In vitro assessment of prebiotic properties of xylooligosaccharides produced by Bacillus subtilis 3610

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In vitro assessment of prebiotic properties of xylooligosaccharides produced by 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 LE I N FO

A B S T R A C T

Keywords: Prebiotic Lactulose Xylooligosaccharides Human fecal inocula In vitro assays

Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic 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 ± 0.5 mM; D2:19.7 ± 0.3 mM) and acetate (D1: 77.5 ± 0.6 mM; D2: 81.0 ± 0.7 mM), while XOS led to the highest production of butyrate (D1: 9.0 ± 0.6 mM; D2: 10.5 ± 0.8 mM) and CO2 (D1: 8.92 ± 0.02 mM; D2: 11.4 ± 0.3 mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS.

1. Introduction

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 of beneficial health effects (Aachary, Gobinath, Srinivasan, & Prapulla, 2015). Yang et al. (2015) reported the XOS effect in reversing changes observed in the human gut microbiota during the development of diabetes. Additionally, due to its minimal recommended dose, 1.4–2.8 g/day (Finegold et al., 2014), XOS are considered price competitive when compared to other prebiotics (Amorim, Silvério, Prather, & Rodrigues, 2019). Besides, XOS also present favorable organoleptic properties, and temperature and acidic stability (Courtin, Swennen, Verjans, & Delcour, 2009). XOS are oligosaccharides composed by a main chain of xylose units linked through (β1,4)-linkages and decorated with several substituent elements, such as acetyl groups, glucuronic acids, arabinose and galactose residues (Coelho, Rocha, Moreira, Domingues, & Coimbra, 2016). Their production through direct fermentation of beechwood xylan by a modified Bacillus subtilis has been previously reported by Amorim, Silvério, Gonçales et al. (2019). Moreover, XOS presented high stability after a static in vitro digestion. However, this method is not elucidative of their prebiotic effect, which depends both on their degree of polymerization (DP) and degree of substitution (Sajib et al., 2018).

Prebiotic compounds have attracted increased attention from academy and industry, as consumers pay more attention to their wellbeing, pivoting their health consciousness to preventive medicine. Thus, the prebiotics global market is expected to increase reaching 7.37 Billion USD by 2023 (MarketsandMarketsTM, 2018). The prebiotic definition was recently updated to “a substrate that is selectively utilized by host microorganisms conferring a health benefit” (Gibson et al., 2017). Prebiotics are indeed attractive compounds due to their multidimensional beneficial effects on both human and animal health, namely on the gastrointestinal tract (e.g. pathogen inhibition, immune modulation), cardiometabolism (e.g. cholesterol lowering), mental health (e.g. energy and cognition) and bones (e.g. enhanced mineral absorption), among others (Gibson et al., 2017; Samanta et al., 2015). Xylooligosaccharides (XOS) have been identified as potentially valuable food and feed prebiotic ingredients (Sajib et al., 2018) and are the only nutraceuticals that can be produced from cheap and abundant lignocellulosic biomass (Samanta et al., 2015). The gut microbiota uses prebiotics to multiply and consequently produce short-chain fatty acids (SCFAs), gases (mainly, hydrogen and carbon dioxide), lactate, and other products (Topping & Clifton, 2001). SCFAs, including acetate, propionate and butyrate, and other compounds such as lactate, are recognized as key metabolites for the intestinal health, influencing others sites distant to the gut (Gibson et al., 2017). Lactate is reported

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Corresponding author. E-mail address: [email protected] (L.R. Rodrigues).

https://doi.org/10.1016/j.carbpol.2019.115460 Received 30 July 2019; Received in revised form 30 September 2019; Accepted 9 October 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Cláudia Amorim, et al., Carbohydrate Polymers, https://doi.org/10.1016/j.carbpol.2019.115460

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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 10 min and the supernatant was further used for HPLC analysis (Section 2.4). Gas samples from the headspace were used to access gas production (Section 2.4). The pH was measured at 48 h and the fermentation broth was withdrawn from the bottles, centrifuged, washed, resuspended in PBS (0.1 M pH 7.0) and stored at −20 °C for DNA extraction and further sequencing analysis (Section 2.5.). Fermentations were run in duplicate, using a blank with no prebiotic addition as negative control.

In vivo studies are expensive and time-consuming, therefore not suitable to be used as a screening tool for selecting prebiotics. The in vitro evaluation of the prebiotic potential of oligosaccharides using fecal inocula and high-throughput sequencing techniques to assess changes on the microbiota are preferable to the use of single or co-cultured microorganisms, since the human gut microbiota is complex and an in vitro evaluation with a limited number of probiotic strains is not representative to understand the prebiotic potential of specific substrates (Gibson et al., 2017). Nonetheless, experimental data on the XOS prebiotic effect regarding in vitro approaches more compatible with the guidelines stated by the updated definition of prebiotic are scarce. In this work, in vitro studies were performed using human fecal inocula and high-throughput sequencing (16S rRNA gene) of microbiota. Lactulose is a well-established and widely accepted prebiotic in the market (Watson et al., 2013), therefore it was used in this work for comparison purposes. The main challenge of this work is to test the hypothesis of XOS produced from beechwood xylan potentially acting as a prebiotic with beneficial effects on human health.

2.4. Analytical techniques The consumption of oligosaccharides was accessed by HPLC as described by Amorim, Silvério, Gonçales et al. (2019), with minor modifications. Mixtures of acetonitrile and water, 68:32 (v/v) (for XOS analysis) and 70:30 (v/v) (for lactulose analysis), were used as mobile phase. Pure lactulose and XOS (DP 2–6, from Megazyme, Bray, Ireland) were used as standards. The production of lactate and SCFAs (acetate, propionate and butyrate) was evaluated according to Fernandes, Rao, and Wolever (2000)), using an HPLC (Knauer, Berlin, Germany) fitted with a KnauerRI detector and an Aminex HPX 87H column (300 mm x 7.8; Biorad, Hercules, CA). Gas samples were analyzed by gas chromatography (GC) using a Bruker Scion 456-GC equipment (Billerica, MA) as described elsewhere (Arantes, Alves, Alfons, Alves, & Sousa, 2018).

2. Experimental 2.1. Prebiotic source XOS were produced by single-step fermentation of beechwood using a cloned Bacillus subtillis 3610 harboring the xylanase gene xyn2 from Trichoderma reesei as previously described (Amorim, Silvério, Gonçales et al., 2019). At the point of maximal XOS production, the cell-free fermentation broth was collected and lyophilized to be further used in the current study as substrate for in vitro batch fermentations. The mixture of linear XOS, DP ranging from 4 to 6, was mainly composed by (1→4)-linked-xylopyranosyl residues with a small amount of branched residues, presenting 3 different fractions: fraction A containing XOS with an average DP of 4 with 5.3% of branching, were the branching points are mainly in disubstituted xylose residues with terminally linked arabinofuranose; fraction B composed of xylopentaose linear oligosaccharides and fraction C presenting an average DP of 6 xylose residues with 2.2% of branching. Commercial lactulose (analytical grade) was obtained from Sigma-Aldrich (St. Louis, MO).

2.5. Microbiota analysis Total DNA from fecal inocula and fecal fermentations at 48 h was extracted from liquid samples using the FastDNA SPIN kit for soil (MP Biomedicals, Solon, OH), according to the manufacturer’s instructions. High-throughput sequencing (16S rRNA gene) by Illumina MiSeq technology 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 Nucleotide Archive under the BioProject accession number PRJEB33616 (Samples accession number: ERS3592390, ERS3592392, ERS3592394, ERS3592396, ERS3592397, ERS3592400, ERS3592401, ERS3592404, ERS3592405, ERS3592406, ERS3592408, ERS3592410, ERS3592412, ERS3592413).

2.2. Fecal inoculum Fecal samples were obtained from two healthy human volunteers who were free of known metabolic and gastrointestinal diseases and did not take any antibiotics, pre- or probiotic supplements for 3 months before the study. The male and female donors aged 26 were non-smokers and consumed non-specific Mediterranean diet. The samples were collected on site, diluted 1/10 (w/w) in anaerobic (100% N2) phosphate-buffered saline solution (PBS, 0.1 M, pH 7.0) and were kept at 4 °C overnight, before inoculation.

2.6. Statistical analysis Differences between products concentrations were checked for significance by ANOVA using Prism 7.0a software (GraphPad. Software. Inc.). Tukey test was used for post hoc comparisons. The differences were considered significant when p < 0.05. 3. Results and discussion

2.3. In vitro batch culture fermentations of oligosaccharides using gut microbiota

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

Static batch culture fermentations were performed at 37 °C during 48 h in serum bottles. The bottles were filled with 40 mL of growth medium at pH 7.0 (peptone water 2 g/L, yeast extract 2 g/L, NaCl 0.1 g/ L, K2HPO4 40 mg/L, KH2PO4 40 mg/L, MgSO4.7H2O 0.01 g/L, CaCl2.6H2O 0.01 g/L, NaHCO3 2 g/L, Tween 80 14.8 ml/L, hemin 5 mg/L, vitamin K1 74.1 μl/L, cysteine HCl 0.5 g/L, bile salts 0.5 g/L, Na2S.9H2O 0.8 mM and resazurine 1 mg/L). The XOS or lactulose solutions were added when required at a final concentration of 10 g/L. Except for the filter-sterilized solutions of vitamin k1 and oligosaccharides, the medium was sterilized by autoclaving and were inoculated with 4.4 mL of fecal inoculum. Anaerobic conditions were maintained by pressurizing the bottles’ headspace with nitrogen up to

Fig. 1 shows the total production of lactate and main SCFAs generated in the colon (≥95% in humans), namely acetate, propionate and n-butyrate, as a result of several bacterial metabolic pathways (Gibson et al., 2017). For both donors, the supplementation with 10 g/L of lactulose or XOS increased significantly (t-test student, α = 0.05) the total production of lactate and SCFAs as expected. These results are well aligned with previous reports on the prebiotic effect of lactulose or XOS using 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). 2

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Fig. 1. Total production of lactate and short chain fatty acids (SCFAs) during 48 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 at 10 g/L. Results are the average of two independent fermentations and triplicate analysis of each sample ± standard deviation.

Fig. 2. Production of lactate and short-chain fatty acids (acetate, propionate and butyrate) after 48 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 at 10 g/L. Results are the average of two independent fermentations and triplicate analysis of each sample ± standard deviation.

Moreover, it seems that the production of lactate and SCFAs is mainly dependent on the addition of prebiotics, rather than the differences between the gut microbiota of the donors (Fig. 1). Nevertheless, further experiments using a larger number of donors should be performed to get a more representative view of the effects of each prebiotic, including individuals from different age range, dietary habits, health backgrounds, among others. The experiments with XOS led to a faster accumulation of lactate and SCFAs from 0 to 12 h, for both donors. This observation was corroborated by the fast consumption of XOS in the same time range. After 24 h of fermentation, XOS presented an utilization of (74 ± 1)% (donor 1, D1) and (71 ± 3)% (donor 2, D2), while lactulose presented an utilization of (50 ± 5)% (D1) and (56 ± 2)% (D2). After 48 h of fermentation, XOS were almost totally consumed, showing an utilization of (92.7 ± 0.8)% and (95.9 ± 0.8)%, for donors 1 and 2, respectively. At this time point, the lactulose utilization was (79 ± 5)% and (74 ± 0.4)% for donors 1 and 2, respectively. The results suggest that the utilization rate is more dependent on the type of prebiotic added, rather than the differences between the gut microbiota of the donors, similarly to the previous observation for SCFAs production. The combination of prebiotics with different fermentation rates can promote fermentation in large parts of the colon, thus potentiating the prebiotic effect (Sajib et al., 2018). Therefore, the supplementation with one or more prebiotic can potentially add value to a product. In comparison to XOS, the use of commercial lactulose led to higher concentrations of lactate and SCFAs after 12 h of fermentation, reaching its maximum difference at 48 h (131 ± 2 mM for the sample from D1, p < 0.0001, and 113.6 ± 0.9 mM, p < 0.0001, for the sample from D2) (Fig. 1). Although the total amount of SCFAs produced is a good indication of the prebiotic potential of the substrates evaluated, the proportion at which they are produced is also important to estimate potential health benefits associated to the consumption of these substrates (Gibson et al., 2017). Fig. 2 shows the production profiles of lactate and SCFAs obtained after 48 h of fermentation by fecal inocula. For both substrates and donors, acetate was the main SCFA accumulated, while propionate was the second most produced. Buruiana et al. (2017); Ruiz et al. (2017) and Dávila, Gullón, Alonso, Labidi, and Gullón (2019)) also reported acetate as being the most abundant SCFA when XOS, from corn stover, olive tree pruning and vine shoots, respectively, were used as substrates for in vitro fermentation with human fecal microbiota. The same evidence was found for lactulose (Ito et al., 1997; Fernandes et al., 2000). Acetate has been reported as beneficial for colorectal cancer prevention (Casanova, Azevedo-Silva, Rodrigues, & Preto, 2018; Ferro

et al., 2016), while propionate plays an important role in the inhibition of cholesterol synthesis and the deposition of adipose tissue, being proposed as a dietary factor to depress appetite and reduce obesity (Arora, Sharma, & Fros, 2011). Interestingly, the supplementation of XOS resulted in the highest production of butyrate (9.0 ± 0.6 mM for samples from D1, and 10.5 ± 0.8 mM for samples from D2), significantly different from the blank for both donors, p < 0.0001. These results are in good agreement with the those reported by Ruiz et al. (2017), that found a butyrate concentration of approximately 14 mM after 48 h of fermentation. Butyrate has been reported as an important metabolite for the maintenance of the intestinal homeostasis and overall health status, exerting several beneficial effects, particularly being associated to the prevention and inhibition of colorectal cancer and diarrhea, and also acting at the extra-intestinal level (Berni Canani et al., 2011; Gonçalves & Martel, 2013). On the other hand, in the experiments with XOS, lactate was undetected after 12 h of fermentation (data not shown), which can be possibly explained by its function as an intermediary in the production of other metabolites, including acetate, butyrate and propionate by other bacterial species (Duncan, Louis, & Flint, 2004). This trend was also observed by Gómez, Míguez, Veiga, Parajó, and Alonso (2015)) and Ruiz et al. (2017). Contrariwise, the addition of lactulose resulted in a high accumulation of lactate (33.7 ± 0.5 mM for samples from D1, and 19.7 ± 0.3 mM for samples from D2) and a reduced production of butyrate. However, since lactate is a precursor of SCFAs, probably increasing the fermentation time would result in the decrease in lactate and further accumulation of SCFAs.

3.2. pH change and ammonia production As previously mentioned, the main health-promoting effects of prebiotic oligosaccharides are associated to the production of SCFA and the subsequent pH drop that promotes the reduction in the pathogenic microbiota and increase in the beneficial bacteria population (Cummings & Macfarlane, 2002). Table 1 shows the final pH and the production of ammonia after 48 h of fermentation of the two substrates under study. The addition of prebiotics led to a pH and ammonia reduction, as expected. The largest pH variation and ammonia reduction were found for experiments with lactulose. Ammonia is related to bad fecal odor 3

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Table 1 pH and ammonia concentration after 48 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 (10 g/L). Results are the average of two independent fermentations and triplicate analysis of each sample ± standard deviation. Assay

Blank Lactulose XOS

Donor 1

Donor 2

pH

Ammonia (mg/ L)

pH

Ammonia (mg/ L)

7.08 ± 0.02a 3.56 ± 0.03b 6.41 ± 0.01c

152 ± 7a 59 ± 1b 131 ± 3ac

7.00 ± 0.03a 3.585 ± 0.05b 6.70 ± 0.30ac

162 ± 5a 49 ± 3b 132 ± 2c*

and high amounts of this compound may contribute to colon carcinogenesis (Visek, 1978). Likewise, Sajib et al. (2018) reported a pH drop to an average of 5.6 or 6.4 pH after 48 h of fermentation, using an in vitro model with human fecal inoculum to ferment arabinoxylans obtained from brewer’s spent grain produced by ultrasound-assisted extraction or by alkaline extraction, respectively. Unlike the in vivo models, the SCFAs formed during fermentation are not absorbed when an in vitro model is used. Therefore, these compounds will greatly alter the medium pH. Although the fermentation medium used in this study is designed to mimic the distal colon pH at baseline, the medium pH was not further controlled throughout the fermentation, which is a limitation of this type of models. In vitro fermentations are regarded as semi-representative models of colonic fermentation and they can be performed by fermentation with pure cultures of selected bacteria, mixed bacterial populations or fecal samples (Macfarlane & Macfarlane, 2007). The use of fecal samples is considered a better approximation to the in vivo models, representing mostly the distal colon microbiota and taking into account complex interactions among bacterial populations (Rycroft, Jones, Gibson, & Rastall, 2001). Moreover, in in vivo models, the produced SCFAs are rapidly absorbed, which limits their measurement (Carlson et al., 2017). Thus, in vitro models are more suitable to study the kinetics of colonic fermentation. However, these models exhibit some limitations, namely the fact that more proximal areas may have a different composition but are hardly accessible with human volunteers. Thus, complex gut models should be used to overcome this issue and further assess prebiotic effects, replicating the different intestinal sections (Payne, Zihler, Chassard, & Lacroix, 2012).

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 of lactulose or XOS at 10 g/L. Results are the average of two independent fermentations and triplicate analysis of each sample ± standard deviation.

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 by the studies performed in vivo with these prebiotics (Bouhnik et al., 2004; Finegold et al., 2014).

3.3. Gas production The oligosaccharides fermentation by fecal inocula can also have an effect on the amount of gases produced. Fig. 3 shows the production of H2 and CO2 during 48 h of fermentation. As expected, larger volumes of CO2 and H2 were obtained from the lactulose and XOS fermentation when comparing to the blank. Carlson et al. (2017) and Buruiana et al. (2017) reported the same trend in gas production using in vitro models with human fecal inoculum and XOS obtained from corn stover. Methane was not detected for both donors’ samples. Indeed, only some people harbour methanogenic archaea which result in methane production (Ghoddusi, Grandison, Grandison, & Tuohy, 2007). Overall, the CO2 production relative to blank was considerably higher in the cultures containing XOS (p < 0.0001 D1 and D2), contrarily to the ones containing lactulose. The maximum production of CO2 was 8.9 ± 0.7 mmol/L at 24 h for samples from D1 and 11 ± 1 mmol/L at 36 h for samples from D2. Since high gas production may result in flatulence problems, which constitute a clinical disincentive to prebiotic use (Cummings, Macfarlane, & Englyst, 2001), it is crucial to evaluate through in vivo models the proper recommended

3.4. Microbiota analysis Although exhibiting different microbial proportions, fecal inocula results obtained for both donors showed as expected, a typical gut microbiota diversity for healthy human adults (Eckburg et al., 2005), being mainly composed by six bacterial phyla, of which Firmicutes relative abundance dominated (43 ± 1% D1; 61.5 ± 0.5% D2) (Table A.1, Supplementary Material). For both donors, adding different oligosaccharides (lactulose or XOS) led to a consistent distinct modulation of the gut microbiota after 48 h of fermentation as compared to the blank (Figs. 4 and 5). The fermentation of lactulose led to a predictable increase in the relative abundance 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 results corroborate the increased production of lactate and SCFAs previously observed and are well aligned with the literature (Watson et al., 2013). Lactobacillus and Bifidobacterium are important genera of commensal 4

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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 a prebiotic solution of lactulose (B) or XOS (C) at 10 g/L.

material). Thus, although the main bacterial growth trends were comparable between the donors, the specific microbiota pattern inferred by the use of a certain prebiotic will depend on the original inoculum. This fact also highlights the need for in vitro and in vivo studies with a representative number and diversity of individuals, as previously discussed. Moreover, the fermentation of lactulose decreased significantly the relative abundance of Bacteriodales (5.1 ± 0.5-fold D1; 5 ± 0-fold D2) (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 this phyla (1.48 ± 0.02-fold D1; 1.50 ± 0.06-fold D2), specially Bacteroides (1.52 ± 0.02-fold D1; 1.79 ± 0.04-fold D2). Microorganisms belonging to the Bacteroides genus are known butyrate-producing bacteria, which in turn is the main source of energy to gut epithelial cells

bacteria widely known for their ability to use complex carbohydrates and to produce lactic acid and SCFAs, which in turn have been associated to human health by several mechanisms. For instance, a reduced amount of SCFAs is associated to a gut microbiota of patients with diabetes, autoimmune disorders, obesity and cancers (Nagpal et al., 2018). Bifidobacterium adolescentis, commonly found in adults (Gibson et al., 2017), was the only Bifidobacterium species identified in the blank, lactulose or XOS fermentation samples for both donors (Table A.1, Supplementary material). Nevertheless, a more diverse community of Lactobacillus species was found. When compared with the blank, a higher diversity of Lactobacillus species was stimulated by lactulose rather than XOS fermentation for both donors. Furthermore, the variety of species is intrinsically related with the donor original microbiota, as shown by the inocula sequencing results (Table A.1, Supplementary 5

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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 a prebiotic solution of lactulose (B) or XOS (C) at 10 g/L.

Gibiino, Binda, & Gasbarrini, 2017), including Escherichia coli whose relative abundance was significantly reduced by the addition of lactulose (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 of methanogenic archaea on the original inoculum may explain its absence on the products profile. For D2, a decrease in the relative abundance of members belonging to Methanobacteriacea family was observed (lactulose: 12.7 ± 0.7-fold; XOS:2 ± 0-fold). The production of CH4 may be comprised below the sensitivity limit of the GC technique, due to the reduced relative abundance of these bacteria on both D2 inoculum and blank samples (Table A.1, Supplementary material). In addition, the largest production of CO2 observed during XOS fermentation, may be related with the stimulation of the Bacteroides growth. The reduction in ammonia production observed during lactulose and XOS fermentations, may also be due to the decrease in the relative abundance of ammonia-producing bacteria, namely E. coli and Clostridium 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,

(Hwang et al., 2017). This observation is in accordance with the results obtained for SCFAs production, namely the highest production of butyrate was observed for XOS fermentation. Thus, the XOS produced by direct fermentation of beechwood xylan appear to be highly selective towards butyrate-producing bacteria, suggesting that these oligosaccharides have potential to be used, for instance, as prebiotic treatment during the active phase of inflammatory bowel disease (IBD). IBD active patients present increased proportions of Bifidobacterium and Lactobacillus on their intestinal microbiota and reduced butyrate-producing bacteria (Wang et al., 2014). Furthermore, Hwang et al. (2017) reported the influence of high-fat diets and low-fiber diets on decreasing members of the Bacteroidales order and butyrate production. On the other hand, the fermentation of both lactulose and XOS led to 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 on the gut microbiota is associated to several intestinal diseases. This phylum comprises several known human pathogens (Rizzatti, Lopetuso,

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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 reduced amounts of Bifidobacterium and Lactobacillus, while XOS produced from beechwood may potentially be used to increase the butyrate concentration and therefore useful for IBD patients’ treatment. 4. Conclusions SCFAs, lactate, ammonia and gas generation, pH variation, and microbiota analysis confirmed for the first time the suitability of the XOS produced by recombinant B. subtilis 3610 from beechwood in a single-step process to present potential functional properties for human health. For both donors’ samples, the fermentation of XOS resulted mainly in the production of acetate, followed by propionate and butyrate, which are important metabolites related to several health benefits. When compared with commercial lactulose, XOS presented the highest production of butyrate and CO2, which was corroborated by the microbiota modulation, namely the increase in the relative abundance of Bacteroides within the bacterial community. This study highlights the microbiota modulation effect promoted by the fermentation of carbo-based substrates with different compositions and structures. This observation suggests distinct potential health applications for lactulose and XOS. Acknowledgments CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from the Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project FoSynBio (POCI-01-0145FEDER-029549), and NewFood (NORTE-01-0246-FEDER-000043). The authors also acknowledge BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115460. References Aachary, A. A., Gobinath, D., Srinivasan, K., & Prapulla, S. G. (2015). Protective effect of xylooligosaccharides from corncob on 1,2-dimethylhydrazine induced colon cancer in rats. Bioactive Carbohydrates and Dietary Fibre, 5, 146–152. Amorim, C., Silvério, S. C., Gonçales, R. F. S., Pinheiro, A. C., Silva, S. P., Coelho, E., et al. (2019). Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610. Carbohydrate Polymers, 205, 176–183. Amorim, C., Silvério, S. C., Prather, K. L. J., & Rodrigues, L. R. (2019). From waste to market: Production and commercial potential of xylooligosaccharides. Biotechnology Advances. https://doi.org/10.1016/j.biotechadv.2019.05.003 (In Press). Arantes, A. L., Alves, J. I., Alfons, J. M., Alves, M. M., & Sousa, D. Z. (2018). Enrichment of syngas-converting communities from a multi-orifice baffled bioreactor. Microbial Biotechnology, 11, 639–646. Arora, T., Sharma, R., & Fros, G. (2011). Propionate, Anti-obesity and satiety enhancing factor? Appetite, 56, 511–515. Berni Canani, R., Di Costanzo, M., Leone, L., Pedata, M., Meli, R., & Calignano, A. (2011). Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World Journal of Gastroenterology, 17, 1519–1528. Bouhnik, Y., Attar, A., Joly, F. A., Riottot, M., Dyard, F., & Flourié, B. (2004). Lactulose ingestion increases faecal bifidobacterial counts: A randomised double-blind study in healthy humans. European Journal of Clinical Nutrition, 58, 462–466. Buruiana, C. T., Gómez, B., Vizireanu, C., & Garrote, G. (2017). Manufacture and evaluation of xylooligosaccharides from corn stover as emerging prebiotic candidates for human health. LWT - Food Science and Technology, 70, 449–459. Carlson, J. L., Erickson, J. M., Hess, J. M., Gould, T. J., & Slavin, J. L. (2017). Prebiotic

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