Phytochemical-induced mucin accumulation in the gastrointestinal lumen is independent of the microbiota

The mucus layer is critical to gastrointestinal health and ecology. Dietary phytochemicals are well documented to stimulate mucus production and secretion, but the underlying mechanism and effects on gut health are poorly understood. We fed germ-free and conventional mice diets containing approximately 0.4% of polyphenols per gram to determine if the phytochemical-induced accumulation of mucin in the gastrointestinal lumen is dependent on the microbiota. In addition, we assess how increased mucin shapes microbial communities in conventional mice. Germ-free mice receiving a pea (Pisum sativuum) seed coat proanthocyanidin-containing diet (PA) had greater levels of fecal mucin compared to the non-proanthocyanidin-containing (NPA) pea seed coat diet control (P < 0.05), confirming that fecal mucin accumulation is independent of the gut microbiota. Conventional mice fed the PA diet and a red osier dogwood (ROD; Cornus sericea) extract diet (DW) had higher mucin levels compared to a control diet without phytochemicals (P < 0.01 and P < 0.05, respectively). The increase in luminal mucin was associated with consistent increases in bacterial taxa belonging to Lachnospiraceae and [Clostridium] leptum species and a decrease in Romboutsia species. We conclude that phytochemicals have the ability to alter gut microbial ecology by increasing the amount of mucin in the gastrointestinal lumen.

162 Numbers assigned to ASVs reflect their total counts from highest to lowest count across samples.
163 Alpha diversity (Observed, Shannon, phylogenetic diversity (PD)) and beta diversity based on a 164 Bray-Curtis dissimilarity index were done with rarefied reads at a count of 8444.
165 Statistical analysis 166 Significance testing and graphing for body weights and fecal mucin were done in 221 14 compared to day 0, whereas the DW group maintained a similar diversity as at day 0 before 222 diet treatment (Fig 2d).

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The phytochemical diets drastically altered the colonic microbiota as determined by 224 differential expression of ASVs using DESeq2 compared to control (Fig 2e-f)

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In this study, we determined that the increase in fecal mucin in response to polyphenol- 296 showed no effect on ileal microbial alpha diversity (Shannon and Simpson) but a prebiotic effect 297 on Lactobacillus species was noted along with no change to growth performance (33). A study in 298 weaned pigs challenged with Escherichia coli k88+ found that 2% and 4% ROD extract diets 299 conferred beneficial effects on growth performance; however, microbial composition was not 300 assessed (16). Phylogenetic diversity in the DW group was maintained at day 14, which could be 301 explained by the increased abundance of Akkermansia municiphila, Parasutterella and 302 Turicibacter, which only appeared in this DW group at day 14 and were not detected in any 303 group at day 0. Although we did not detect these microbes in the sequencing data of Control and 304 PA groups at day 14, they may have been present below our detection limit for 16s rRNA 305 sequencing. The ROD extract effectively reduced the abundance of some species thereby 329 rich environments (37) and a potential marker of stability in the gut. The competitive advantage 330 gained by mucolytic bacteria may have altered the nutrient-rich niches in the gut that genera like 331 Romboutsia depend on for growth. The mucus layer supports microbial niches by directly 332 providing glycans for energy and indirectly through cross-feeding from one microbe to another 333 (5). Future research should focus on characterizing the bioactive compounds promoting luminal 334 mucin accumulation, as well as identifying the type and source of the accumulating mucins (1).
335 Further knowledge of these interactions will provide the foundational framework necessary to 336 understand how the mucus layer contributes to host-microbe stability and health.

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The production and maintenance of the mucus layer is a vital part of intestinal 339 homeostasis. It has become clear that host mucus provides a foundation of host derived glycans 340 that supports mutualism and commensalism among microbes in the gut. Understanding how 341 phytochemicals influence the viscoelasticity of the mucus layer will help to determine the best 342 therapeutic use of phytochemicals to promote health. This research provides insight into 343 establishing the mechanisms involved in the ability of mucus to stabilize gut ecology and control 344 microbial communities. Further studies are required to determine the specific phytochemical 345 compounds and structure that induce mucus secretion and/or disrupt mucin binding and 346 formation.