MicroRNAs as potential bioactive components present in cheese byproducts

Whey Protein Isolate (WPI), a byproduct of cheesemaking, is largely consumed by athletes due to its high protein content. Its counterpart, whey protein phospholipid concentrate (WPPC) is not used in human nutrition but has been suggested as a promising source of bioactive compounds. However, its composition is poorly documented, in particular, in its microRNA (miRNA) content. They are small non-coding RNAs regulate post-transcriptionally gene expression. A growing number of studies are showing that they can survive digestion and be taken up by human intestinal cells, potentially regulating gene expression in consumers. MiRNAs were detected in milk, cheese, and butter. The objective of this study was to ascertain the presence of miRNAs in WPPC and WPI and establish their miRNA profiles.

Four dairy companies provided the WPPC and WPI. RNAs were extracted using the miRNeasy Serum/Plasma Kit. Library construction, small RNA sequencing, and sequencing data analysis were performed using the VAHTS Small RNA Library Prep Kit for Illumina_v2. The annotation of the sequences was performed using the Bos taurus ARS-UCD2.0 genome and miRBase to identify known miRNAs. Nine miRNAs detected in milk were also quantified by RT-qPCR in triplicate, using a TaqMan® Advanced miRNA cDNA Synthesis kit. Predictions of the function of the top 20 most abundant miRNAs were performed using OmicsNet.

A comparison of WPPCs sequencing data revealed that they shared 338 known miRNAs out of approximatively 600 identified. Among the top 20 most abundant, 12 were in common with the most abundant miRNAs in milk. miR-148a was extremely abundant (76%) in WPPC. They were predicted, insilico, to influence pathways, like cell-cell junction, cytoskeleton organization, focal adhesion, involved in epithelial permeability, as well as pathways regulating inflammation, lipid metabolism, or vascular function. Due to the low yield of WPI extraction, we obtained sequences from only one sample showing 377 known miRNAs. Therefore, WPI miRNome must be considered with caution. However, RT-qPCR, requiring less miRNAs, detected miRNAs in all WPPC (n=4) and WPI (n=4) showing that certain miRNAs (miR-92a-3p) had the similar abundance in WPPC and WPI whereas others (miR-148a-3p, let-7a-5p and miR-21-5p) presented higher abundance in WPPC. Conversely, miR-375-3p seems to be more abundant in WPI.

In conclusion, this study confirms the presence of miRNAs in WPPC and WPI, with a higher miRNA content in WPPC than WPI.  This suggests that milk processing can result in varying losses of miRNAs depending on the treatment applied and the specific miRNAs in question. We determined for the first time the miRNome of WPPC, which present a very high abundance of miR-148a. In literature, these miRNAs are predicted to impact the cell functions of the consumers and therefore present food bioactives that can impact consumers’ health; this needs to be experimentally validated.