Role of Intestinal Microbiota in Ulcerative Colitis
Role of Intestinal Microbiota in Ulcerative Colitis Role of Intestinal Microbiota in Ulcerative Colitis
1040 J. Holck et al. / Process Biochemistry 46 (2011) 1039–1049 Fig. 1. (A) Schematic illustration of sugar beet pectin structural elements. (B) Schematic flow sheet for biorefining strategy indicating the enzymatic treatments and membrane separation steps. Finally sugar beet pectin contains minor amounts of rhamnogalacturonan II (RGII), that has a backbone similar to HG substituted with side chains of a complex structure and composition [7]. The combination of shorter HG chain length, high degree of acetylation and the large amount of side chains contributes to the poor gelling properties of sugar beet pectin [1]. Several studies have indicated health promoting effects of pectin derived compounds. These effects include (i) prebiotic effects that selectively increase growth and/or activity of bacteria considered to be probiotic such as Bifidobacterium and Lactobacillus strains [8–12], (ii) modification of the ratio between subgroups of the phyla Bacteroidetes and Firmicutes [13], and (iii) a decrease in the growth of potentially pathogenic bacteria like clostridium [11]. It has also been reported that certain pectin derived oligosaccharides reduce the binding of E. coli Shiga-like toxin in human colonic adenocarcinoma cell receptors [14], induce apoptosis in human colonic adenocarcinoma cells [15] and prostate cancer cells [18], inhibit proliferation of several types of cancer along with antioxidant effects [16], and increase urinary excretion of toxic metals [17]. A common theme in numerous papers regarding health promoting effects of pectin is the dependency of size of the applied poly- and oligosaccharides [12,14,16]. Nevertheless, the studies assessing the putative bioactivity of pectin have been performed using mixtures of heterogeneous pectin derived fractions with a relatively broad size distribution [9–11,18,19]. Several studies based on the pectic oligosaccharides generated by Olano-Martin et al. [20] revealed that a low methoxyl fraction with a binominal molecular weight distribution of approximately 3.8 kDa and 0.97 kDa (roughly corresponding to DP21–23 and DP5–6) showed better potential as a health promoting candidate than a high methoxyl fraction with a molecular weight of approximately 3.5 kDa (corresponding to DP20–21). The work by Manderson et al. [8] and Mandalari et al. [9] indicated that pectic oligosaccharides with DP
Table 1 List of enzymes. Enzyme Source pH optimum Temperature optimum J. Holck et al. / Process Biochemistry 46 (2011) 1039–1049 1041 EC number Family Reference Pectin lyase Aspergillus nidulans 7.0 n.a. 4.2.2.10 PL 1 [29] �-Galactosidase-1 Aspergillus niger 4.0–4.5 60 ◦ C 3.2.1.23 GH 35 Megazyme �-Galactosidase-2 Kluyveromyces lactis n.a. n.a. 3.2.1.23 GH 2 n.a. �-Galactanase Aspergillus niger 4.0–4.5 50 ◦ C 3.2.1.89 GH 53 Megazyme �-Arabinofuranosidase Aspergillus aculeatus 4.0–4.5 30 ◦ C 3.2.1.55 – [50] �-Arabinanase Aspergillus aculeatus 5.5 30 ◦ C 3.2.1.99 GH 43 [50] RGI lyase Bacillus licheniformis 8.0–8.5 60–65 ◦ C 4.2.2.– – Unpublished data phy to obtain the structural elements and avoids the use of organic solvents. To assess the possible effect of different chain length, the study includes evaluation of the biological activity of homogalacturonan oligosaccharides DP4 and DP5 based on their capacity to alter the ratio between Bacteroidetes and Firmicutes, which represent the dominant bacterial phyla in the human intestine. This is assessed by in vitro bacterial fermentation using human faecal samples. 2. Materials and methods 2.1. Substrate Sugar beet pectin was obtained from Danisco A/S (Nakskov, Denmark). The pectin had been prepared by sequential acid extraction with nitric acid from sugar beet pulp, involving removal of insoluble cellulose, ultrafiltration, and diafiltration with a 50 kDa cutoff essentially as described by Buchholt et al. [1], except that precipitation in isopropanol was replaced by spray drying. Degree of methoxylation and acetylation were determined to be 59% and 20%, respectively. 2.2. Chemicals Ammonium formate, Trizma-HCl, sodium acetate, acetic acid, hepes, manganese chloride, d-glucuronic acid, d-galactose, d-arabinose, d-fucose, l-rhamnose monohydrate, d-galacturonic acid monohydrate, and �-cyano-4-hydroxy-cinnamic acid were purchased from Sigma–Aldrich (Steinheim, Germany) d-glucose and d-xylose were purchased from Merck (Darmstadt, Germany). Trifluoroacetic acid was from Riedel-de Haën (Seelze, Germany). High resolution Source 15Q anion exchange resin and chromatography columns were obtained from GE Healthcare Biosciences (Uppsala, Sweden). All chemicals used were analytical grade. 2.3. Enzymes Pectin lyase was produced by fermentation essentially as described by Stratton et al. [28]. The Pichia pastoris clone transformed with the pectin lyase gene AN2569.2 was obtained from the Fungal Genetic Stock Center as described by Bauer et al. [29]. Rhamnogalacturonase I from Bacillus licheniformis was cloned and expressed in P. pastoris (unpublished results). Galactanase and �-galactosidase-1 were purchased from Megazyme (Bray, Co. Wicklow, Ireland), �-galactosidase-2 was purchased from Sigma–Aldrich (Steinheim, Germany). 1 and 2 denotes the origin of gene, either Aspergillus niger (1) or Kluyveromyces lactis (2). Arabinanase and �-l-arabinofuranosidase were obtained from Novozymes (Bagsvaerd, Denmark) (Table 1). 2.4. Biorefining strategy The sugar beet pectin was separated into homogalacturonan (HG) and rhamnogalacturonan I (RGI) by sequentially applying monocomponent enzymes addressed towards certain structures in the pectin molecule, followed by removal of the released oligomers by membrane separation (Fig. 1B). Pectin lyase was employed to catalyze the cleavage of methoxylated homogalacturonan by �-elimination. This gave rise to a double bond in between carbon 4 and 5 of galacturonic acid in the nonreducing end of one of the cleavage products. In turn, this allowed the assessment of the molar level of released products in the HG permeate by UV spectroscopy. Next, enzymes active towards arabinan and galactan side chains of the RGI were added to catalyze the release of arabinose and galactose oligomers and monomers. These products were separated into the side chain (SC) permeate (Fig. 1). Finally RGI lyase was added to the retentate to catalyze the cleavage of the remaining RGI backbone by �-elimination (Fig. 1) again introducing a detectable double bond. The released RGI oligomers were separated into the RGI permeate. Any unprocessed material was found in the final retentate. 2.5. Membrane reactions and separation All reactions and separations were performed in a 200 mL stirred membrane reactor model 8200 (Millipore, Billerica, MA) equipped with a 3 kDa regenerated cellulose membrane (Millipore, Billerica, MA), connected to compressed nitrogen for flux regulation. The reactor contents were mixed by magnetic stirring using a RCT basic magnetic stirrer (IKA, Germany). Temperature control was maintained using a Julabo ED5 water bath (Julabo, Germany). 2.6. Pectin lyase treatment A 3% (w/w) SB pectin solution in 50 mM Tris buffer pH 8 was treated with 0.5% E/S (enzyme/substrate) pectin lyase and incubated at 50 ◦ C for 15 h in a stirred and thermostatically controlled reactor. The reaction mixture was transferred to the membrane reactor and filtration was performed at 50 ◦ C and 2 bars. The retentate was diafiltrated twice in 0.7 sample volumes of water. All permeate was pooled and stored at 5 ◦ C until further use. 2.7. Side chain degrading treatment A 1.4% (w/w) RGI retentate solution in 50 mM acetate buffer pH 5 was treated with a mixture of 0.2% �-galactosidase-1, 4.7% �-galactosidase-2, 0.2% galactanase, 0.8% arabinofuranosidase, and 0.8% arabinanase (all percentages in % E/S) and incubated in a membrane reactor at 45 ◦ C for 16 h. The enzymes were dosed according to a tentative estimate of the available concentration of the specific structural substrate element and the individual enzyme activity. Filtration was performed at 50 ◦ C and 2 bars. The retentate was diafiltrated once in 1 sample volume of water. All permeate was pooled and stored at 5 ◦ C until further use. 2.8. RGI lyase treatment Prior to the enzymatic treatment, RGI lyase was incubated in 100 mM MnCl2 for 0.5 h for activation [30]. A 0.75% (w/w) RGI backbone solution in 50 mM Hepes buffer pH 7.8 was treated with 0.5% E/S RGI lyase and incubated in a membrane reactor at 60 ◦ C for 2 h. Filtration was performed at 60 ◦ C and 2 bars. The retentate was diafiltrated twice in 1 sample volume of water. All permeate was pooled and stored at 5 ◦ C until further use. 2.9. Alkaline deesterification Prior to ion exchange chromatography, methoxyl- and acetyl groups were removed from pectic substances using cold alkaline conditions. The low temperature was used in order to prevent spontaneous �-elimination [31]. Sample temperature was lowered to approx. 5 ◦ C and cold 2 M NaOH was added under stirring until pH 13 and a final concentration of 40 mM NaOH was reached. Samples were incubated for 24 h. After incubation pH was adjusted using 1 M acetate buffer pH 6.5 and 2 M HCl. Samples were diluted to conductivity under or equal to the conductivity of the ion exchange buffer. 2.10. Ion exchange chromatography Anion separation was performed at room temperature with an ÄKTA purifier 100 work station equipped with a P-900 pump, UV-900 monitor, pH/C monitor, and Frac-950 fraction collector, all controlled by UNICORN software. A HR16/10 column with a 23 mL packed bed of high resolution Source 15Q was used for separation. The elution of the unsaturated oligosaccharides was monitored by the UV absorption at 235 nm and subsequently quantified by use of the molar extinction coefficient 4600 M −1 cm −1 [32]. Baseline correction was made with a blank run. Elution was performed at a flow rate of 10 mL/min with water and ammonium formate as eluents. Before injection the column was equilibrated with 20 mM ammonium formate for two column volumes (CV). After injection the column was washed with 20 mM ammonium formate for three CV, followed by elution with a gradient specified below. After elution the column was regenerated with 1000 mM ammonium formate for two CV and with water for three CV. To obtain the oligomers in the HG permeate, a sample was injected on the column and eluted using a linear gradient
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1040 J. Holck et al. / Process Biochemistry 46 (2011) 1039–1049<br />
Fig. 1. (A) Schematic illustration <strong>of</strong> sugar beet pect<strong>in</strong> structural elements. (B) Schematic flow sheet for bioref<strong>in</strong><strong>in</strong>g strategy <strong>in</strong>dicat<strong>in</strong>g the enzymatic treatments and membrane<br />
separation steps.<br />
F<strong>in</strong>ally sugar beet pect<strong>in</strong> conta<strong>in</strong>s m<strong>in</strong>or amounts <strong>of</strong> rhamnogalacturonan<br />
II (RGII), that has a backbone similar to HG substituted<br />
with side cha<strong>in</strong>s <strong>of</strong> a complex structure and composition [7]. The<br />
comb<strong>in</strong>ation <strong>of</strong> shorter HG cha<strong>in</strong> length, high degree <strong>of</strong> acetylation<br />
and the large amount <strong>of</strong> side cha<strong>in</strong>s contributes to the poor gell<strong>in</strong>g<br />
properties <strong>of</strong> sugar beet pect<strong>in</strong> [1].<br />
Several studies have <strong>in</strong>dicated health promot<strong>in</strong>g effects <strong>of</strong> pect<strong>in</strong><br />
derived compounds. These effects <strong>in</strong>clude (i) prebiotic effects that<br />
selectively <strong>in</strong>crease growth and/or activity <strong>of</strong> bacteria considered<br />
to be probiotic such as Bifidobacterium and Lactobacillus stra<strong>in</strong>s<br />
[8–12], (ii) modification <strong>of</strong> the ratio between subgroups <strong>of</strong> the<br />
phyla Bacteroidetes and Firmicutes [13], and (iii) a decrease <strong>in</strong> the<br />
growth <strong>of</strong> potentially pathogenic bacteria like clostridium [11]. It<br />
has also been reported that certa<strong>in</strong> pect<strong>in</strong> derived oligosaccharides<br />
reduce the b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> E. coli Shiga-like tox<strong>in</strong> <strong>in</strong> human colonic<br />
adenocarc<strong>in</strong>oma cell receptors [14], <strong>in</strong>duce apoptosis <strong>in</strong> human<br />
colonic adenocarc<strong>in</strong>oma cells [15] and prostate cancer cells [18],<br />
<strong>in</strong>hibit proliferation <strong>of</strong> several types <strong>of</strong> cancer along with antioxidant<br />
effects [16], and <strong>in</strong>crease ur<strong>in</strong>ary excretion <strong>of</strong> toxic metals [17].<br />
A common theme <strong>in</strong> numerous papers regard<strong>in</strong>g health promot<strong>in</strong>g<br />
effects <strong>of</strong> pect<strong>in</strong> is the dependency <strong>of</strong> size <strong>of</strong> the applied poly- and<br />
oligosaccharides [12,14,16]. Nevertheless, the studies assess<strong>in</strong>g the<br />
putative bioactivity <strong>of</strong> pect<strong>in</strong> have been performed us<strong>in</strong>g mixtures<br />
<strong>of</strong> heterogeneous pect<strong>in</strong> derived fractions with a relatively broad<br />
size distribution [9–11,18,19]. Several studies based on the pectic<br />
oligosaccharides generated by Olano-Mart<strong>in</strong> et al. [20] revealed<br />
that a low methoxyl fraction with a b<strong>in</strong>om<strong>in</strong>al molecular weight<br />
distribution <strong>of</strong> approximately 3.8 kDa and 0.97 kDa (roughly correspond<strong>in</strong>g<br />
to DP21–23 and DP5–6) showed better potential as a<br />
health promot<strong>in</strong>g candidate than a high methoxyl fraction with<br />
a molecular weight <strong>of</strong> approximately 3.5 kDa (correspond<strong>in</strong>g to<br />
DP20–21). The work by Manderson et al. [8] and Mandalari et al.<br />
[9] <strong>in</strong>dicated that pectic oligosaccharides with DP
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