Effects of the thawing rate and heating temperature on immunoglobulin A and lysozyme activity in human milk

Abstract

Background: The percentage of infants receiving frozen human milk (HM) is increasing. The effects of thawing and warming on the secretory immunoglobulin A (SIgA) level and lysozyme activity in frozen HM should be investigated to identify optimal methods for preserving immune factors in frozen HM.

Methods: Milk samples were collected from 40 mothers with healthy full-term infants who had been lactating for one to six months. The baseline samples were analyzed within 24 h after collection, and the other samples were frozen at -18 °C before analyses. We compared two methods: placing the container overnight in a refrigerator at 4 °C before warming (slow thawing) and immediately thawing in warm water after removing the sample from the freezer (rapid thawing). Additionally, we investigated the effects of the warming temperature by comparing room temperature (25 °C) and physiological temperature (37 °C). The SIgA concentrations and lysozyme activities in the milk samples were determined using ELISA kits and fluorometric lysozyme activity assay kits, respectively.

Results: The SIgA concentrations and lysozyme activity in frozen HM were 16.5-52.1% and 16.8-39.3% lower than those in fresh HM, respectively. The SIgA concentrations in frozen HM were stable during slow thawing at 37 °C (p = 0.072) compared with those in fresh HM. The SIgA concentrations and lysozyme activity were maintained at significantly higher levels during slow thawing than during rapid thawing at 25 °C (p = 0.002 and p < 0.001, respectively). Slow thawing preserved higher SIgA concentrations and lysozyme activity than rapid thawing at 37 °C, but the difference was not significant.

Conclusions: The SIgA level in HM frozen at -18 °C for two months was stable after overnight thawing in the refrigerator (4 °C for 12 h) before warming to 37 °C compared with that in fresh milk. The thawing of HM in the refrigerator overnight (and then warming to 25 °C or 37 °C for 30 min) has the potential to preserve the SIgA concentration and lysozyme activity to a greater extent than heating immediately after removal from the freezer. Broader temperature ranges should be analyzed to determine the temperature that minimizes the losses in SIgA concentration and lysozyme activity in HM.

Keywords: Human milk; Immunoglobulin A; Lysozyme activity; Thawing rate; Warming temperature.

Fig. 1

Diagram of the study design. Lys lysozyme’ R25 rapid thawing at 25 °C’ R37 rapid thawing at 37 °C’ SIgA secretory immunoglobulin A’ ST slow thawing’ S25 slow thawing at 25 °C’ S37 slow thawing at 37 °C

Fig. 2

Comparison of the SIgA concentrations between fresh and frozen human milk samples. R25 rapid thawing at 25 °C, R37 rapid thawing at 37 °C, S25 slow thawing at 25 °C, S37 slow thawing at 37 °C, SIgA secretory immunoglobulin A. The data were analyzed by one-way analysis of variance with Tukey’s HSD (honest significance) pairwise comparisons and are presented as the means and 95% confidence intervals (CIs). * p < 0.05 and ** p < 0.001

Fig. 3

Comparison of lysozyme activities between fresh and frozen human milk samples. R25 rapid thawing at 25 °C, R37 rapid thawing at 37 °C, S25 slow thawing at 25 °C, S37 slow thawing at 37 °C. The data were analyzed using the Kruskal–Wallis test with Dwass-Steel-Critchlow-Fligner pairwise comparisons. The lysozyme activities are presented as the medians and percentiles (25^th, 75^th). * p < 0.05 and ** p < 0.001