Preservation of Anti-cytomegalovirus Activity in Human Milk Following High-Pressure Processing Compared to Holder Pasteurization

Abstract

Pasteurized donor human milk is recommended for hospitalized preterm infants when mother's own milk is unavailable. Our aim was to compare the antiviral activity of human milk processed by Holder pasteurization (HoP) or high-pressure processing (HPP) against representative enveloped and non-enveloped viruses including cytomegalovirus and hepatitis A virus. Expressed milk from 20 donors collected from the Ontario Milk Bank was combined into 10 pools, each from two unique donors. Each pool was processed by HoP (62.5°C, 30 min) or HPP (500 MPa, 8 min, 4°C) and subsequently inoculated with cytomegalovirus or hepatitis A virus to achieve a final concentration of 5-log plaque-forming units/mL. Plaque reduction assays were used to quantify detectable virus after 30 min incubation (room temperature). Post hoc experiments using a 4 h incubation time were conducted if reductions were detected at 30 min. Irrespective of processing, cytomegalovirus concentrations declined in all pools after 30 min incubation (P < 0.0001). Milk processed by HoP exhibited significantly less reduction compared to raw milk (P = 0.0069). In post hoc experiments, anti-cytomegalovirus activity was maintained at 4 h, with high inter-pool variability. Hepatitis A virus concentration remained unchanged after 30 min incubation in raw and processed milk. Anti-cytomegalovirus activity in human milk is preserved following HoP and HPP, persisting up to 4 h post-inoculation; anti-hepatitis A virus activity was not observed in raw or processed milk. Further research is needed to understand how HoP or promising alternative processing methods affect the antiviral activity of donated milk, given its potential importance to recipient infants.

Keywords: Holder pasteurization; antiviral; cytomegalovirus (CMV); donor human milk; hepatitis A virus (HAV); high-pressure processing (HPP).

FIGURE 1

Study flow diagram. The flow chart demonstrates sample collection, processing, and experimental design. HoP, Holder pasteurization; HPP, high-pressure processing.

FIGURE 2

Recovered titers of CMV from inoculated milk following incubation at room temperature. Mean CMV log PFU/mL for raw, Holder pasteurization (HoP), and high-pressure processing (HPP) treated pools. Each bar represents the mean of the pools plated in duplicate after 0 min, 30 min, and 4 h incubations (room temperature, ∼22°C) with error bars representing the standard deviation; dashed line represents the limit of detection. Statistical analyses were conducted using linear mixed effect models with post hoc pairwise comparisons. Different letters denote statistical significance (P < 0.05).

FIGURE 3

Distribution of anti-CMV activity per pool up to 4 h. Individual donor human milk pools (panels) exhibit varying degrees of reduction in detectable CMV after 30 min and 4 h incubations. Plaque reduction assays were plated in duplicate; the dashed line represents the limit of detection. HoP, Holder pasteurization; HPP, high-pressure processing.