Effect of N-ethylmaleimide as a blocker of disulfide crosslinks formation on the alkali-cold gelation of whey proteins

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Abstract

N-ethylmaleimide (NEM) was used to verify that no new disulfide crosslinks were formed during the fascinating rheology of the alkali cold-gelation of whey proteins, which show Sol-Gel-Sol transitions with time at pH > 11.5. These dynamic transitions involve the formation and subsequent destruction of non-covalent interactions between soluble whey aggregates. Therefore, incubation of aggregates with NEM was expected not to affect much the rheology. Experiments show that very little additions of NEM, such as 0.5 mol per mol of protein, delayed and significantly strengthened the metastable gels formed. Interactions between whey protein aggregates were surprisingly enhanced during incubation with NEM as inferred from oscillatory rheometry at different protein concentrations, dynamic swelling, Trp fluorescence and SDS-PAGE measurements.

Figures

  • Fig 1. Elastic modulus during the alkali cold gelation of 8 wt% pre-heated WPI solutions (68.5˚C for 2 h) at different ratio of [NEM]/[WPI] at pH 11.84 at 25˚C, at (a) low NEM concentrations, and at (b) high concentrations.
  • Table 1. Characteristic parameters of alkali cold gelation with NEM of pre-heated WPI aggregates with different [NEM]/[WPI] ratios. Errors show one standard deviation.
  • Fig 2. WPI concentrations dependence of G’max and G”max at different concentration of NEM. (a-c) the aggregates were pre-heated for 2 h at 68.5˚C, (d-f) the aggregates were pre-heated for 24 h. Red (G”max, squares) and blue (G’max, triangles) straight lines are the best-fit power-law equations considering two regimes. Black dash lines show G’L at different conditions; continuous purple lines for G’max are the best fit regressions considering the percolation model Eq 3 using [WPI]c,low.
  • Table 2. Regression parameters using two power-law regimes, Eqs 1 and 2, and using a percolation model, Eq 3. Uncertainties given are the standard error of the regression parameters.
  • Fig 3. The diameter profiles of 1 wt% WPI 24 h aggregates at different pH (unbuffered) and room temperature, without NEM (empty symbols, dashed lines) and with NEM at RNEM/WPI = 0.5 (solid symbols, continuous lines). Dotted line shows comparable results from the literature for 2 h preheated WPI aggregates at pH 12.3 [12].
  • Fig 4. Relative maximum fluorescence intensity after 1 h incubation at different pH, using unheated WPI, and 24 h pre-heated WPI with (RNEM/WPI = 0.5) and without NEM. Data normalized with the fluorescence intensity of aggregates without NEM at pH 9.3. Final [WPI] = 0.03 wt%, [ANS] = 0.05 mM, excitation wavelength λex = 295 nm, emission wavelength λem = 464 nm. Lines show the best-fit sigmoidal curves to determine the pKa.
  • Fig 5. Relative maximum tryptophan fluorescence intensity of native WPI after 1 h at different pH, using unheated WPI, and 24 h pre-heated WPI with (RNEM/WPI = 0.5) and without NEM. Data normalized with the fluorescence intensity of aggregates without NEM at neutral pH. Final [WPI] = 0.005 wt%, excitation wavelength λex = 295 nm; the maximum emission intensity λem between 330–360 nm is used.
  • Fig 6. Zeta-potential of native WPI and 2 h WPI aggregates with (RNEM/WPI = 0.5) and without NEM at different pH conditions. Error bars show the SD of 4–7 measurements within 15 min.

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Lei, Z., Chen, X. D., & Mercadé-Prieto, R. (2016). Effect of N-ethylmaleimide as a blocker of disulfide crosslinks formation on the alkali-cold gelation of whey proteins. PLoS ONE, 11(10). https://doi.org/10.1371/journal.pone.0164496

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