Effects of apples and specific apple components on the cecal environment of conventional rats: role of apple pectin

Abstract

Background: Our study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota.

Results: Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum Bacteroidetes, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum Firmicutes, and specific species belonging to the Clostridium Cluster XIVa.Quantitative real-time PCR confirmed a lower amount of given Bacteroidetes species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of Clostridium coccoides (belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals.

Conclusions: Our findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and beta-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.

Figure 1

PCA analysis of samples from Experiment A. Principal Component Analysis of DGGE profiles of bacterial rRNA genes present in fecal samples from rat fed with control diet (green) or 10 g apples a day (red), respectively. A: Uninitiated animals. The amount of variability accounted for by Factor X is 25.0%, by Factor Y 16.2% and by Factor Z 13.6%. B: DMH initiated animals. The amount of variability accounted for by Factor X is 31.6%, by Factor Y 14.3% and by Factor Z 12.0%. Comparison of initiated and uninitiated animals by PCA revealed no grouping related to DMH initiation.

Figure 2

PCA analysis of samples from Experiment B. Principal Component Analysis of DGGE profiles of bacterial rRNA genes present in fecal samples from rat fed with control diet (red) or pectin diet (green), respectively. A: Pectin in diet constituted 3.3%. The amount of variability accounted for by Factor X is 25.5%, by Factor Y 19.6% and by Factor Z 13.8%. B: Pectin in diet constituted 0.33%. The amount of variability accounted for by Factor X is 36.4%, by Factor Y 22.1%, and by Factor Z 10.7%.

Figure 3

Cluster analysis of samples from Experiment C. Pearson correlation analysis of universal DGGE gel profiles from cecal content of rats fed with either control- or 7% pectin diet for four weeks. Bands indicated by arrows represents Anaeroplasma (1), Clostridium sp. (2), Clostridiales (3), Bacteroides sp. (4, 6 and 7), and Alistipes (5). Metric scale indicates degree of similarity in percent.

Figure 4

Quantitative PCR of samples from Experiment C. Relative amount of target gene in samples from animals in the control group (black), the pectin-fed group (white) and the apple-fed group (gray). Target genes encoded either 16S rRNA from Bacteroides spp. (a), Lactobacillus (b), Bifidobacterium (c), Clostridium coccoides (d) or the butyryl-coenzyme A CoA transferase. DNA amount in the control group was set to 100%. Error bars represent standard errors of the means. Asterisks indicate a significant difference from the control group; P < 0.05 (*) or P < 0.01 (**).