.6
Control 4 J cm-2 8 J cm-2 12 J cm-2 16 J cm-2
80 60
40 20 0 Phosphate buffer, pH 6.6 Acetate buffer, pH 4.6
Fig 2. β-lg detection (%) by Proteon β-lg detection kit in pulsed light treated samples
(Da) 14400 20100 30000
Std. L1 L2 L3 L4 L5 L6 Std.
45000 66000 97000 Fig 3. Native-PAGE of β-lg in acetate and phosphate buffer nottreated (L1)(L2), treated at 8 J cm-2 (L3)(L4) and 16 J cm-2 (L5)(L6), respectively.
Further studies Additional investigations should be focused on the molecular alteration that this novel technology causes on the β-lg structure (e.g. β-lg digestibility). The effect of PL on complex matrices (milk or dairy products) will be studied to assess the behaviour of β-lg in the presence of other natural components. Those studies should be carried on keeping in mind the PL effects on sensory quality. References Elmnasser, N., Dalgalarrondo, M., Orange, N., Bakhrouf, A., Haertlè, T., Federighi, M., et al. (2008). Effect of pulsed-light treatments on milk proteins and lipids. Journal of Agricultural and Food Chemistry, 56, 1984–1991.
2.- Pulsed light decreases the detection by IgG of isolated β-lg in solutions at pHs next to the milk and whey ones. 3.- However, the increase of IgE binding inhibition is not proportional to the decrease of detection by IgG. That means that the reduction in the detection of β-lg by commercial methods (usually based on IgG recognition) is not a warranty of the same reduction in the allergenic potential of the protein (due to differences of the IgG and IgE epitopes recognition). 4.- At pH 6.6, β-lg seems to be more susceptible to PL treatment showing the highest values of IgE inhibition and the lowest IgG detection. In contrast, at pH 4.6, β-lg is more stable and presents more resistance to PL treatment. 5.-
Native-PAGE points out that there is a conformational change in the β-lg structure when PL treatments are applied.
6.- PL technology shows promising results to decrease the allergenicity of the β-lg.
Acknowledgments Authors thank Basque Government (Economic Development and Competitiveness Department) for funding the JAN4U (IE13-356) project (Etortek program) and granting Janire Orcajo as research student. We also thank to Dr. Marja-Leena Laukkanen from VTT Technical Research Centre (Finland) for kindly donate recombinant IgE.