All reagents were of analytical grade and purchased from Sigma-Aldrich (Sigma-Aldrich, Steinheim, Germany) unless otherwise noted.
Characterization of pectinolytic enzymes
Pectin lyase (PL) and polygalacturonase (PG) were produced as monocomponent enzymes by fermentations as described in de Silva et al. (Silva et al. 2011) using Pichia pastoris clones transformed with the PL gene AN2569.2 and the PG gene AN4372.2, both from Aspergillus nidulans, as described in Bauer et al. (Bauer et al. 2006). The protein concentrations in the enzyme solutions following sterile filtration and ultrafiltration were determined by the bicinchoninic acid assay with bovine serum albumin as a standard as described by the manufacturer (Thermo Fisher Scientific, Rockford, IL).
PL activity was assayed by incubating 100 µL of enzyme sample, diluted 10 times in 200 mM N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), pH 8.6, with 100 µL 1 g/L citrus pectin (Sigma-Aldrich, Steinhein, Germany) in 200 mM CHES, pH 8.6, at 37 °C in triplicate. The reaction was followed for 60 min in an Infinite200 Microplate Reader (Tecan, Salzburg, Austria) by recording the absorbance at 235 nm. A standard curve of 0–1.5 mg/L PL was used to express the amount of enzyme in the samples as percentages of the fed amount. A measured activity corresponding to 0.012 mg enzyme, corresponding to 2 % (w/w) of the fed amount, was considered a positive presence of enzyme in the given section.
PG was assayed by a modified ruthenium red assay (Torres et al. 2011; Ortiz et al. 2014). Briefly, samples were diluted twice in 200 mM acetate buffer (pH 4.5) and 60 µL diluted sample was added to 20 µL 4 g/L polygalacturonic acid (Sigma-Aldrich) in acetate buffer at pH 4.5 at 600 RPM and 20 °C in triplicate. After 60 min of incubation, the enzymatic reaction was stopped by adding 920 µL 100 mg/mL ruthenium red followed by mixing at 1000 RPM for 5 min and centrifugation for 5 min at 5000g. Controls were made by addition of diluted sample after addition of ruthenium red. A standard curve of 0–1.375 mg/L PG was used to express the amount of enzyme in the samples as percentages of the fed amount. A measured activity corresponding to 0.012 mg enzyme, corresponding to 2 % of the fed amount, was considered a positive presence of enzyme in the given section.
Pepsin in the stomach supernatant was assayed by incubating 50 µL supernatant in 250 µL 0.06 M HCl with 20 g/L hemoglobin in triplicate. The reaction was run for 37 °C at 900 RPM and terminated after 60 min with 500 µL of 5 % w/v trichloracetic acid. After 10 min of mixing, the samples were centrifuged for 2 min at 14,000g and 100 µL of the supernatant was measured at 280 nm in an Infinite200 microplate reader (Tecan, Salzburg, Austria). Controls were made by adding the trichloracetic acid before the gastric sample. Units were defined as nmol tyrosine released per minute using an extinction coefficient of 1250 M−1cm−1. Pepsin concentration was calculated by comparing the activity to a standard curve of commercial pepsin [P7125, #SLBB6557V] (Sigma-Aldrich).
Animals
Twenty pigs were purchased from a commercial farm in Denmark at 30 days of age, 2 days after weaning. The animals were of mixed breed, had been given a wheat based creep feed since 7 days of age, had not been given antibiotics or growth promoters and weighed ~7 kg. The pigs were fed a commercial weaning feed, Porkido™, 24 h before the start of the experiments and were not fed on the day of the experiment.
All handling of animals was performed by trained personnel and veterinarians, fulfilling the regulations from the Danish Ministry of Justice.
Experimental design
FiberBind (KMC a.m.b.a, Brande, Denmark), the dried residue following starch removal from potatoes, was ground to ~300 µm particle size and 2000 mg was weighed off into a syringe. To avoid catalysis outside the animals, all reagents were kept on ice: 5 min before feeding, 19 mL of ice-cold water was drawn into the syringe and mixed thoroughly on ice. Immediately before use, 1 mL of 0.6 g/L of each PL and PG was drawn into the syringe, resulting in a final concentration of 0.03 g/L of each enzyme e.g. an enzyme-to-substrate ratio of 0.03 % (w/w). Control animals received no enzyme. The reaction was initiated by orally placing a rubber tube in stomach of each animal by which they were given the mixture. Animals were sacrificed by an overdose of pentobarbital and jugular bleeding in duplicate at 20, 40, 60, 120 and 180 min after feeding. Immediately after death, the pigs were necropsied and the entire contents of the stomach and small intestine was extracted and put on dry ice, followed by storage at −20º C until further analysis. Before extraction of the content from the small intestine, the entire intestine was laid out and divided into three equally long sections; the contents of which is hence referred to as Duo, Jeje and Ile.
Determination of reaction progress
All handling of samples was done on ice. Sample weight was noted, followed by measurement of pH using a pH-meter and dry matter by overnight heating at 105 °C. Subsequently, samples were spun at 21,000g for 10 min at 4 °C. Filtration was avoided due to occasional low sample volume. The supernatant was then measured for the activity of PL and PG as well as pepsin activity. The polymeric carbohydrate content in the remaining supernatant was precipitated by addition of isopropanol to a final concentration of 70 % (v/v) after which it was centrifuged at 5000g for 5 min. The supernatant was discarded and the pellet was dried in a vacuum drier and weighed.
The chemical characteristics of GRG are the monosaccharides rhamnose, galacturonic acid and galactose as well as a molecular size larger than 100 kDa. As such, the measure of reaction progress was then the amount of the monosaccharides rhamnose, galactose and galacturonic acid as well as the amount of carbohydrate with a molecular mass larger than 100 kDa (CH100) in the IPA-precipitated residue. Rhamnose and galacturonic acid in particular are unique to pectin.
Carbohydrate composition
Monosaccharide composition analysis was done by a modified NREL sulphuric acid hydrolysis (Sluiter et al. 2011). In brief, ~5 mg of finely ground carbohydrate sample was added to 300 µL 72 % sulfuric acid, followed by 60 min incubation in a water bath at 30 °C. Next, 8.4 mL of water was added and samples were autoclaved at 121 °C for 60 min. Monosaccharides were then quantified on a HPAEC-PAD system fitted with a Dionex CarboPac PA1 analytical column (2 × 250 mm) combined with a CarboPac PA1 precolumn (4 × 50 mm) by eluting with 0.25–500 mM NaOH at 1 mL/min (Ravn et al. 2015). Fucose, rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid were included as standards. Glucosamine and galactosamine were included to account for monosaccharides from the host. Monosacharides concentrations were calculated from the standard curves, giving the concentration in the hydrolyzed sample and, in turn, the total mass of monosaccharide in the intestinal content.
Size determinations were done by dissolving carbohydrate samples at 3 g/L in 0.1 M sodium acetate, pH 6, with 0.02 % (w/v) sodium azide followed by filtering with a 0.22 µm filter. Samples where then injected in Shodex OHpak SB-806 HQ (8.0 × 300 mm) column (Showa Denko KK, Kawasaki, Japan), and eluted with 0.1 M sodium acetate (pH 6). The injection volume was 100 µL and the flow rate was 0.5 mL/min at 30 °C on a system consisting of a P680 HPLC pump and an ASI-100 automated sample injector using a refractive index detector Shodex RI-101 (Showa Denko KK). Pullulan standards of 1.3, 10, 110, 400 and 800 kDa were used. As the precipitate was not always fully soluble, the concentration of CH100 was calculated from a standard curve of fully soluble GRG. This was then expressed as the mass of CH100 in the intestinal content.
In vitro reaction kinetics
The action of each enzyme in the stomach was evaluated by an in vitro model of the piglet stomach. 1000 mg of FiberBind (300 µm particle size) was added to 30 mL of water with 0.32 mg/mL pepsin which was adjusted to pH 2 with 475 µL 1 M HCl. This was preheated in a 45 °C water bath for 5 min, after which 0.3 mg of either PG or PL was added. Controls were without any enzyme. To stop reactions, tubes were shaken vigorously on ice and immediately centrifuged for 5 min at 5000g at 5 °C. 10 mL of supernatant was then filtered in a Whatman 40 filter and precipitated with 23.3 mL isopropyl alcohol, after which the supernatant was discarded and the pellet was dried in a vacuum drier and weighed. CH100 could be used as the sole reaction parameter, since the in vitro reactions did not contain obscuring compounds such as the in vivo ones.
Data presentation and statistics
For the in vivo data, means are presented in graphs. Linear regression and squared Pearson correlation coefficients were used to relate rhamnose, galacturonic acid, galactose and CH100 with one another and to examine relationship with incubation time and PG. To ease interpretation, data is generally presented as the cumulative amount in the entire GIT, e.g. the sum of the four sections in each animal. ANCOVA was used to examine the effect of time and enzyme addition on the accumulated release of the four main parameters. When appropriate, the data is also expressed as a percentage of the theoretically available amount, calculated from Strube et al. (Strube et al. 2015).
In vitro data was fitted with a modified Monod equation of the form.
$${\text{CH1}}00 = {\text{a}} + ({\text{b }}*{\text{ t}}/({\text{c}} + {\text{t}})),$$
where t is time, a is the value of CH100 at t = 0, b is the maximum value with value at t = 0 subtracted, c is the time at which half of the maximum release is attained.
