Article Cite This: J. Agric. Food Chem. 2019, 67, 7755−7764
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Utilization of Complex Pectic Polysaccharides from New Zealand Plants (Tetragonia tetragonioides and Corynocarpus laevigatus) by Gut Bacteroides Species Manuela Centanni,‡ Susan M. Carnachan,† Tracey J. Bell,† Alison M. Daines,† Simon F. R. Hinkley,† Gerald W. Tannock,‡,§,∥ and Ian M. Sims*,†,∥ †
Ferrier Research Institute, Victoria University of Wellington, 69 Gracefield Road, Lower Hutt 5040, New Zealand Department of Microbiology and Immunology and ∥Microbiome Otago, Department of Microbiology and Immunology, University of Otago, Post Office Box 56, Dunedin 9054, New Zealand § Riddet Institute Centre of Research Excellence, Palmerston North 4442, New Zealand
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‡
S Supporting Information *
ABSTRACT: Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries ( Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostly homogalacturonan (64.4%) and rhamnogalacturonan I (5.8%), with side chains of arabinan (8.1%), galactan (2.2%), and type II arabinogalactan (7.1%); karaka berry polysaccharides comprised homogalacturonan (21.8%) and rhamnogalacturonan I (10.0%), with greater proportions of side chains (arabinan, 15.6%; galactan, 23.8%; and type II arabinogalactan, 19.3%). Screening of gut commensal Bacteroides showed that six were able to grow on the NZ spinach extract, while five were able to grow on the karaka berry extract. Analysis of the polysaccharides remaining after fermentation, by size-exclusion chromatography and constituent sugar analysis, showed that the Bacteroides species that grew on these two substrates showed preferences for the different pectic polysaccharide types. Our data suggest that, to completely degrade and utilize the complex pectin structures found in plants, members of Bacteroides and other bowel bacteria work as metabolic consortia. KEYWORDS: NZ spinach, karaka berry, pectic polysaccharides, Bacteroides, gut microbiota
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INTRODUCTION The majority of dietary fiber originates from the cell walls of fruit, vegetables, and grains in the human diet.1 The polysaccharides (pectic polysaccharides, hemicelluloses, and cellulose) that make up these plant cell walls pass through the small intestine and reach the colon largely unaltered, where they are completely or partially fermented by the trillions of bacterial cells (the gut microbiota) that inhabit the colon. As demonstrated by the epidemiological studies conducted by Burkitt, Cleave, Campbell, Painter, Trowell, and Walker in Africa decades ago (reviewed by Cummings and Engineer),2 dietary fiber is clearly important in regulating intestinal transit time and influences the prevalence of some non-communicable diseases of humans (bowel transit/constipation, diverticulosis, and bowel cancer).3−6 Much more research needs to be directed to solving the question of how the gut microbiota “works” to degrade and ferment the diversity of dietary fibers present in food.7 Then, dietary interventions could provide rational and reliable remedies for gut diseases and conditions where gut dysbiosis may have an etiological role.8 Pectic polysaccharides are a highly diverse family of acidic polysaccharides representing the major “soluble” fiber in many fruits and vegetables. They are comprised of backbones of homogalacturonan (HG), a linear polymer of →4]-α-D-GalpA[1→, and rhamnogalacturonan I (RG-I), which is comprised of a repeating disaccharide of →4]-α-D-GalpA-[1→2]-α-L-Rhap[1→.9 The RG-I backbone is branched at the rhamnosyl © 2019 American Chemical Society
residues with side chains of arabinan, galactan, or arabinogalactan (types I and II). HG and RG-I, together with the various neutral side chains, make up the majority of pectic polysaccharides. Rhamnogalacturonan II (RG-II) is a lowabundance pectic polysaccharide that accumulates in many processed foods as a result of its resistance to degradation.10−12 This structurally complex molecule is comprised of a branched HG backbone with conserved side chains containing a variety of sugars and glycosyl linkage types.9 Members of the human gut microbiota belonging to the genus Bacteroides are of particular interest with regards to the hydrolysis and fermentation of pectic polysaccharides, although certain Firmicutes also show some capacity to utilize pectin.13,14 The genomes of the Bacteroides species encode a wide range of glycosyl hydrolases and polysaccharide lyases.15 Two recent papers have studied in detail the enzyme pathways involved in the degradation of pectic polysaccharides by the Bacteroides species.16,17 These and other reports18−21 used purified pectic polysaccharide components as substrates. Pectic polysaccharides with varying degrees of methylesterification, including homogalacturonan, galactan, arabinan, and arabinogalactans, are available commercially, while RG-I has been Received: Revised: Accepted: Published: 7755
April 17, 2019 June 18, 2019 June 19, 2019 June 19, 2019 DOI: 10.1021/acs.jafc.9b02429 J. Agric. Food Chem. 2019, 67, 7755−7764
Article
Journal of Agricultural and Food Chemistry
lyophilized fractions were resuspended in water, filtered under vacuum to remove any remaining insoluble material, and lyophilized. Bacteroides Growth Assay on Pectic Polysaccharide Substrates. The growth of type strains of 15 Bacteroides species commonly found in the human gut microbiota (>90% of humans;30,31 Table S1 of the Supporting Information) on the different pectic polysaccharide substrates was determined using the procedure as described previously.32 Briefly, cultures of the different bacterial strains were grown under anaerobic conditions for 18 h at 37 °C in the appropriate medium following DSMZ or ATCC protocols. To assess their ability to utilize different substrates, pure cultures of each species were used to inoculate (1%, v/v) basal medium32,33 containing 2 g L−1 of the specific pectic polysaccharide. Three technical replicates and two biological replicates were carried out for each species studied. Optical density (A600) was measured after 24 and 48 h of anaerobic incubation at 37 °C. Spent culture supernatants were collected after centrifugation of samples at 14500g for 5 min and stored at −80 °C for subsequent carbohydrate analysis. Unpaired t tests were performed to compare the optical densities of bacterial cultures grown in the presence and absence of each carbohydrate substrate, using GraphPad Prism, version 7.0b (GraphPad Software, La Jolla, CA, U.S.A.; www.graphpad.com), considering a p value of
