Fibre fermentation and pig faecal microbiota composition are affected by the interaction between sugarcane fibre and (poly)phenols in vitro

Figures & data

Table 1. TPC and TEAC of raw samples and digested samples after in vitro digestion.

		In vitro digested sample			IB^a	CA^b
Analysis	Raw sample	supernatant	precipitate	Total (supernatant + precipitate)
TPC	(mg GAE)	(mg GAE)	(mg GAE)	(mg GAE)	(%)	(%)
	0.2g PRSE (29.78 ± 0.45)	9.95 ± 0.48	1.47 ± 0.23	11.42 ± 0.27	87.1 ± 2.3 a	12.9 ± 2.3 c
	0.27g SCFiber (0.94 ± 0.02)	0.14 ± 0.05	0.80 ± 0.22	0.94 ± 0.23	15.3 ± 4.2 c	84.7 ± 4.2 a
	0.5g PRSE + SCFiber (28.79 ± 0.17)	12.34 ± 0.85	7.64 ± 1.52	19.98 ± 1.77	61.9 ± 5.1 b	38.0 ± 5.1 b
TEAC	(mg TE)	(mg TE)	(mg TE)	(mg TE)	(%)	(%)
	0.2g PRSE (29.10 ± 0.11)	11.03 ± 2.84	2.09 ± 0.72	13.12 ± 3.56	84.1 ± 1.5 a	15.9 ± 1.5 b
	0.27g SCFiber (0.61 ± 0.02)	0.13 ± 0.02	0.56 ± 0.07	0.69 ± 0.07	18.5 ± 2.9 b	81.5 ± 2.9 a
	0.5g PRSE + SCFiber (31.63 ± 0.94)	27.65 ± 3.99	8.51 ± 1.91	36.16 ± 5.90	76.5 ± 5.7 a	23.5 ± 5.7 b

Samples were subjected to the in vitro digestion phases. The pre-digested samples and the in vitro digested samples were collected and their TPC and TEAC were measured. Results are expressed as means ± SD (n = 3). Statistical significance (p < 0.05) of the IB^a and CA^b between samples is indicated by different letters within each column.

^aIntestinal bio-accessibility (IB) (%) = A/(A + B) ×100 and ^bColonic availability (CA) (%) = B/(A + B) ×100, where: A = TPC or TEAC of in vitro digesta supernatant after in vitro digestion; B = TPC or TEAC of in vitro digesta precipitate after in vitro digestion.

Figure 1. Alterations in the faecal bacterial community in different treatments. (a) Principal coordinates analysis (PCoA) plot showing the nonparametric microbial interdependence test (NMIT) distance at genus level to investigate the differences in microbiota profile between treatments using the time-based microbiota composition data. (b) Taxonomic cladogram showing the significantly discriminative taxa associated with different treatments by linear discriminant analysis (LDA) effect size (LEfSe) analysis (Kruskal–Wallis sum-rank test α < 0.05; LDA score > 2.00). The significantly discriminative taxa associated with each treatment is represented by different colour shading and the non-significant bacterial taxa is represented by the yellow dots.

Table 2. Firmicutes/Bacteroidetes ratio of different samples after 24 h of in vitro colonic fermentation.

Sample	F/B ratio^a
Blank	10.8 ± 2.1 a
PRSE	8.8 ± 0.3 a
SCFiber	4.7 ± 1.1 b
PRSE + SCFiber	4.2 ± 1.5 b

Samples were subjected to the digestion phases and pre-digested insoluble materials from the small intestinal digestion stage were carried forward to the colonic fermentation stage. Firmicutes/Bacteroidetes ratio (F/B ratio) of blank, PRSE, SCFiber, and PRSE + SCFiber at the end of 24 h in vitro colonic fermentation. Results are expressed as means ± SD (n = 3). Different letters in each row indicate significant differences at p < 0.05 compared between different samples by Tukey’s HSD test.

^aF/B ratio = Firmicutes/Bacteroidetes ratio.

Figure 2. Changes in the relative abundances of the faecal microbial community in different treatments during the in vitro colonic fermentation. Relative abundances plot of the major bacterial genera (relative abundance > 0.5%) of blank, PRSE, SCFiber, and PRSE + SCFiber resulting from independent fermentation of each treatment at the 0, 3, 6, 12, and 24 h of in vitro colonic fermentation. Genera grouped as “Other” were those with relative abundance < 0.5%.

Figure 3. Concentration of short chain fatty acids produced throughout the in vitro colonic fermentation. (a) Total SCFA, (b) acetic acid, (c) propionic acid, (d) butyric acid, (e) iso-butyric acid, (f) valeric acid and (g) iso-valeric acid content (mmol/L fermenta) of blank, PRSE, SCFiber, and PRSE + SCFiber at the 0, 3, 6, 12, and 24 h of in vitro colonic fermentation. Each treatment was subjected to in vitro digestion and the digested precipitate was proceeded to in vitro colonic fermentation. Statistical significance (p < 0.05) of the results between different treatments is indicated by different letters in each plot and results are expressed as means ± SD (n = 3).

Figure 4. Correlations between specific bacterial genera and SCFA in different treatments. Spearman’s correlation heatmap of (a) blank, (b) PRSE, (c) SCFiber, and (d) PRSE + SCFiber showing the relationship between the changes in the relative abundances of major bacterial genera (relative abundance > 0.5%) and the changes in SCFA contents in different treatments. Positive correlations are indicated by green colours (r values > 0.6, FDR adjusted p < 0.05), while negative correlations are represented by pink colours (r values < –0.6, FDR adjusted p < 0.05). Other correlation coefficients are indicated by the colour scheme. Statistically significant correlations are marked with an asterisk ‘*’ (FDR adjusted p < 0.05).

Table 3. Predicted metabolic pathways using PICRUSt2 analysis related to the significantly correlated pairs of bacterial genera and SCFAs from Spearman’s correlation result.

Treatment	SCFAs	Pathway ID	Pathway description	^aRelative function abundance (%)
				Lactobacillus
Blank	acetate	ARG + POLYAMINE-SYN	superpathway of arginine and polyamine biosynthesis	0.027 ± 0.034	–
		ARGSYN-PWY	L-arginine biosynthesis I (via L-ornithine)	0.159 ± 0.197	–
		PWY0-1261	anhydromuropeptides recycling I	0.032 ± 0.047	–
		PWY-2941	L-lysine biosynthesis II	0.202 ± 0.217	–
		PWY-5088	L-glutamate degradation VIII (to propanoate)	0.003 ± 0.003	–
		PWY-5154	L-arginine biosynthesis III (via N-acetyl-L-citrulline)	0.021 ± 0.027	–
		PWY-5345	superpathway of L-methionine biosynthesis (by sulfhydrylation)	0.015 ± 0.019	–
		SULPHATE-CYS-PWY	superpathway of sulphate assimilation and cysteine biosynthesis	0.014 ± 0.018	–
	propionate	P108-PWY	pyruvate fermentation to propanoate I	0.022 ± 0.022	–
		PWY-5088	L-glutamate degradation VIII (to propanoate)	0.003 ± 0.003	–
		PWY-7013	L-1,2-propanediol degradation	0.040 ± 0.070	–
		THREOCAT-PWY	superpathway of L-threonine metabolism	0.005 ± 0.007	–
				Dorea
	butyrate	CENTFERM-PWY	pyruvate fermentation to butanoate	0.031 ± 0.030
		P163-PWY	L-lysine fermentation to acetate and butanoate	0.008 ± 0.005
		PWY-6590	acetobutylicum acidogenic fermentation	0.022 ± 0.018
				Terrisporobacter	Sarcina
PRSE	propionate	P108-PWY	pyruvate fermentation to propanoate I	0.026 ± 0.021	0.035 ± 0.027
		PWY-5088	L-glutamate degradation VIII (to propanoate)	0.003 ± 0.004	0.008 ± 0.006
		PWY-7013	(S)-propane-1,2-diol degradation	0.006 ± 0.006	0.015 ± 0.015
		THREOCAT-PWY	superpathway of L-threonine metabolism	0.005 ± 0.006	0.012 ± 0.014
				Lactobacillus	Solobacterium
	butyrate	CENTFERM-PWY	pyruvate fermentation to butanoate	0.018 ± 0.021	0.025 ± 0.030
		P162-PWY	L-glutamate degradation V (via hydroxyglutarate)	0.007 ± 0.006	0.011 ± 0.002ta
		P163-PWY	L-lysine fermentation to acetate and butanoate	0.025 ± 0.022	0.015 ± 0.013
		PWY-6590	acetobutylicum acidogenic fermentation	0.029 ± 0.033	0.023 ± 0.023
				Oscillospira	Rikenellaceae RC9 gut group
PRSE + SCFiber	propionate	P108-PWY	pyruvate fermentation to propanoate I	0.010 ± 0.010	0.093 ± 0.141
		PWY-5088	L-glutamate degradation VIII (to propanoate)	0.001 ± 0.001	0.007 ± 0.007
		PWY-7013	(S)-propane-1,2-diol degradation	0.004 ± 0.002	–
		THREOCAT-PWY	superpathway of L-threonine metabolism	0.003 ± 0.002	–
				Ruminococcaceae UCG-002
	butyrate	CENTFERM-PWY	pyruvate fermentation to butanoate	0.062 ± 0.055
		P162-PWY	L-glutamate degradation V (via hydroxyglutarate)	0.030 ± 0.021
		P163-PWY	L-lysine fermentation to acetate and butanoate	0.022 ± 0.017
		PWY-5677	succinate fermentation to butanoate	0.079 ± 0.050
		PWY-6590	acetobutylicum acidogenic fermentation	0.032 ± 0.027

Only functional compositions of the OTUs where taxonomic annotation matched the bacterial genera that were significantly correlated with individual SCFAs were reported.

^aRelative function abundance (%) represents the percentage of contribution of individual taxon to the overall pathway abundance. It was calculated by averaging the relative function abundance of all samples in each of the treatment groups (i.e. 0, 3, 6, 12 and 24 h in blank, PRSE, SCFiber, and PRSE + SCFiber, respectively.