Cooked Broccoli Alters Cecal Microbiota and Impacts Microbial Metabolism of Glucoraphanin in Lean and Obese Mice

Abstract

Scope: Brassica vegetables contain unique compounds known as glucosinolates (GSLs), which, when hydrolyzed by plant or microbial myrosinase, form bioactive isothiocyanates (ITCs) that offer health benefits to the host. The present study evaluated the impact of cooked broccoli (broccoli myrosinase inactivated) consumption on cecal microbial metabolism of glucoraphanin (GRP) in lean and obese mice and characterized the changes in cecal microbiota following broccoli-containing diets.

Methods and results: Twenty lean and 20 diet-induced obese (DIO) mice were randomized to consume control or cooked broccoli supplemented diets for 7 days. Cooked broccoli consumption increased ex vivo microbial GRP hydrolysis by cecal contents collected from lean and obese mice, led to increased production of sulforaphane (SF), sulforaphane-cysteine (SF-CYS), total ITC, and colonic NAD(P)H: Quinone Oxidoreductase (NQO1) activity. Further investigation revealed increased abundance of health-promoting gut microbiota, including Lachnospiraceae NK4A136 group and Dubosiella newyorkensis, following broccoli-containing diets. The Peptococcaseae family, the Blautia genus, and an amplicon sequence variation (ASV) from the Oscillospiraceae family exhibited negative correlation with total ITC production.

Conclusion: These finding suggest that cooked broccoli consumption enhances microbial GRP hydrolysis to produce more bioactive ITCs and inform future strategies toward altering microbial GSL metabolism to promote gut health in both lean and obese individuals.

Keywords: broccoli; glucoraphanin; gut microbiome; microbial metabolites; mouse; sulforaphane.

FIGURE 1

Microbial metabolites of glucoraphanin. Concentrations of SF, SF‐CYS, SF‐NIT, and total ITC after incubation of glucoraphanin with mouse cecal content were quantified using LC‐MS (for SF, SF‐CYS, and SF‐NIT) and HPLC‐DAD (for total ITC) (n = 5 per treatment group). Concentrations lower than the LOD of the analytical method were labeled as ND. The LOD of SF, SF‐CYS, and SF‐NIT with LC‐MS are 0.18, 0.10, and 0.20 µM, respectively. The LOD of total ITC with LC‐DAD is 1.00 µM. Data are mean ± SEM. LOD, limit of detection; ND, not detected; SF, sulforaphane; SF‐CYS, sulforaphane‐cysteine conjugate; SF‐NIT, sulforaphane‐nitrile; total ITC, total isothiocyanates. Unpaired parametric t test was performed for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant; dotted lines represent the LOD.

FIGURE 2

Colonic NAD(P)H: quinone oxidoreductase (NQO1) activity (n = 5 per treatment group). Results are absorbance change at 440 nm/min/µg of protein. Data are mean ± SEM. Unpaired parametric t test was performed for statistical analysis. **p < 0.01.

FIGURE 3

Alpha‐ and beta‐diversity of cecal microbiota in HFCB, HFD, LFCB, and LFD fed mice (n = 9–10 per treatment group). (A) Alpha‐diversity, quantified by the Observed, Chao1, Shannon, and Simpson index; the boxes represent the interquartile range (IQR) of alpha‐diversity, with the horizontal line inside the box indicating the median. Whiskers extent to the smallest and largest values within 1.5 times the IQR, while points outside this range represent outliers. The difference between treatment groups was analyzed using the Kruskal–Wallis test with post hoc pairwise Wilcoxon test; (B) beta‐diversity (Bray–Curtis distances) calculated and visualized using principal coordinate analysis (PCoA). Permutational multivariate analysis of variance (MANOVA) was used to identify differences between intervention groups. HFCB, high‐fat diet with cooked broccoli; HFD, high‐fat diet; LFCB, low‐fat diet with cooked broccoli; LFD, low‐fat diet.

FIGURE 4

(A) Number of ASVs assigned to phylum, family, genus, and species level after PacBio sequence. (B–E) Cecal microbiota compositional changes in treatment groups (n = 9–10 per group) at the phylum level. Kruskal–Wallis test with post hoc Dunn's multiple comparison test was performed for identifying taxa change at the phylum level (Figure 4C–E). *p < 0.05. ASV, amplicon sequence variation; FB ratio: Firmicutes to Bacteroidota ratio; HFCB, high‐fat diet with cooked broccoli; HFD, high‐fat diet; LFCB, low‐fat diet with cooked broccoli; LFD, low‐fat diet.

FIGURE 5

Cecal microbiota compositional changes in treatment groups (n = 9–10 per group). (A) ASV abundance impacted by background diet (LFD vs. HFD), cooked broccoli supplementation (broccoli‐supplemented diets [LFCB + HFCB] vs. background diets [LFD + HFD]), and the interaction between the two factors (top 20% significant ASVs). Color gradients represent the direction and magnitude of the estimated effects on ASVs abundance, with blue indicating an increase and red indicating a decrease. The effects of background diets, cooked broccoli, and their interaction on the abundance of individual ASVs were analyzed using negative binomial generalized linear models (R, v4.2.1). (B, C) Comparison of abundance of individual ASV across four intervention groups. The boxes represent the interquartile range (IQR), with the horizontal line inside the box indicating the median abundance. Whiskers extent to the smallest and largest values within 1.5 times the IQR, while points outside this range represent outliers. Pairwise multilevel comparison using pairwise.adonis was performed (R, pairwiseAdonis, v0.4). ASV, amplicon sequence variation; CB, cooked broccoli; HFCB, high‐fat diet with cooked broccoli; HFD, high‐fat diet; LFCB, low‐fat diet with cooked broccoli; LFD, low‐fat diet. Asterisk (*) in panel A and letters in panels B–C denote statistically significant differences. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

Correlations between cecal microbiota and GSL microbial metabolites. Pearson correlations of microbial ASV, genera, and families with GSL microbial metabolite concentrations (cor.test function; R, stats, v4.2.1) including best‐fit line (geom_smooth function; ggplot; linear regression method) and standard error of best fit (shaded portions). Pearson's product‐moment correlation coefficient, denoted as “correlation”, and p value are indicated for each taxon‐metabolite pair. ASV, amplicon sequence variation; GSL, glucosinolate; SF, sulforaphane; SF‐CYS, sulforaphane‐cysteine; SF‐NIT, sulforaphane‐nitrile; Total ITC, total isothiocyanates.