Abstract
Pasteurization of donated human milk preserves it for storage and makes it safe for feeding, but at the expense of its composition, nutritional values and functions. Here, we aimed to investigate the impact of Holder Pasteurization (HoP) and High Pressure Processing (HPP) methods on miRNA in human milk and to evaluate impact of these changes on miRNA functions. Milk samples obtained from women in 50th day of lactation (n = 3) were subjected either to HoP, HPP or remained unpasteurized as a control. Subsequently, miRNA was isolated from whole material and exosomal fraction and sequenced with Illumina NextSeq 500. Sequencing data were processed, read counts were mapped to miRNA and analyzed both quantitatively with DESeq2 and functionally with DIANA mirPath v.3. While HPP caused statistically insignificant decrease in number of miRNA reads compared to unprocessed material, HoP led to 82-fold decrease in whole material (p = 0.0288) and 302-fold decrease in exosomes (p = 0.0021) not leaving enough reads for further analysis. Changes in composition of miRNA fraction before and after HPP indicated uneven stability of individual miRNAs under high pressure conditions, with miR-30d-5p identified as relatively stable and miR-29 family as sensitive to HPP. Interestingly, about 2/3 of unprocessed milk miRNA content consists of only 10 distinct miRNAs with miR-148a-3p at the top. Functional analysis of most abundant human milk miRNAs showed their involvement in signaling pathways, cell communication, proliferation and metabolism that are obviously important in rapidly growing infants. Functions of miRNAs which suffered the greatest depletion during HPP were similar to roles of the majority of unprocessed human milk's miRNA, which indicates that those functions may be weakened although not completely lost. Our findings indicate that HPP is less detrimental to human milk miRNAs than HoP and should be considered in further research on recommended processing procedures for human milk banks.
Highlights
Breast milk is uniquely tailored for the human infant, both in terms of nutritional composition and in the non-nutritive bioactive factors that promote survival and healthy development [1]
Our findings indicate that High Pressure Processing (HPP) is less detrimental to human milk miRNAs than Holder Pasteurization (HoP) and should be considered in further research on recommended processing procedures for human milk banks
On average sequencing depth was slightly lower in samples subjected to HoP, but in all cases this was considered sufficient to warrant further analysis (Fig 1A, S1 Table)
Summary
Breast milk is uniquely tailored for the human infant, both in terms of nutritional composition and in the non-nutritive bioactive factors that promote survival and healthy development [1] It influences development and maturation of infant’s organs and tissues in many ways. The role of milk miRNAs is a subject of ongoing research [6] with two competing hypotheses: functional stating that they are capable of exerting regulatory impact on infants’ organism and nutritional claiming that they are a source of nutrition. The former hypothesis requires that miRNA are uptaken in the intestines rather than degraded by digestive fluids. Title et al in 2015 found no sign of miRNA uptake from ingested milk in mice [7], Baier et al [8] provided evidence that even amounts of miRNA absorbed from 0.25 l of cow’s milk are sufficient to alter human gene expression and Wang et al observed changes in bovine miRNA concentrations after ingestion of dietary products [9]
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