Lauren D. Wu; Angela Zivkovic; Sarah Totten; L. Renee Ruhaak; Carlito B. Lebrilla

University of California, Davis, CA

Novel Aspect
The stability of oligosaccharides in human breast milk during sample handling and storage is assessed for the first time.

Human milk oligosaccharides (HMOs) are one of the more abundant bioactives in human milk. They are now known to influence the development of healthy bacteria in the infant’s gut. Recently, our group has developed a TOF-MS based analytical method, including an extensive library, for the in-depth characterization of a mother’s HMO profile. Using this method, we have shown variations in HMO composition between different mothers, but limited effort has been made to assess sample stability during storage and sample handling. Therefore, we here assess the effects of long-term storage (>1 year) and multiple freeze-thaw cycles on the HMO profile of a donor milk sample.

Fresh breast milk (BM) was obtained from a single donor mother and was aliquoted and stored at -80°C. The HMOs from one aliquot were extracted immediately (BM Fresh), while another was stored for 1 year (BM Storage). BM Fresh was processed under consecutive freeze-thaw cycles in the same day it was received, with HMO separation and purification following each thaw cycle. HMOs were analyzed using nano-LC/MS and were stored for one year to observe the stability of purified compounds. BM Storage was processed under the same conditions and analyzed alongside the HMOs purified one year earlier. Upon data processing, absolute abundances were compared between samples analyzed within the same instrumental run, and relative abundances were assessed between runs.

Preliminary Results/Abstract

Using the obtained three data sets (BM fresh, BM Fresh at year 1, and BM Storage), we were able to observe oligosaccharide stability after freeze-thaw cycles, purified HMO stability, and long-term storage including its effect on freeze-thaw cycles. We first focused on the effects of unstable temperature conditions, where we compared a fresh milk sample to multiple freeze-thaw cycles using absolute abundance in ion counts and percent relative abundances. There was a significant difference in absolute abundance of total sialylated compounds in two of the cycles, but no distinct pattern. The purified HMOs were saved to test the stability of purified compounds after one year. There were no significant differences between each freeze-thaw cycle.

We then tested storage stability by comparing the HMOs from a fresh milk sample to the same milk that was stored frozen. These samples were analyzed one year apart using the same instrument parameters. Because of the interday instrument variation, we only assessed relative abundances. Fucosylation and sialylation in purified HMOs and extracted HMOs from frozen samples were more abundant than HMOs extracted from fresh milk, while non-fucosylated neutrals were lower in abundance.

To observe how storage affects the compound stability during freeze-thaw cycles, we extracted HMOs from frozen milk samples and processed them in the same conditions. There was no distinct trend of HMOs increasing or decreasing in relative or absolute abundances between the first freeze-thaw cycle to each subsequent set. The compounds in this batch seemed to remain relatively stable even after one year of storage.

Although there is some variation in sialylation during freeze-thaw cycles, HMOs do not exhibit a distinct pattern where oligosaccharides can be assumed to be degrading. While other biomolecules are known to change after unstable sample handling and storage conditions, these factors are insignificant to HMO analysis.