Effects of High-Pressure Processing, UV-C Irradiation and Thermoultrasonication on Donor Human Milk Safety and Quality

Abstract

Holder pasteurization (HoP) is the current recommended treatment for donor human milk. Although this method inactivates microbial contaminants, it also negatively affects various milk components. High-pressure processing (HPP, 400, 500, and 600 MPa), ultraviolet-C irradiation (UV-C, 2,430, 3,645, and 4,863 J/L) and thermoultrasonication (TUS, 1,080 and 1,620 kJ/L) were investigated as alternatives to thermal pasteurization (HoP). We assessed the effects of these methods on microbiological safety, and on concentration and functionality of immunoglobulin A, lactoferrin, lysozyme and bile salt-stimulated lipase, with LC-MS/MS-based proteomics and activity assays. HoP, HPP, TUS, and UV-C at 4863 J/L, achieved >5-log₁₀ microbial reduction. Native protein levels and functionality showed the highest reduction following HoP, while no significant reduction was found after less intense HPP and all UV-C treatments. Immunoglobulin A, lactoferrin, and lysozyme contents were also preserved after low intensity TUS, but bile salt-stimulated lipase activity was significantly reduced. This study demonstrated that HPP and UV-C may be considered as suitable alternatives to HoP, since they were able to ensure sufficient microbial inactivation while at the same time better preserving the bioactive components of donor human milk. In summary, our results provide valuable insights regarding the evaluation and selection of suitable processing methods for donor human milk treatment, which may replace HoP in the future.

Keywords: antimicrobial proteins; bacteria inactivation; bacteriostatic properties; donor human milk; non-thermal processing; proteomics.

FIGURE 1

Schematic representation of the experimental approach used. K12, SE, and EC stand for E. coli K12, S. epidermidis and E. cloacae, respectively. Two independent experiments (biological replicates) were performed while all analyses were performed in technical duplicate for each sample.

FIGURE 2

Native milk serum protein concentration as determined with a BCA assay. The results are presented as mean ± standard deviation of two independent experiments and all analyses were performed in technical duplicate. UN represents the untreated values. *Indicates significant differences to untreated samples (p < 0.05).

FIGURE 3

Hierarchical cluster analysis and heatmap showing the changes in the protein profile after HPP, UV-C, TUS, and HoP, based on iBAQ values (log₁₀ scale from 2 to 12 according to color bar). Proteins are labeled by their UniProt ID. Functional categories (enzyme, immune, transport, and other) were based on GO annotation of biological function. Two independent experiments (biological replicates) were performed while all analyses were performed in technical duplicate for each sample. UN represents the untreated values. HPP; 400 MPa for 5, 10, and 30 min, 500 MPa for 1.5, 2 × 1.5, 3, and 5 min, 600 MPa for 1.5, 2 × 1.5, 3, and 5 min, UV-C; 2430J/L, 3645J/L, and 4863L/L,TUS; 9_40W, 6_60W, and 9_60W.

FIGURE 4

Effect of HPP, UV-C, TUS and HoP on the IgA (A), LTF (B), and LYZ (C) content. The retention values were calculated based on the iBAQ intensities obtained by LC-MS/MS analysis. Untreated values were set at 100% (dotted line). The results are presented as mean ± standard deviation of two independent experiments and all analyses were performed in technical duplicate.*Indicates significant differences to untreated samples (p < 0.05).

FIGURE 5

BSSL retention after HPP, UV-C, TUS, and HoP, based on iBAQ intensities and a lipase activity assay. Untreated values were set at 100% (dotted line). The results are presented as mean ± standard deviation of two independent experiments and all analyses were performed in technical duplicate.*Indicates significant differences to untreated samples (p < 0.05).

FIGURE 6

Growth rate per hour of S. aureus and E. coli in untreated, HPP, UV-C, TUS, and HoP DHM samples. The results are presented as mean ± standard deviation of two independent experiments and all analyses were performed in technical duplicate. UN represents the untreated values. *Indicates significant differences to untreated samples (p < 0.05).

FIGURE 7

Correlation matrix of S. aureus and E. coli growth rates and IgA, LTF and LYZ iBAQ values. Each box contains an r value (Pearson correlation coefficient).