Mounting evidence supports the potential of dietary bioactives to reduce chronic disease risk. N-trans-caffeoyltyramine (NCT) and N-trans-feruloyltyramine (NFT) have been hypothesized to drive regulation of gut permeability, but these components have not yet been studied in the context of the human gut microbiome. This work examined whether purified NCT and NFT, or a hemp hull product containing NCT and NFT (Brightseed® Bio Gut Fiber™), can impact the gut microbiome using an in vitro fermentation assay. Representative human gut microbiomes were treated with Bio Gut Fiber™ or NCT and NFT and compared to starch and methylcellulose, as controls, in vitro. Stronger changes were exerted by Bio Gut Fiber™, NCT, and NFT. Communities treated with Bio Gut Fiber™ saw increased productivity and diversity. We found a dose-dependent effect of NCT and NFT on microbial communities. Here, we describe novel potential for hemp-derived bioactives to shape the gut microbiome.

Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. Here, we have characterized the protein composition and N -glycome of a yeast-synthesized and commercially available whey protein ingredient and compared this novel ingredient to whey protein isolate powder derived from bovine milk. Despite strong similarities in protein composition, we found that the N -glycome significantly differs between the two protein sources, reflecting the biosynthetic machinery of the production systems. Furthermore, the diversity of proteins found in yeast-synthesized whey protein were lower relative to bovine whey protein, despite both being predominantly β-lactoglobulin. Finally, to understand whether these differences in N- glycome profiles may affect the human gut microbiome, we compared these proteins in an in vitro fecal fermentation model. The two whey protein sources generated significant differences among three representative gut microbiomes in vitro , most likely due to differences in N -glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of synthesized proteins and how these differences impact the human gut microbiome. IMPORTANCE: Recent advances in food technology have led to the production of animal-free products from yeast that are traditionally derived from animals, such as milk proteins. These new processes raise important questions about the use of synthetic proteins as a replacement for traditionally sourced protein, especially in the context of the gut microbiome. Importantly, yeast produce N -glycans comprised primarily of mannose, while animals synthesize structurally and compositionally complex N -glycan structures. Given these differences, we characterized a new, yeast-derived whey protein ingredient and compared it to bovine whey protein. We found that yeast-derived whey protein differs in its impact on human gut microbiomes because of differences in N -glycan structures, despite similarity in protein composition. These findings raise important questions as to whether these differences in synthetic proteins lead to significant changes to the gut microbiome in vivo , and whether this may impact the utility of these novel ingredients.

Post‐translational modifications (PTMs) such as acetylation and phosphorylation have been implicated in contractile dysfunction and muscle wasting. Moreover, mounting evidence demonstrates that histone deacetylase (HDAC) inhibitors attenuate muscle wasting in animal models of disuse and muscular dystrophy. Historically, lysine acetylation was studied in the regulation of gene transcription, where for instance HDACs remove acetyl groups on lysine residues of histone proteins to suppress gene expression. However, recent evidence has shown that non‐histone proteins are also regulated by acetylation; functioning in protein stability, intracellular signaling, mitochondrial function and muscle contraction. Despite our current understanding for lysine acetylation and protein phosphorylation in muscle, how these PTMs cross‐talk to regulate muscle physiology remains unclear. In our attempt to understand acetylation‐phosphorylation cross‐talk, we examined the role for HDAC inhibitors on protein kinase C delta (PKCδ) and delta/theta (PKCδ/θ) phosphorylation. PKC is composed of multiple isoforms and its activation has been linked to muscle disease and dysfunction. Myotubes were treated with or without a pan‐HDAC inhibitor (trichostatin A; TSA) in the absence or presence of a pathological agonist (i.e. PMA). Phorbol 12‐myristate 13‐acetate (PMA) increased phosphorylation of PKCδ and PKCδ/θ. Interestingly, PMA‐induced PKCδ and PKCδ/θ phosphorylation was amplified in myotubes treated with the pan‐HDAC inhibitor TSA. Class selective HDAC inhibitors confirmed these findings and further demonstrated that inhibition of class I HDACs (HDAC1, 2, 3) was sufficient to increase PKCδ and PKCδ/θ phosphorylation. It should be noted that total PKCδ and PKCδ/θ protein expression increased with class I HDAC inhibition concomitant to the increase in protein phosphorylation. These data suggest that HDAC inhibition increases total PKC gene/protein expression that can then be phosphorylated. Finally, we report that the HDAC 1/2 selective inhibitor Romidepsin also increased total and phosphorylated PKCδ and PKCδ/θ in response to PMA. Genetic knockdown studies are currently underway to elucidate the specific HDAC that regulates PKC gene/protein expression, while chromatin immunoprecipitation (ChIP) is being used to determine HDAC binding to the PKC promoter. Lastly, elucidating a functional consequence for HDAC inhibitor‐mediated regulation of PKCδ and PKCδ/θ protein expression and phosphorylation in muscle could yield significant insight into muscle wasting and function. Support or Funding Information: This work is supported by the USDA NIFA (Hatch‐NEV00727, Hatch‐NEV00767), the Dennis Meiss & Janet Ralston Fund for Nutri‐epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459) and the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496) and NSF EPSCOR Track II (OIA‐1826801) to B.S.F.

Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. To address this, we have characterized the protein composition and N -glycome of a yeast-synthesized whey protein ingredient isolated from commercially available ice cream and compared this novel ingredient to whey protein powder isolate derived from bovine milk. We found that despite strong similarities in protein composition, the N -glycome significantly differs between these protein sources, reflecting the biosynthetic machinery of the production systems. Further, the composition profile and diversity of proteins found in the synthetic whey protein were lower relative to bovine whey protein, despite both being predominantly composed of β-lactoglobulin. Finally, to understand whether these differences in N- glycome profiles affected the human gut microbiome, we tested these proteins in an in vitro fecal fermentation model. We found that the two whey protein sources generated significant differences among three distinct microbial compositions, which we hypothesize is a product of differences in N -glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of these proteins, and how these differences impact the human gut microbiome.

Three novel endo-β-N-acetylglucosaminidases (AVUL01, BCAC01, and BFIN01) classified as members of the glucoside hydrolase (GH) family 18 were identified from human fecal samples and then cloned and characterized for their ability to hydrolyze two distinct classes of N-glycans. Endo-β-N-acetylglucosaminidases (ENGases) are known for the hydrolysis of chitin and the N,N′-diacetylchitobiose core of N-linked glycans, depending on the glycan architecture. N-glycans have shown bioactivity as substrates in the human gut microbiome for microbes that encode ENGases, thus demonstrating their ecological relevance in the gut. However, distinct types of N-glycan structures, for example, oligomannosidic or complex, have been shown to enrich different microbes within the human gut. Novel advances in food technology have commercialized animal-derived dietary proteins with oligomannosidic instead of traditionally complex N-glycans using precision fermentation. This indicates that there is an unmet need to identify the classes of N-glycans that gut-derived ENGases act upon to determine whether these novel proteins alter gut ecology. AVUL01, BCAC01, and BFIN01 all demonstrated activity on exclusively oligomannosidic N-glycans from RNase B and bovine lactoferrin; however, they failed to show activity on complex or α-1,3-core fucosylated high-mannose N-glycans derived from fetuin and horseradish peroxidase, respectively. These results suggest that α-1,3 core fucosylation and complex N-glycan architecture inhibit the activity of AVUL01, BCAC01, and BFIN01. Furthermore, BFIN01 performed significantly better than BCAC01, resulting in a greater amount of N-glycans, suggesting that certain ENGases may possess enhanced specificity and kinetics as an evolutionary strategy to compete for resources.

The N- glycome profiles purified from dietary bovine whey, egg white, pea, soy protein isolates and a recently commercialized animal-free whey is described. Purified glycoproteins resulting from centrifugation and ethanol precipitation of protein powder supplements were treated with peptide- N- glycosidase F (PNGase F) to release protein-bound N -glycans. Once released from the protein, N -glycans were labeled by procainamide labeling, purified via cotton-hydrophilic interaction liquid chromatography (HILIC), and analyzed using HILIC high performance liquid chromatography equipped with a fluorescence detector and a quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). A total of 33, 33, 10, and 10 N- glycan structures were identified from bovine whey, egg, soy, and pea glycoprotein isolates, respectively. The type of N- glycans per glycoprotein source were highly predictable, likely attributed to differences in biosynthetic glycosylation pathways. Mammalian glycoprotein sources favored a combination of complex and hybrid glycan configurations while the plant proteins were dominated by oligomannosidic N -glycans. Bovine whey glycoprotein isolate contained the most diverse N- glycans by monosaccharide composition as well as structure, while plant sources such as pea and soy glycoprotein isolates contained an overlap of oligomannosidic N- glycans. The results suggest N- glycan structure and composition is dependent on the host organism rather than protein sequence homology, likely driven by the differences in N- glycan biosynthetic pathways.