Effect of the Various Steps in the Processing of Human Milk in the Concentrations of IgA,IgM,and Lactoferrin

Introduction

The Human Milk Banks (HMB) are specialized hospital services that are in charge of promoting, protecting, and supporting human lactation. These services are responsible for collecting, processing, and storing donated human milk and providing it to premature newborn infants who have a very low birth weight. ¹ Human milk immune components are unique and are absent in milk formula and cow's milk. Some of these components are immunoglobulins, of which IgA and IgM are the most abundant. ² Lactoferrin is found in high concentrations, and although it is not considered an immune protein, it helps newborns fight against infectious diseases. This protein binds iron in human milk, a required ion for bacterial metabolism. ³

The objective of human milk processing at the HMB is to eliminate any possible pathogen present in the milk before its administration to infants. ¹ Donated human milk, obtained from mothers, is usually subjected to freezing temperatures for storage, then thawed and pasteurized at 62.5°C for 30 min, and finally frozen again before it is provided to infants. This process, however, has been described to reduce the immunological and nutritional components of human milk.^4,5 The reduction of the biological activity of immune proteins of human milk (immunoglobulins and lactoferrin, among other proteins) has been attributed to the pasteurization process. ⁶ A reduction of IgA levels from 28.9% to 63.9%, of IgM up to 100%, and of lactoferrin from 35.3% to 78.6% also occurred.^2,4,7,8 Human milk during freezing and thawing, and the time duration that human milk remains frozen also affect immunoglobin levels.^9,10 The objective of this study was to measure the concentrations of IgA, IgM, and lactoferrin sequentially in donor milk at three critical points in time during the processing procedure.

Materials and Methods

The study was performed at the HMB at the Hospital Nacional Pedro de Bethancourt, Antigua Guatemala, from October 2013 through July 2014.

Fifty human milk samples were collected with consent from donors at the HMB. A volume of 150 mL was obtained by using a mechanical pump. All milk containers were identified and processed according to the HMB Procedure Manual. ¹¹ Three critical points of time were selected in the processing sequence for study (Freezing/thawing I, Pasteurization, and Freezing/thawing II). An aliquot of 5 mL was obtained at the time of milk collection (baseline) and after each of the three critical points of time. Each sample was transported at 4°C to the laboratory where all the samples were analyzed. Samples were centrifuged at 3,500 rpm and 4°C for 10 min to obtain human milk whey. IgA and IgM concentrations were immediately measured. Whey samples were stored at −20°C until testing for lactoferrin. IgA and IgM were measured by using a turbidimetric assay. Briefly, a goat IgG anti-human IgA or anti-human IgM was added to the milk whey and incubated for 15 min at 37°C. Optical density was read by using an A25 photometric system (Byosystem, S. A.), and immunoglobulin levels were calculated by comparing OD sample readings with the OD of five standards of known IgA or IgM concentrations. Lactoferrin was determined by using a commercially available ELISA test (Calbiochem™). Whey samples were diluted to 1:500,000 (expected lactoferrin concentration from 20 mg/dL to 1,000 mg/dL), and 100 μL was added to the microwell plate. Lactoferrin standard, dilution buffer, anti-lactoferrin biotinylated conjugate, avidin-peroxidase, and substrate were used according to the manufacturer's instructions. IgA, IgM, and lactoferrin concentrations were reported in mg/dL.

Results

Milk samples from 50 different donors were analyzed. Grubb's test for outliers yielded a final sample size of 43 for IgA, 40 for IgM, and 32 for lactoferrin. IgA, IgM, and lactoferrin mean concentrations obtained during the whole process had a significant reduction (p < 0.001). Levels from the pre-pasteurization freezing/thawing procedure were compared with the baseline by using the Fisher LSD test, and no significant difference was established for either IgA (p = 0.160) or lactoferrin (p = 0.345), but for IgM there was a significant reduction (p = 0.016). Reduction of biological activity from freezing/thawing I and pasteurization was significant for IgA and lactoferrin (p < 0.001), but not for IgM (p = 0.124). Mean concentrations were compared from pasteurization and freezing/thawing II, and significant difference was observed for IgA (p < 0.001) but not for IgM (p = 0.280) and lactoferrin (0.813) (Table 1).

Discussion

Concentrations of IgA, IgM, and lactoferrin in human milk samples were determined during processing the milk donations. Baseline mean concentrations of IgA, IgM, and lactoferrin were comparable to the previously reported values.^{2,4,7,8,12–17} The effect of the entire sequence of processing on the concentrations of IgA, IgM, and lactoferrin was found to be a significant reduction in all three components (p < 0.001). This reduction effect was less for IgA (30.0%), than IgM (36.0%) and lactoferrin (70.0%). The partial eta square (ηp²) for the three proteins was above 0.8, reflecting a strong effect on the whole process.

Pasteurization was confirmed as the most influential processing phase in protein destruction for IgA and lactoferrin (p < 0.001). In this process, previous reports identified the temperature used as the determining factor in the reduction of biological activity.^2,4,6,8,18 It has also been suggested that protein aggregation can explain lactoferrin reduction when determined by immune methods. ¹⁹ IgM was the protein least affected by pasteurization (17.4% reduction), followed by IgA (17.6% reduction), and lactoferrin was the protein that was most affected (66.8% reduction). The rates of reduction for IgA and lactoferrin were similar to those reported in the literature for 62.5°C. The mean concentration of IgM after pasteurization was 27.1 mg/dL (SD: 7.1 mg/dL), in contrast with previous studies that reported the total destruction of IgM after pasteurization.^3,20 Both freezing/thawing critical points have a less important effect in reducing the concentration of lactoferrin (p > 0.05).^21,22 IgA was significantly reduced by the second process (p < 0.001), and IgM concentration suffered a significant reduction in the first process (p = 0.016). This effect on IgM has not previously been reported. Lactoferrin concentration between pasteurization and freezing/thawing II showed an increase from 77.4 mg/dL (SD: 62.1 mg/dL) to 79.2 mg/dL (SD: 60.2 mg/dL), which was a 2.3% increase. This variation, although it was not statistically significant, can be explained partially because the method that was used required a high dilution of milk samples introducing a large technical error ²³ and the intrinsic variability of the samples that was from different donors.

Conclusion

The study of the effect of the different procedures of the handling of donor milk in the HMB demonstrated a significant reduction of biological activity of the immune components, with the pasteurization procedure having the most major effect.