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Carbohydrate Polymers

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In vitro assessment of prebiotic properties of xylooligosaccharides producedby Bacillus subtilis 3610

Cláudia Amorim, Sara C. Silvério, Beatriz B. Cardoso, Joana I. Alves, Maria Alcina Pereira,Lígia R. Rodrigues*CEB-Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal

A R T I C L E I N F O

Keywords:PrebioticLactuloseXylooligosaccharidesHuman fecal inoculaIn vitro assays

A B S T R A C T

Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, invitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate theprebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610.The fermentation of lactulose led to the highest production of lactate (D1: 33.7 ± 0.5mM; D2:19.7 ± 0.3mM)and acetate (D1: 77.5 ± 0.6mM; D2: 81.0 ± 0.7mM), while XOS led to the highest production of butyrate (D1:9.0 ± 0.6mM; D2: 10.5 ± 0.8mM) and CO2 (D1: 8.92 ± 0.02mM; D2: 11.4 ± 0.3mM). Microbiota analysisshowed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase inBifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS.

1. Introduction

Prebiotic compounds have attracted increased attention fromacademy and industry, as consumers pay more attention to their well-being, pivoting their health consciousness to preventive medicine.Thus, the prebiotics global market is expected to increase reaching 7.37Billion USD by 2023 (MarketsandMarketsTM, 2018). The prebiotic de-finition was recently updated to “a substrate that is selectively utilizedby host microorganisms conferring a health benefit” (Gibson et al.,2017). Prebiotics are indeed attractive compounds due to their multi-dimensional beneficial effects on both human and animal health,namely on the gastrointestinal tract (e.g. pathogen inhibition, immunemodulation), cardiometabolism (e.g. cholesterol lowering), mentalhealth (e.g. energy and cognition) and bones (e.g. enhanced mineralabsorption), among others (Gibson et al., 2017; Samanta et al., 2015).

Xylooligosaccharides (XOS) have been identified as potentially va-luable food and feed prebiotic ingredients (Sajib et al., 2018) and arethe only nutraceuticals that can be produced from cheap and abundantlignocellulosic biomass (Samanta et al., 2015). The gut microbiota usesprebiotics to multiply and consequently produce short-chain fatty acids(SCFAs), gases (mainly, hydrogen and carbon dioxide), lactate, andother products (Topping & Clifton, 2001). SCFAs, including acetate,propionate and butyrate, and other compounds such as lactate, arerecognized as key metabolites for the intestinal health, influencingothers sites distant to the gut (Gibson et al., 2017). Lactate is reported

as a precursor of different SCFAs, such as propionate and butyrate,which are widely known to promote a prebiotic effect (Flint, Duncan,Scott, & Louis, 2015).

XOS have been the focus of several studies given their wide range ofbeneficial health effects (Aachary, Gobinath, Srinivasan, & Prapulla,2015). Yang et al. (2015) reported the XOS effect in reversing changesobserved in the human gut microbiota during the development of dia-betes.

Additionally, due to its minimal recommended dose, 1.4–2.8 g/day(Finegold et al., 2014), XOS are considered price competitive whencompared to other prebiotics (Amorim, Silvério, Prather, & Rodrigues,2019). Besides, XOS also present favorable organoleptic properties, andtemperature and acidic stability (Courtin, Swennen, Verjans, & Delcour,2009).

XOS are oligosaccharides composed by a main chain of xylose unitslinked through (β1,4)-linkages and decorated with several substituentelements, such as acetyl groups, glucuronic acids, arabinose and ga-lactose residues (Coelho, Rocha, Moreira, Domingues, & Coimbra,2016). Their production through direct fermentation of beechwoodxylan by a modified Bacillus subtilis has been previously reported byAmorim, Silvério, Gonçales et al. (2019). Moreover, XOS presentedhigh stability after a static in vitro digestion. However, this method isnot elucidative of their prebiotic effect, which depends both on theirdegree of polymerization (DP) and degree of substitution (Sajib et al.,2018).

https://doi.org/10.1016/j.carbpol.2019.115460Received 30 July 2019; Received in revised form 30 September 2019; Accepted 9 October 2019

⁎ Corresponding author.E-mail address: lrmr@deb.uminho.pt (L.R. Rodrigues).

Carbohydrate Polymers 229 (2020) 115460

Available online 17 October 20190144-8617/ © 2019 Elsevier Ltd. All rights reserved.

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In vivo studies are expensive and time-consuming, therefore notsuitable to be used as a screening tool for selecting prebiotics. The invitro evaluation of the prebiotic potential of oligosaccharides using fecalinocula and high-throughput sequencing techniques to assess changeson the microbiota are preferable to the use of single or co-culturedmicroorganisms, since the human gut microbiota is complex and an invitro evaluation with a limited number of probiotic strains is not re-presentative to understand the prebiotic potential of specific substrates(Gibson et al., 2017). Nonetheless, experimental data on the XOS pre-biotic effect regarding in vitro approaches more compatible with theguidelines stated by the updated definition of prebiotic are scarce.

In this work, in vitro studies were performed using human fecalinocula and high-throughput sequencing (16S rRNA gene) of micro-biota. Lactulose is a well-established and widely accepted prebiotic inthe market (Watson et al., 2013), therefore it was used in this work forcomparison purposes.

The main challenge of this work is to test the hypothesis of XOSproduced from beechwood xylan potentially acting as a prebiotic withbeneficial effects on human health.

2. Experimental

2.1. Prebiotic source

XOS were produced by single-step fermentation of beechwood usinga cloned Bacillus subtillis 3610 harboring the xylanase gene xyn2 fromTrichoderma reesei as previously described (Amorim, Silvério, Gonçaleset al., 2019). At the point of maximal XOS production, the cell-freefermentation broth was collected and lyophilized to be further used inthe current study as substrate for in vitro batch fermentations. Themixture of linear XOS, DP ranging from 4 to 6, was mainly composed by(1→4)-linked-xylopyranosyl residues with a small amount of branchedresidues, presenting 3 different fractions: fraction A containing XOSwith an average DP of 4 with 5.3% of branching, were the branchingpoints are mainly in disubstituted xylose residues with terminallylinked arabinofuranose; fraction B composed of xylopentaose linearoligosaccharides and fraction C presenting an average DP of 6 xyloseresidues with 2.2% of branching. Commercial lactulose (analyticalgrade) was obtained from Sigma-Aldrich (St. Louis, MO).

2.2. Fecal inoculum

Fecal samples were obtained from two healthy human volunteerswho were free of known metabolic and gastrointestinal diseases and didnot take any antibiotics, pre- or probiotic supplements for 3 monthsbefore the study. The male and female donors aged 26 were non-smo-kers and consumed non-specific Mediterranean diet. The samples werecollected on site, diluted 1/10 (w/w) in anaerobic (100% N2) phos-phate-buffered saline solution (PBS, 0.1 M, pH 7.0) and were kept at4 °C overnight, before inoculation.

2.3. In vitro batch culture fermentations of oligosaccharides using gutmicrobiota

Static batch culture fermentations were performed at 37 °C during48 h in serum bottles. The bottles were filled with 40mL of growthmedium at pH 7.0 (peptone water 2 g/L, yeast extract 2 g/L, NaCl 0.1 g/L, K2HPO4 40mg/L, KH2PO4 40mg/L, MgSO4.7H2O 0.01 g/L,CaCl2.6H2O 0.01 g/L, NaHCO3 2 g/L, Tween 80 14.8 ml/L, hemin5mg/L, vitamin K1 74.1 μl/L, cysteine HCl 0.5 g/L, bile salts 0.5 g/L,Na2S.9H2O 0.8mM and resazurine 1mg/L). The XOS or lactulose so-lutions were added when required at a final concentration of 10 g/L.Except for the filter-sterilized solutions of vitamin k1 and oligo-saccharides, the medium was sterilized by autoclaving and were in-oculated with 4.4 mL of fecal inoculum. Anaerobic conditions weremaintained by pressurizing the bottles’ headspace with nitrogen up to

170 K Pa.Liquid samples were collected at different time points (0, 6, 12, 24,

36 and 48 h), centrifuged at 4000 x g for 10min and the supernatantwas further used for HPLC analysis (Section 2.4). Gas samples from theheadspace were used to access gas production (Section 2.4). The pHwas measured at 48 h and the fermentation broth was withdrawn fromthe bottles, centrifuged, washed, resuspended in PBS (0.1M pH 7.0)and stored at −20 °C for DNA extraction and further sequencing ana-lysis (Section 2.5.). Fermentations were run in duplicate, using a blankwith no prebiotic addition as negative control.

2.4. Analytical techniques

The consumption of oligosaccharides was accessed by HPLC as de-scribed by Amorim, Silvério, Gonçales et al. (2019), with minor mod-ifications. Mixtures of acetonitrile and water, 68:32 (v/v) (for XOSanalysis) and 70:30 (v/v) (for lactulose analysis), were used as mobilephase. Pure lactulose and XOS (DP 2–6, from Megazyme, Bray, Ireland)were used as standards.

The production of lactate and SCFAs (acetate, propionate and bu-tyrate) was evaluated according to Fernandes, Rao, and Wolever(2000)), using an HPLC (Knauer, Berlin, Germany) fitted with a Knauer-RI detector and an Aminex HPX 87H column (300mm x 7.8; Biorad,Hercules, CA).

Gas samples were analyzed by gas chromatography (GC) using aBruker Scion 456-GC equipment (Billerica, MA) as described elsewhere(Arantes, Alves, Alfons, Alves, & Sousa, 2018).

2.5. Microbiota analysis

Total DNA from fecal inocula and fecal fermentations at 48 h wasextracted from liquid samples using the FastDNA SPIN kit for soil (MPBiomedicals, Solon, OH), according to the manufacturer’s instructions.High-throughput sequencing (16S rRNA gene) by Illumina MiSeqtechnology were performed at the RTL Genomics (Lubbock, TX).Detailed description of the procedure was previously described(Salvador et al., 2019). All the samples were analyzed in duplicate.Submission of the FASTQ files was done at the European NucleotideArchive under the BioProject accession number PRJEB33616 (Samplesaccession number: ERS3592390, ERS3592392, ERS3592394,ERS3592396, ERS3592397, ERS3592400, ERS3592401, ERS3592404,ERS3592405, ERS3592406, ERS3592408, ERS3592410, ERS3592412,ERS3592413).

2.6. Statistical analysis

Differences between products concentrations were checked for sig-nificance by ANOVA using Prism 7.0a software (GraphPad. Software.Inc.). Tukey test was used for post hoc comparisons. The differenceswere considered significant when p < 0.05.

3. Results and discussion

3.1. Production of lactate and short-chain fatty acids (SCFAs), andsubstrate consumption

Fig. 1 shows the total production of lactate and main SCFAs gen-erated in the colon (≥95% in humans), namely acetate, propionate andn-butyrate, as a result of several bacterial metabolic pathways (Gibsonet al., 2017). For both donors, the supplementation with 10 g/L oflactulose or XOS increased significantly (t-test student, α=0.05) thetotal production of lactate and SCFAs as expected. These results are wellaligned with previous reports on the prebiotic effect of lactulose or XOSusing human fecal inocula (Buruiana, Gómez, Vizireanu, & Garrote,2017; Carlson, Erickson, Hess, Gould, & Slavin, 2017; Ehara et al.,2016; Mao et al., 2014; Reis et al., 2014; Sajib et al., 2018).

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http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592390http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592392http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592394http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592396http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592397http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592400http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592401http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592404http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592405http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592406http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592408http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592410http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592412http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERS3592413

Moreover, it seems that the production of lactate and SCFAs ismainly dependent on the addition of prebiotics, rather than the dif-ferences between the gut microbiota of the donors (Fig. 1). Never-theless, further experiments using a larger number of donors should beperformed to get a more representative view of the effects of eachprebiotic, including individuals from different age range, dietary habits,health backgrounds, among others.

The experiments with XOS led to a faster accumulation of lactateand SCFAs from 0 to 12 h, for both donors. This observation was cor-roborated by the fast consumption of XOS in the same time range. After24 h of fermentation, XOS presented an utilization of (74 ± 1)%(donor 1, D1) and (71 ± 3)% (donor 2, D2), while lactulose presentedan utilization of (50 ± 5)% (D1) and (56 ± 2)% (D2). After 48 h offermentation, XOS were almost totally consumed, showing an utiliza-tion of (92.7 ± 0.8)% and (95.9 ± 0.8)%, for donors 1 and 2, re-spectively. At this time point, the lactulose utilization was (79 ± 5)%and (74 ± 0.4)% for donors 1 and 2, respectively. The results suggestthat the utilization rate is more dependent on the type of prebioticadded, rather than the differences between the gut microbiota of thedonors, similarly to the previous observation for SCFAs production. Thecombination of prebiotics with different fermentation rates can pro-mote fermentation in large parts of the colon, thus potentiating theprebiotic effect (Sajib et al., 2018). Therefore, the supplementationwith one or more prebiotic can potentially add value to a product.

In comparison to XOS, the use of commercial lactulose led to higherconcentrations of lactate and SCFAs after 12 h of fermentation, reachingits maximum difference at 48 h (131 ± 2mM for the sample from D1,p < 0.0001, and 113.6 ± 0.9mM, p < 0.0001, for the sample fromD2) (Fig. 1).

Although the total amount of SCFAs produced is a good indicationof the prebiotic potential of the substrates evaluated, the proportion atwhich they are produced is also important to estimate potential healthbenefits associated to the consumption of these substrates (Gibsonet al., 2017). Fig. 2 shows the production profiles of lactate and SCFAsobtained after 48 h of fermentation by fecal inocula. For both substratesand donors, acetate was the main SCFA accumulated, while propionatewas the second most produced. Buruiana et al. (2017); Ruiz et al.(2017) and Dávila, Gullón, Alonso, Labidi, and Gullón (2019)) alsoreported acetate as being the most abundant SCFA when XOS, fromcorn stover, olive tree pruning and vine shoots, respectively, were usedas substrates for in vitro fermentation with human fecal microbiota. Thesame evidence was found for lactulose (Ito et al., 1997; Fernandes et al.,2000). Acetate has been reported as beneficial for colorectal cancerprevention (Casanova, Azevedo-Silva, Rodrigues, & Preto, 2018; Ferro

et al., 2016), while propionate plays an important role in the inhibitionof cholesterol synthesis and the deposition of adipose tissue, beingproposed as a dietary factor to depress appetite and reduce obesity(Arora, Sharma, & Fros, 2011).

Interestingly, the supplementation of XOS resulted in the highestproduction of butyrate (9.0 ± 0.6mM for samples from D1, and10.5 ± 0.8mM for samples from D2), significantly different from theblank for both donors, p < 0.0001. These results are in good agree-ment with the those reported by Ruiz et al. (2017), that found a bu-tyrate concentration of approximately 14mM after 48 h of fermenta-tion. Butyrate has been reported as an important metabolite for themaintenance of the intestinal homeostasis and overall health status,exerting several beneficial effects, particularly being associated to theprevention and inhibition of colorectal cancer and diarrhea, and alsoacting at the extra-intestinal level (Berni Canani et al., 2011; Gonçalves& Martel, 2013).

On the other hand, in the experiments with XOS, lactate was un-detected after 12 h of fermentation (data not shown), which can bepossibly explained by its function as an intermediary in the productionof other metabolites, including acetate, butyrate and propionate byother bacterial species (Duncan, Louis, & Flint, 2004). This trend wasalso observed by Gómez, Míguez, Veiga, Parajó, and Alonso (2015))and Ruiz et al. (2017).

Contrariwise, the addition of lactulose resulted in a high accumu-lation of lactate (33.7 ± 0.5mM for samples from D1, and19.7 ± 0.3mM for samples from D2) and a reduced production ofbutyrate. However, since lactate is a precursor of SCFAs, probably in-creasing the fermentation time would result in the decrease in lactateand further accumulation of SCFAs.

3.2. pH change and ammonia production

As previously mentioned, the main health-promoting effects ofprebiotic oligosaccharides are associated to the production of SCFA andthe subsequent pH drop that promotes the reduction in the pathogenicmicrobiota and increase in the beneficial bacteria population(Cummings & Macfarlane, 2002). Table 1 shows the final pH and theproduction of ammonia after 48 h of fermentation of the two substratesunder study.

The addition of prebiotics led to a pH and ammonia reduction, asexpected. The largest pH variation and ammonia reduction were foundfor experiments with lactulose. Ammonia is related to bad fecal odor

Fig. 1. Total production of lactate and short chain fatty acids (SCFAs) during48 h of fecal inocula growth from donors 1 and 2 in the absence of prebiotics(blank) or in a medium enriched with a prebiotic solution of lactulose or XOS at10 g/L. Results are the average of two independent fermentations and triplicateanalysis of each sample ± standard deviation.

Fig. 2. Production of lactate and short-chain fatty acids (acetate, propionateand butyrate) after 48 h of fecal inocula growth from donors 1 and 2 in theabsence of prebiotics (blank) or in a medium enriched with a prebiotic solutionof lactulose or XOS at 10 g/L. Results are the average of two independent fer-mentations and triplicate analysis of each sample ± standard deviation.

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and high amounts of this compound may contribute to colon carcino-genesis (Visek, 1978). Likewise, Sajib et al. (2018) reported a pH dropto an average of 5.6 or 6.4 pH after 48 h of fermentation, using an invitro model with human fecal inoculum to ferment arabinoxylans ob-tained from brewer’s spent grain produced by ultrasound-assisted ex-traction or by alkaline extraction, respectively.

Unlike the in vivomodels, the SCFAs formed during fermentation arenot absorbed when an in vitro model is used. Therefore, these com-pounds will greatly alter the medium pH. Although the fermentationmedium used in this study is designed to mimic the distal colon pH atbaseline, the medium pH was not further controlled throughout thefermentation, which is a limitation of this type of models. In vitro fer-mentations are regarded as semi-representative models of colonic fer-mentation and they can be performed by fermentation with pure cul-tures of selected bacteria, mixed bacterial populations or fecal samples(Macfarlane & Macfarlane, 2007). The use of fecal samples is con-sidered a better approximation to the in vivo models, representingmostly the distal colon microbiota and taking into account complexinteractions among bacterial populations (Rycroft, Jones, Gibson, &Rastall, 2001).

Moreover, in in vivo models, the produced SCFAs are rapidly ab-sorbed, which limits their measurement (Carlson et al., 2017). Thus, invitro models are more suitable to study the kinetics of colonic fermen-tation. However, these models exhibit some limitations, namely the factthat more proximal areas may have a different composition but arehardly accessible with human volunteers. Thus, complex gut modelsshould be used to overcome this issue and further assess prebiotic ef-fects, replicating the different intestinal sections (Payne, Zihler,Chassard, & Lacroix, 2012).

3.3. Gas production

The oligosaccharides fermentation by fecal inocula can also have aneffect on the amount of gases produced. Fig. 3 shows the production ofH2 and CO2 during 48 h of fermentation.

As expected, larger volumes of CO2 and H2 were obtained from thelactulose and XOS fermentation when comparing to the blank. Carlsonet al. (2017) and Buruiana et al. (2017) reported the same trend in gasproduction using in vitro models with human fecal inoculum and XOSobtained from corn stover. Methane was not detected for both donors’samples. Indeed, only some people harbour methanogenic archaeawhich result in methane production (Ghoddusi, Grandison, Grandison,& Tuohy, 2007).

Overall, the CO2 production relative to blank was considerablyhigher in the cultures containing XOS (p < 0.0001 D1 and D2), con-trarily to the ones containing lactulose. The maximum production ofCO2 was 8.9 ± 0.7mmol/L at 24 h for samples from D1 and11 ± 1mmol/L at 36 h for samples from D2. Since high gas productionmay result in flatulence problems, which constitute a clinical disin-centive to prebiotic use (Cummings, Macfarlane, & Englyst, 2001), it iscrucial to evaluate through in vivo models the proper recommended

dose of prebiotic consumption.The results obtained suggest that the recommended daily dose of

XOS should be lower than that of lactulose, which is corroborated bythe studies performed in vivo with these prebiotics (Bouhnik et al.,2004; Finegold et al., 2014).

3.4. Microbiota analysis

Although exhibiting different microbial proportions, fecal inocularesults obtained for both donors showed as expected, a typical gutmicrobiota diversity for healthy human adults (Eckburg et al., 2005),being mainly composed by six bacterial phyla, of which Firmicutesrelative abundance dominated (43 ± 1% D1; 61.5 ± 0.5% D2) (TableA.1, Supplementary Material).

For both donors, adding different oligosaccharides (lactulose orXOS) led to a consistent distinct modulation of the gut microbiota after48 h of fermentation as compared to the blank (Figs. 4 and 5). Thefermentation of lactulose led to a predictable increase in the relativeabundance of bacteria from the Lactobacillus (640 ± 10-fold D1;200 ± 50-fold D2) and Bifidobacterium genera (2.3 ± 0.1-fold D1;4.1 ± 0.4-fold D2) (Table A.1, Supplementary Material). These resultscorroborate the increased production of lactate and SCFAs previouslyobserved and are well aligned with the literature (Watson et al., 2013).Lactobacillus and Bifidobacterium are important genera of commensal

Table 1pH and ammonia concentration after 48 h of fecal inocula growth from donors 1and 2 in the absence of prebiotics (blank) or in a medium enriched with aprebiotic solution of lactulose or XOS (10 g/L). Results are the average of twoindependent fermentations and triplicate analysis of each sample ± standarddeviation.

Assay Donor 1 Donor 2

pH Ammonia (mg/L)

pH Ammonia (mg/L)

Blank 7.08 ± 0.02a 152 ± 7a 7.00 ± 0.03a 162 ± 5a

Lactulose 3.56 ± 0.03b 59 ± 1b 3.585 ± 0.05b 49 ± 3b

XOS 6.41 ± 0.01c 131 ± 3ac 6.70 ± 0.30ac 132 ± 2c*

Fig. 3. Production of H2 and CO2 by fecal inocula from donors 1 (A) and 2 (B)in the absence of prebiotics (blank) or enriched with a prebiotic solution oflactulose or XOS at 10 g/L. Results are the average of two independent fer-mentations and triplicate analysis of each sample ± standard deviation.

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bacteria widely known for their ability to use complex carbohydratesand to produce lactic acid and SCFAs, which in turn have been asso-ciated to human health by several mechanisms. For instance, a reducedamount of SCFAs is associated to a gut microbiota of patients withdiabetes, autoimmune disorders, obesity and cancers (Nagpal et al.,2018).

Bifidobacterium adolescentis, commonly found in adults (Gibsonet al., 2017), was the only Bifidobacterium species identified in theblank, lactulose or XOS fermentation samples for both donors (TableA.1, Supplementary material). Nevertheless, a more diverse communityof Lactobacillus species was found. When compared with the blank, ahigher diversity of Lactobacillus species was stimulated by lactuloserather than XOS fermentation for both donors. Furthermore, the varietyof species is intrinsically related with the donor original microbiota, asshown by the inocula sequencing results (Table A.1, Supplementary

material). Thus, although the main bacterial growth trends were com-parable between the donors, the specific microbiota pattern inferred bythe use of a certain prebiotic will depend on the original inoculum. Thisfact also highlights the need for in vitro and in vivo studies with a re-presentative number and diversity of individuals, as previously dis-cussed.

Moreover, the fermentation of lactulose decreased significantly therelative abundance of Bacteriodales (5.1 ± 0.5-fold D1; 5 ± 0-foldD2) (Figs. 4 and 5), in particular of Bacteroides (5.8 ± 0.8-fold D1;6 ± 0-fold D2) (Table A.1, Supplementary material). On the contrary,the fermentation of XOS stimulated the growth of bacteria from thisphyla (1.48 ± 0.02-fold D1; 1.50 ± 0.06-fold D2), specially Bacter-oides (1.52 ± 0.02-fold D1; 1.79 ± 0.04-fold D2). Microorganismsbelonging to the Bacteroides genus are known butyrate-producing bac-teria, which in turn is the main source of energy to gut epithelial cells

Fig. 4. Relative abundance of different bacteria after 48 h of in vitro fermentation by fecal inocula from D1 in the absence of prebiotics (blank) (A) or enriched with aprebiotic solution of lactulose (B) or XOS (C) at 10 g/L.

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(Hwang et al., 2017). This observation is in accordance with the resultsobtained for SCFAs production, namely the highest production of bu-tyrate was observed for XOS fermentation.

Thus, the XOS produced by direct fermentation of beechwood xylanappear to be highly selective towards butyrate-producing bacteria,suggesting that these oligosaccharides have potential to be used, forinstance, as prebiotic treatment during the active phase of in-flammatory bowel disease (IBD). IBD active patients present increasedproportions of Bifidobacterium and Lactobacillus on their intestinalmicrobiota and reduced butyrate-producing bacteria (Wang et al.,2014). Furthermore, Hwang et al. (2017) reported the influence ofhigh-fat diets and low-fiber diets on decreasing members of the Bac-teroidales order and butyrate production.

On the other hand, the fermentation of both lactulose and XOS ledto a predictable reduction in the relative percentages of Proteobacteria(lactulose: 29 ± 4-fold D1, 36 ± 1-fold D2; XOS: 2.1 ± 0.3-fold D1,2.2 ± 0.1-fold D2) (Figs. 4 and 5). The abundance of Proteobacteria onthe gut microbiota is associated to several intestinal diseases. Thisphylum comprises several known human pathogens (Rizzatti, Lopetuso,

Gibiino, Binda, & Gasbarrini, 2017), including Escherichia coli whoserelative abundance was significantly reduced by the addition of lactu-lose (32.0 ± 5-fold D1; 750 ± 50-fold D2) and XOS (12 ± 4-fold D1;28 ± 8-fold D2) (Table A.1, Supplementary material).

Concerning the production of CH4, in the case of D1, the lack ofmethanogenic archaea on the original inoculum may explain its ab-sence on the products profile. For D2, a decrease in the relative abun-dance of members belonging toMethanobacteriacea family was observed(lactulose: 12.7 ± 0.7-fold; XOS:2 ± 0-fold). The production of CH4may be comprised below the sensitivity limit of the GC technique, dueto the reduced relative abundance of these bacteria on both D2 in-oculum and blank samples (Table A.1, Supplementary material). Inaddition, the largest production of CO2 observed during XOS fermen-tation, may be related with the stimulation of the Bacteroides growth.The reduction in ammonia production observed during lactulose andXOS fermentations, may also be due to the decrease in the relativeabundance of ammonia-producing bacteria, namely E. coli andClostridium sp. (lactulose: 4.2 ± 0.8-fold D1, 1.9 ± -0.6-fold D2; XOS:3 ± -1-fold D1, 2.5 ± 0.25-fold D2) (Richardson, McKain, & Wallace,

Fig. 5. Relative abundance of different bacteria after 48 h of in vitro fermentation by fecal inocula from D2 in the absence of prebiotics (blank) (A) or enriched with aprebiotic solution of lactulose (B) or XOS (C) at 10 g/L.

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2013) (Table A.1, Supplementary material).Attending to the results herein reported, lactulose appears to be a

more suitable prebiotic to treat gut dysbiosis associated with reducedamounts of Bifidobacterium and Lactobacillus, while XOS produced frombeechwood may potentially be used to increase the butyrate con-centration and therefore useful for IBD patients’ treatment.

4. Conclusions

SCFAs, lactate, ammonia and gas generation, pH variation, andmicrobiota analysis confirmed for the first time the suitability of theXOS produced by recombinant B. subtilis 3610 from beechwood in asingle-step process to present potential functional properties for humanhealth. For both donors’ samples, the fermentation of XOS resultedmainly in the production of acetate, followed by propionate and buty-rate, which are important metabolites related to several health benefits.When compared with commercial lactulose, XOS presented the highestproduction of butyrate and CO2, which was corroborated by the mi-crobiota modulation, namely the increase in the relative abundance ofBacteroides within the bacterial community.

This study highlights the microbiota modulation effect promoted bythe fermentation of carbo-based substrates with different compositionsand structures. This observation suggests distinct potential health ap-plications for lactulose and XOS.

Acknowledgments

CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 andSFRH/BD/132324/2017) from the Portuguese Foundation for Scienceand Technology (FCT). The study received financial support from FCTunder the scope of the strategic funding of UID/BIO/04469/2019 unit;COMPETE 2020 (POCI-01-0145-FEDER-006684), through nationalfunds and where applicable co-financed by the FEDER, within thePT2020 Partnership Agreement; the Project FoSynBio (POCI-01-0145-FEDER-029549), and NewFood (NORTE-01-0246-FEDER-000043). Theauthors also acknowledge BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fundunder the scope of Norte2020 - Programa Operacional Regional doNorte.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.carbpol.2019.115460.

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In vitro assessment of prebiotic properties of xylooligosaccharides produced by Bacillus subtilis 3610IntroductionExperimentalPrebiotic sourceFecal inoculumIn vitro batch culture fermentations of oligosaccharides using gut microbiotaAnalytical techniquesMicrobiota analysisStatistical analysis

Results and discussionProduction of lactate and short-chain fatty acids (SCFAs), and substrate consumptionpH change and ammonia productionGas productionMicrobiota analysis

ConclusionsAcknowledgmentsSupplementary dataReferences