Soluble xyloglucan generates bigger bacterial community shifts than pectic polymers during in vitro fecal fermentation

Xyloglucans and pectic polymers can be obtained from a variety of plants ubiquitous in the human diet, however, their fermentability in the colon and consequent nutritional benefits are poorly understood.

Carbohydrate Polymers 206 (2019) 389–395 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Soluble xyloglucan generates bigger bacterial community shifts than pectic polymers during in vitro fecal fermentation T Thaisa Moro Cantu-Junglesa,b, Geórgia Erdmman Nascimentoa, Xiaowei Zhangb, ⁎ ⁎⁎ Marcello Iacominia, Lucimara M.C Cordeiroa, , Bruce R Hamakerb, a b Departamento de Bioquímica e Biologia Molecular, Universidade Federal Paraná, Curitiba, Brazil Whistler Center for Carbohydrate Research and Department of Food Science, Purdue University, West Lafayette, USA A R T I C LE I N FO A B S T R A C T Keywords: In vitro fecal fermentation Pectin Xyloglucan Short chain fatty acids Prebiotics Xyloglucans and pectic polymers can be obtained from a variety of plants ubiquitous in the human diet, however, their fermentability in the colon and consequent nutritional benefits are poorly understood Here, we evaluated metabolite profiles and bacterial shifts during in vitro fecal fermentations of two isolated pectic polymers and a xyloglucan Depending on their chemical structure, pectic polymers were more acetogenic or propiogenic Xyloglucan fermentation also resulted in elevated propionate if compared to FOS Bacteroides plebeius, B uniformis, Parabacteroides distasonis and bacterial groups such as Blautia, Lachnospira, Clostridiales and Lachnospiraceae, presented distinct abundances on each dietary fiber ferment PCA and heat map analysis showed that major microbiota shifts occurred during xyloglucan fermentation, but not pectin fermentation These data suggest that uncommon carbohydrate structures (i.e isolated, soluble xyloglucan) in the diet hold the potential to generate larger shifts in microbiota communities than commonly consumed fibers (i.e pectins) Introduction Dietary fiber is the part of the plant material resistant to enzymatic digestion in the small intestine and, thus, arrives intact in the colon where may be fermented by resident bacteria (Codex Alimentarius Commission, 2009) Saccharification and fermentation of some dietary fibers in the large intestine result in the generation of beneficial metabolites such as short chain fatty acids (mainly acetate, butyrate and propionate) and/or lead to bacterial shifts that are of biological significance to the human health (den Besten et al., 2013; Holscher, 2017) It was proposed that depending on specific structural features of a dietary fiber, a distinct fermentation profile would be observed (Hamaker & Tuncil, 2014; Holscher, 2017) Dietary fiber composition in higher plants varies among different plant types and cell types within individual plants In general, most dicotyledonous and non-graminaceous monocotyledons species possess xyloglucans as the predominant hemicellulose These species are also relatively rich in pectins, which make up ∼35% of cell wall dry mass (Carpita & Gibeaut, 1993; OchoaVillarreal, Aispuro-Hernández, Vargas-Arispuro, & Ángel MartínezTéllez, 2012; Willats, Knox, & Mikkelsen, 2006) Xyloglucans are strongly associated with cellulose and thus ⁎ entrapped in the insoluble cell wall matrix When isolated, however, these polymers became water soluble This soluble form is hardly found in the intact plant cell wall and consequently rarely consumed in a regular diet Xyloglucans have a (1→4)-linked β-D-Glcp backbone with α-D-Xylp substituents attached at O-6 that may be further substituted at O-2 by β-D-Galp units (Buckeridge, 2010) Besides that, a variety of sugars, such as fucose, arabinose and galacturonic acid can be found linked to the α-D-Xylp residues and the β-D-Galp units may be Oacetylated at different positions (Hsieh & Harris, 2009; Kiefer, York, Darvill, & Albersheim, 1989) Pectic polymers are defined as a class of polymers rich in galacturonic acid (GalA) and are usually water soluble Three major pectic polymers are recognized: homogalacturonans (HG), Rhamnogalacturonans I (RGI) and Rhamnogalacturonans II Homogalacturonan is a linear polymer consisting of (1→4)-linked α-DGalpA, whilst rhamnogalacturonan I consists of the repeating disaccharide [→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→] to which a variety of different glycan chains (arabinan, galactan and/or arabinogalactans) are attached to the Rha residues Finally, rhamnogalacturonan II has a backbone of HG rather than RG, with complex side chains attached to the to the GalpA residues (Willats et al., 2006) Corresponding author at: CP 19.046, CEP 81.531-980 Curitiba, PR, Brazil Corresponding author at: 745 Agriculture Mall Drive, West Lafayette, IN 47907, USA E-mail addresses: lucimaramcc@ufpr.br (L.M.C Cordeiro), hmakerb@purdue.edu (B.R Hamaker) ⁎⁎ https://doi.org/10.1016/j.carbpol.2018.11.011 Received 29 May 2018; Received in revised form 22 October 2018; Accepted November 2018 Available online 10 November 2018 0144-8617/ © 2018 Elsevier Ltd All rights reserved Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al containing 50 mM 4-methyl-valeric acid (used as an internal standard for SCFA analysis, No 277827- G, Sigma-Aldrich Inc., St Louis, Mo., USA), meta-phosphoric acid (5%) and copper sulfate (1.56 mg/mL); mixed and centrifuged at 13,000 rpm for 10 An aliquot (0.2 μL) was injected into a GC-FID 7890 A (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a fused silica capillary column (NukolTM, Supelco No 40369-03 A, Bellefonte, Pa., USA) The initial oven temperature was held at 50 °C for min, ramped to 70 °C at a rate of 10 °C/min, to 85 °C at a rate of °C/min, to 110 °C at a rate of °C/ min, to 290 °C at a rate of 30 °C/min, and finally held at 290 °C for Helium was used as a carrier gas at a constant flow rate of mL/ through the column Quantification was performed based on relative peak area of each short chain fatty acid in a fatty acid external standard (Volatile Free Acid Mix, 10 mM, 46975-U, Supelco), adjusting the quantity of each compound based on that of the internal standard Although ubiquitous in plants, there are no studies in the literature comparing the gut fermentability of pectins vs xyloglucans, and in forms commonly consumed (soluble pectins) vs uncommon (soluble xyloglucans) Thus, we aimed to investigate the fermentation profile by the human gut microbiota of previously isolated and characterized pectins and a xyloglucan and investigate how gut commensal bacteria differently respond to each polymer type Materials and methods 2.1 Dietary fibers Two pectic polymers previously obtained and characterized by Cantu-Jungles et al (2014) were utilized: a HG together with a RGI highly branched by type I arabinogalactans (HG + AGI) and the isolated RGI highly branched by type I arabinogalactans (AGI) Fraction HG + AGI had a monosaccharide composition of Ara: Gal: Glc:GalA in a 23:21:2:54 ratio The relative richness of HG versus AGI in HG + AGI calculated based on the ratio of uronic acid/(Ara + Gal) was of 1.3 and 79% of the uronic acids in the homogalacturonan were methyl-esterified Fraction AGI was composed by Ara: Gal: Rha: GalA in a ratio of 47.8:31.5:10.7:10 (Cantu-Jungles et al., 2014) The xyloglucan was obtained from tucumã pulp as previously described (Cantu-Jungles, Iacomini, Cipriani, & Cordeiro, 2017) It was mainly composed by Fuc:Xyl:Gal:Glc in a ratio of 6.2:19.4: 12: 62.4 (Cantu-Jungles, Iacomini et al., 2017) Regarding the monosaccharide composition reported above for all fractions, the uronic acid content is reported in weight %, while neutral sugars are reported in molar amount Fructooligosaccharides (FOS - No F8052, Sigma-Aldrich Inc., St.Louis, Mo., USA) was used as a positive control 2.3 Bacterial shifts during the in vitro fecal fermentation Metagenomic DNA was extracted from mL of fecal ferments using FastDNA SPIN Kit for Feces (MP Biomedicals, Laboratories, Carlsbad, CA) according to the manufacturer's instructions DNA integrity was determined using a 1% agarose gel with ethidium bromide, and DNA concentrations were verified using the Nanodrop ND-1000 (Nanodrop Technology) DNA samples were stored at −20 C until sequencing Sequencing was performed at the DNA Services Facility at the University of Illinois, Chicago DNA was PCR amplified with primers 515 F and 806R (Caporaso et al., 2012), targeting the V4 variable region of bacterial and archaeal small subunit (SSU) ribosomal RNA (rRNA) gene Metagenomic sequencing was performed on an Illumina MiSeq system The identity of bacterial populations was analyzed using QIIME software and R statistical software was used to carry out principal components analysis (PCA) (Caporaso et al., 2010) 2.2 In vitro fecal fermentation Results Batch fecal fermentation was performed as the methodology of Lebet et al (Lebet, Arrigoni, & Amadò, 1998) with some minor modifications (Rose, Patterson, & Hamaker, 2010) The total carbohydrate in the samples was determined by the phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) Each substrate (50 mg equivalent carbohydrate) was weighed in test tubes for triplicate analysis Hydration of samples was performed by adding mL of carbonate-phosphate buffer pH 6.8 ± 0.1 to each tube Fructooligosaccharides and tubes without any added carbohydrate were used as positive and negative controls, respectively Fecal samples were obtained from healthy volunteers who were on their Western routine diet and had not taken antibiotics within the previous months Fecal samples were collected in plastic bags that were sealed after removing the air, transported on ice, and immediately placed inside an anaerobic chamber (10% H2, 5% CO2, and 85% N2; BactronEZ, SHEL LAB, Cornelius, OR) where all further procedures were performed within h after collection Fecal samples were pooled together, and the fecal slurry prepared by homogenization with carbonate-phosphate buffer pH 6.8 ± 0.1 in a ratio of 1:3 (w/v) and further strained through layers of cheesecloth The hydrated carbohydrate sample and the controls were then inoculated with the filtrate (1 mL) Tubes were sealed and incubated at 37 °C in a shaking water bath Immediately after incubation and after 4, 8, 12, and 24 h of fermentation, assigned tubes were removed from the water bath, and total gas volume was measured by plunger displacement of the syringe Finaly, pH was measured and a copper sulfate solution (400 μL of 2.75 mg/mL) was then used as an antimicrobial agent, stopping microbial activity Previously obtained and characterized pectic polymers (AGI and HG + AGI) and a xyloglucan (XYG) were submitted to an in vitro fecal fermentation model Profiles of generated metabolites (gas, pH and SCFA) were analyzed at 4, and 12 h of fermentation and compared to the negative (Blank) and positive (FOS) controls Fig shows the amount of gas produced and pH alterations during in vitro fermentation Gas production was higher for pectic polymers than for the tested xyloglucan at 12 h fermentation Drops in pH were also more intense during fermentation of AGI and HG + AGI than the XYG Moreover, intense pH drops occurred up to h in pectic ferments and FOS, while pH drops in XYG lasted longer, up to the h fermentation point (Fig 1) These are indicative that XYG presented a slower fermentation rate compared to the tested pectins and FOS Regarding total SCFA, a similar production (61.6 ± 3.8 mM/50 mg carbohydrate) was observed for pectic polymers and xyloglucan at 12 h fermentation (Fig 2) However, distinct SCFA were produced in higher amounts depending on the polymer fermented Fermentation of HG + AGI led to the highest acetate production at 12 h, around 22% greater than in XYG ferments On the other hand, propionate was more effectively produced during XYG and AGI fermentation, resulting in a final concentration ∼ 31% higher than that observed in HG + AGI ferments Both xyloglucan and pectic polymers led to low butyrate production compared to the positive control (FOS) (Fig 2) To further explore bacterial preferences for each tested dietary fiber, we performed DNA sequencing in the initial inoculum and all 12-hour ferments (Figs and 4) In the initial inoculum and the blank, Firmicutes was the predominant phylum, making up 60 ± 1.3 and 56 ± 1.9% of the reads, respectively (Fig 3) The abundance of Firmicutes was significantly reduced in all dietary fiber ferments tested (Fig 3) Conversely, the abundance of Bacteroidetes was significantly higher in pectic and xyloglucan ferments, which presented a similar 2.2.1 Short chain fatty acid quantification To quantify short chain fatty acid (SCFA) contents, aliquots (400 μL) from fermented samples were combined with 100 μL of a mixture 390 Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al Fig Gas production (mL/50 mg carbohydrate) and pH variations during in vitro fecal fermentation of AGI, HG + AGI and XYG compared to FOS and the Blank Although total Bacteroidetes population was similar in all pectic and xyloglucan ferments (Fig 3), differences in community composition emerged at lower phylogenetic levels (Fig 4) Bacteroides plebeius was higher in AGI (13.7 ± 2.4%) than HG + AGI (8.8 ± 2.4%) or XYG (6.5 ± 0.8%) ferments (Fig 4) On the other hand, Bacteroides Bacteroidetes population of 47 ± 3.6, 41 ± 4.7 and 44 ± 1.4% for AGI, HG + AGI and XYG, respectively, than in the initial inoculum and the blank Finally, in FOS ferments, a greater abundance of Actinobacteria was observed compared to the initial inoculum and the blank (9 ± 0.3%) (Fig 3) Fig Total SCFA, acetate, propionate and butyrate production (mM/50 mg carbohydrate) during in vitro fecal fermentation of AGI, HG + AGI and XYG compared to FOS and the Blank 391 Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al Fig Composition of different bacterial phyla of samples at 12 h fermentation as determined by 16S rRNA gene sequencing Only taxa greater than 0.8% total read composition were included in this graph In the Firmicutes phyla relevant differences were found in some bacteria from Clostridiales order that were significant higher in XYG ferments (3.2 ± 0.1%) than in pectic ferments (2.4 ± 0.1% in both AGI and HG + AGI) (Fig 4) Within Clostridiales, some bacteria from Lachnospiraceae family were also higher in XYG (15.3 ± 0.4%) than pectic ferments (11.8 ± 1.1% in AGI and 12.2 ± 1.5% in HG + AGI) Conversely, Blautia, a genus that also belongs to the Lachnospiraceae uniformis population was highly stimulated in detriment of other Bacteroides species in the presence of XYG, but not pectic ferments Indeed, B uniformis was the predominant specie in XYG ferments constituting 20 ± 0.7% of the total reads, a value 10 times higher than its population in the initial inoculum Parabacteroides distasonis was also significantly higher in XYG (3.7 ± 0.3%) than pectic ferments (1.2 ± 0,1% in AGI and 1.0 ± 0.2% in HG + AGI) (Fig 4) Fig Microbial composition of samples at 12 h fermentation as determined by 16S rRNA gene sequencing Only taxa greater than 1% total read composition were included in this graph 392 Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al Fig Heat map analysis from 16S rRNA gene sequencing of the initial inoculum and fiber ferments at 12 h explained 83.1% of the variations between the different bacterial communities Both the analyses indicated that great bacterial community shifts occurred during xyloglucan fermentation but were not so intense in pectic polymers fermentation The latter, as well as the positive control (FOS) ferments, preserved a microbial composition more similar to that of the original inoculum and the blank than xyloglucan ferments (Figs and 6) family, was higher in pectic ferments (6.5 ± 0.6% in AGI and 5.4 ± 0.3% in HG + AGI) than XYG (4.1 ± 0.5%) (Fig 4) While a similar bacterial composition in most Firmicutes could be observed within pectic ferments, Lachnospira genera was significantly higher in HG + AGI (4.1 ± 1.4%) than AGI (0.6 ± 0.0%) (Fig 4) Heat map and PCA analysis were conducted to investigate general relationships among the bacterial communities from the different dietary fiber ferments (Figs and 6) Hierarchical differences in heat map analysis showed that while the initial inoculum, blank, FOS, HG + AGI and AGI ferments samples formed one major cluster, the xyloglucan ferments were present in a secondary, distinct cluster (Fig 5) In PCA analysis (Fig 6), initial inoculum, blank, FOS, HG + AGI and AGI ferments and XYG were distinctly separated by the first principal component axis (PC1), rather than the PC2, which accounted for 64.9% of the total variations PC2 accounted for only 18.2% of the variance in the bacterial communities Overall, the PC1 and PC2 axes Discussion and conclusions Structural features of a polymer such as the molecular weight, the type of monosaccharides released after digestion, the degree of polymerization and substitution, and other physical features that allows the access of bacterial enzymes to the saccharides units may influence the fermentation profile of a fiber in the colon, including its rate of digestion (Hamaker & Tuncil, 2014; Holscher, 2017) Thus, more complex 393 Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al Wolever, 2004) and its contribution to increase propionate in AGI ferments cannot be discarded The distinct metabolites profiles generated by each dietary fiber tested, the microbial communities also varied accordingly, especially at low phylogenetic levels Indeed, shifts in gut microbiota composition promoted by dietary fiber fermentation were shown to occur in very specific species or strains (Chung et al., 2016) In our study, B plebeius, P distasonis and bacterial groups such as Blautia, Lachnospira, Clostridiales and Lachnospiraceae, presented a distinct abundance on each dietary fiber ferment Moreover, a substantial increase in B uniformis population to the detriment of other Bacteroides species was evident in XYG ferments B uniformis is among the most abundant organisms in the microbiota of individuals in Western countries, and may be beneficial as it has been shown to exhibit the potential to ameliorate metabolic and immune dysfunctions associated with obesity (FernándezMurga & Sanz, 2016; Gauffin Cano, Santacruz, Moya, & Sanz, 2012) A work of Larsbrink et al (2014) showed that xyloglucans can be used as energy source by most of B uniformis strains in pure cultures Our results reveal that even in the competitive environment of the fecal complex microbiota, these xyloglucans were efficiently fermented by B uniformis Within pectic polymers (AGI and HG + AGI) only minor differences in few bacterial groups were observed depending on the chemical structure of the pectic polymer fermented While Bacteroides plebeius was higher in AGI, Lachnospira genera was higher in HG + AGI This data corroborates previous studies, which showed that some bacteria such as Faecalibacterium prausnitzii and Bacteroides spp respond differently depending on the pectin source and chemical structure (Hamaker & Tuncil, 2014; Lopez-Siles et al., 2012) PCA and heat map analysis indicate that pectic polymers lead to microbial composition that resembles more of the initial inoculum than the XYG ferments Pectic polymers are present in its natural soluble form in a variety of foods such as fruits and vegetables usually present in a healthy diet (Wikiera, Irla, & Mika, 2014) The common presence of soluble pectin in the diet could explain the higher similarity in composition of the microbiota of pectic ferments to the initial inoculum Xyloglucans, however, although ubiquitous in foods, are usually present embed in the plant’s cellulosic cell wall matrix (Hsieh & Harris, 2009; Scheller & Ulvskov, 2010) that may be difficult the access to bacterial fermentation In our study, the xyloglucan was isolated with alkali and was applied in the fermentation experiment in its water-soluble form (Cantu-Jungles, Iacomini et al., 2017), and was accessible to bacteria This is an uncommon form of xyloglucans, and it is plausible that their fermentation would drive to a very distinct fermentation profile different from initial inoculum Overall, the results presented indicate that the pectic polymers and xyloglucan, during their in vitro fermentation, drive different characteristic metabolites profiles and bacterial communities’ profiles Such an understanding of how isolated dietary fibers are fermented and affect microbial composition will be key to design new food ingredients capable of modulating the intestinal microbiota in a targeted way Fig Principal component analysis (PCA) from 16S rRNA gene sequencing of the initial inoculum and fiber ferments at 12 h polymers are expected to be slower fermented than simple structures The delayed fermentation profile of tested dietary fibers in this study (xyloglucan > > pectins > FOS) supports this hypothesis A slower fermentation rate indicates that SCFA are prone to be delivered in all portions of the large intestine, including the distal colon The absence of fermentable carbohydrates in more distal parts of the large intestine, may lead to bacterial fermentation of proteins and consequent generation of detrimental metabolites such as hydrogen sulfide, phenols, and ammonia (Blachier, Mariotti, Huneau, & Tome, 2007; MacFarlane, Gibson, & Cummings, 1992) Thus, one could speculate that xyloglucan and tested pectins should be able to exert a positive effect with fermentation occurring more distally in the colon in comparison to FOS Regarding short chain fatty acid production, each dietary fiber produced a unique metabolite profile during in vitro fecal fermentation While FOS was highly butyrogenic, HG + AGI was more acetogenic, and the XYG and AGI more propiogenic While fermentation results of xyloglucans is lacking in the literature, it was previously demonstrated that pectic polymers from different sources are extensively fermented by the colonic gut microbiota in vitro (Cantu-Jungles, Cipriani, Iacomini, Hamaker, & Cordeiro, 2017; Gulfi, Arrigoni, & Amadò, 2005; Jonathan et al., 2012; Licht et al., 2010; Min et al., 2015; Titgemeyer, Bourquin, Fahey, & Garleb, 1991) However, these studies were conducted with the homogalacturonan (HG) alone (Cantu-Jungles, Cipriani et al., 2017; Gulfi et al., 2005; Jonathan et al., 2012) or non-characterized pectic structures (Licht et al., 2010; Min et al., 2015; Titgemeyer et al., 1991) In many plant types, however, type I rhamnogalacturonans branched by arabinans, galactans and/or type I arabinogalactan (AGI) can be found together with portions of HG (Willats et al., 2006) Here we have shown that a HG together with a rhamnogalacturonan I highly branched by type I arabinogalactan (HG + AGI), was specifically acetogenic with a low butyrate production similar to what was found in other reports of HG alone (Cantu-Jungles, Cipriani et al., 2017; Gulfi et al., 2005; Jonathan et al., 2012) However, when AGI was isolated from HG, with butyrate production kept low, acetate production was reduced, and propionate production was raised Aguirre, Bussolo de Souza, and Venema, (2016) also have shown that fermentation of type II arabinogalactan (AGII) produced high propionate rates in the microbiota from obese subjects, similar to what was found in our study for rhamnogalacturonan I branched by type I arabinogalactan (AGI) Indeed, in a study using mono and disaccharides, Mortensen, Holtug, and Rasmussen, (1988) showed that propionate production occurs during in vitro fecal incubation with a variety of sugars, including arabinose, which is present in both type I and type II arabinogalactans Moreover, ingestion of L-rhamnose, a sugar present in the main chain of RGI to which AGI is attached, was shown to increase propionate in the serum of healthy volunteers (Vogt, Pencharz, & Acknowledgments This research was supported by Projeto Universal (Process 477971/ 2012-1) provided by CNPq foundation (Brazil), by PRONEXCarboidratos and CAPES The author Thaisa Moro Cantu-Jungles received from CNPq Foundation a fellowship for a year of study at Whistler Center for Carbohydrate Research at Purdue University (process 208166/2014-9) and for posdoctoctoral studies (Process 150235/ 2017-8) References Aguirre, M., Bussolo de Souza, C., & Venema, K (2016) The gut microbiota from lean and obese subjects contribute differently to the fermentation of arabinogalactan and 394 Carbohydrate Polymers 206 (2019) 389–395 T Moro Cantu-Jungles et al Holscher, H D (2017) Dietary fiber and prebiotics and the gastrointestinal microbiota Gut Microbes, 8(2), 172–184 Hsieh, Y S Y., & Harris, P J (2009) Xyloglucans of monocotyledons have diverse structures Molecular Plant, 2(5), 943–965 Jonathan, M C., van den Borne, J J G C., van Wiechen, P., Souza da Silva, C., Schols, H A., & Gruppen, H (2012) In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans Food Chemistry, 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during their in. .. ubiquitous in plants, there are no studies in the literature comparing the gut fermentability of pectins vs xyloglucans, and in forms commonly consumed (soluble pectins) vs uncommon (soluble xyloglucans)