High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation

Abstract

Background: Donor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins.

Objectives: We aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins.

Methods: Pooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300-500 MPa at 16-19°C (due to adiabatic heating) for 1-9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay.

Results: Treatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor.

Conclusion: Compared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Keywords: antibody; breast milk; enzyme; infant nutrition; microbial inactivation; mother’s milk; premature infant; preterm infant; protease; thermal processing.

FIGURE 1

Overall experimental design. A large volume (>50 L) of donor human milk was collected from a large number of donors and separated into two arms of the study. One division was inoculated with bacterial species of interest and the other remained uninoculated. Milk was aliquoted into plastic pouches and inoculated on three separate occasions with various pathogenic bacteria at a concentration of 7 log CFU/mL. Uninoculated milk was similarly aliquoted into pouches. Samples were shipped on dry ice to the HPP processing facility and returned after HPP treatments (HPP: 300–500 MPa, 1–9 min; n = 3–5 processing trials per treatment). Concurrently, the same pool of milk was treated by HoP (n = 3) in our laboratory. Samples from the inoculated arm of the study were enumerated for surviving bacteria, whereas the concentrations of bioactive proteins were measured in samples from the uninoculated arm. HoP, Holder pasteurization; HPP, high-pressure processing.

FIGURE 2

Microbial inactivation (log CFU/mL) of vegetative bacteria in human milk treated with high-pressure processing [HPP: 300–500 MPa (circle represents 300 MPa, diamond represents 350 MPA, triangle represents 400 MPa, square represents 450 MPa, and inverted triangle represents 500 MPa), 1–9 min]. The concentrations of each bacterium were measured in both raw and processed samples. (A) Cronobacter sakazakii ATCC BAA-894. (B) Listeria monocytogenes ATCC 35152, (C) Enterococcus faecium ATCC 8459, and (D) Staphylococcus aureus cocktail (138-CPS and 146-CPS). Data represented as the mean ± standard error (n = 3–4 independent replicates). ∗ indicates that the 95% CI was above the 5-log reduction target.

FIGURE 3

Bacterial spore inactivation (log CFU/mL) in human milk treated with high-pressure processing (HPP: 300–500 MPa, 1–9 min). (A) Bacillus subtilis spore cocktail (NRRL B-354 and NRRLB-356) and (B) Paenibacillus spp. spore cocktail (P. macerans NRRL B-14029 and P. polymyxa NRRL B-510). Data represented as the mean ± standard error (n = 3 independent replicates). ∗ indicates significantly (P < 0.05) different from the travel control based on one-sided t-tests.

FIGURE 4

Antibody concentrations ((A): IgA, (B): IgG, and (C): IgM) in raw, HPP (300–500 MPa, 1–9 min), and HoP-treated donor milk. HPP treatments are labeled on the x-axis with the pressure followed by treatment time (for example, 300-1 indicates 300 MPa, 1 min). Experiments were performed in triplicate, and results were expressed as mean ± SD. The black bar represents the raw milk. The orange bar represents the HoP-treated milk. Blue bars indicate HPP treatments that resulted in a significantly reduced concentration of detectable antibody compared with raw milk (one-sided t-test; P < 0.05). ∗ indicates donor milk treated with HPP with significantly higher retention of antibodies compared with HoP (one-sided t-test; P < 0.05).

FIGURE 5

Lactoferrin (A), elastase (B), and polymeric immunoglobulin receptor (PIGR) (C) concentration in raw milk, HPP-treated donor milk (300–500 MPa, 1–9 min), and HoP-treated donor milk. These proteins were grouped together because they were preserved better by HPP than HoP. Experiments were performed in triplicate, and results were expressed as mean ± SD. The black bar represents the raw milk. The orange bar9 represents the HoP-treated milk. Blue bars indicate HPP treatments that resulted in a significantly reduced concentration of detectable protein compared with raw milk (one-sided t-test; P < 0.05). ∗ indicates donor milk treated with HPP with significantly higher retention of proteins compared with HoP (one-sided t-test; P < 0.05).

FIGURE 6

Lysozyme (A), osteopontin (B), α-lactalbumin, (C) vascular endothelial growth factor (VEGF), and (D) concentration in raw milk, HPP-treated donor milk (300–500 MPa, 1–9 min), and HoP-treated donor milk. These proteins are grouped together because they were preserved similarly by HoP and HPP. Experiments were performed in triplicate, and results were expressed as mean ± SD. The black bar represents the raw milk. The orange bar represents the HoP-treated milk. Blue bars indicate HPP treatments that resulted in a significantly reduced concentration of detectable protein compared with raw milk (one-sided t-test; P < 0.05). ∗ indicates donor milk treated with HPP with significantly higher retention of protein compared with HoP (one-sided t-test; P < 0.05).

FIGURE 7

Bile salt-stimulated lipase (BSSL) activity in samples in raw milk, HPP-treated donor (300–500 MPa, 1–9 min), and HoP-treated donor milk. Experiments were performed in triplicate, and results were expressed as mean ± SD. The black bar represents the raw milk. The orange bar represents the HoP-treated milk. Blue bars indicate HPP treatments that resulted in a significantly reduced concentration of detectable BSSL activity compared with raw milk (one-sided t-test; P < 0.05). ∗ indicates donor milk treated with HPP with significantly higher retention of BSSL activity compared with HoP (one-sided t-test; P < 0.05).