Citrus pectin

Esterase-mediated uronic acid formation was monitored continuously using the K-URONIC kit (Megazyme, Ireland). Kinetic measurements were performed in 96-well plates using a FLUOstar Omega (BMG LABTECH, Germany) in 200 μL reactions containing 50 mM sodium phosphate, 2 μL uronate dehydrogenase, and 16 μL NAD⁺. The buffer pH was at or close to the enzymes’ respective pH optima, due to substrate instability at higher pH, and where > 75% of maximal enzyme activity is maintained: pH 7.5 for O. terrae and S. usitatus enzymes and pH 6.5 for S. linguale enzymes. The substrates BnzGlcA, AllylGlcA, MeGlcA, and MeGalA (Additional file 1: Figure S2) (Carbosynth, UK) were dissolved in 100% dimethyl sulfoxide (DMSO); all reactions contained ≤ 10% DMSO. Kinetic assays were performed at least in duplicate at 25 °C using enough enzyme to ensure ≥ 2-fold change in substrate turnover versus auto-hydrolysis rates. pH-dependency profiles were generated with 2 mM BnzGlcA in a three-component buffer containing 25 mM acetic acid, 25 mM 2-(N-morpholino)ethanesulfonic acid, and 50 mM Tris–HCl, covering pH 4.5–9.5 [36 (link)].
Acetyl esterase activity was assayed using 4-nitrophenyl acetate (pNP-Ac; Sigma Aldrich) and 1,2,3,4-tetra-O-acetyl-β-d-xylopyranose (TetAcXyl; Carbosynth) (Additional file 1: Figure S2). pNP release was detected at λ₄₀₅ and quantified using an extinction coefficient 18.7 mM⁻¹cm⁻¹. Acetate release from TetAcXyl were measured using the K-ACET kit (Megazyme). Pectin methyl esterase activity was assayed with poly-d-galacturonic acid methyl ester (Carbosynth) and citrus peel pectin (Sigma Aldrich) in reactions containing 0.2% (w/v) pectin and 1 mg/mL CE15 enzyme. Reactions were collected at 30 min and 24 h, filtered through a 10 kDa Amicon spin filter, and methanol release through NAD⁺ reduction using alcohol oxidase (Pichia pastoris, Sigma Aldrich) and formaldehyde dehydrogenase (Pseudomonas sp., Sigma) as previously described [37 (link)]. Nonlinear data were fitted to the Michaelis–Menten equation using GraphPad Prism (GraphPad, US). In nonsaturable cases, k_cat/K_m values were determined by linear regression.

Animals. Male mice, 35 gm wild-type C57BL/6 (Jackson Laboratory, Bar Harbor, ME,), were anesthetized prior to euthanasia. The care of the mice was consistent with guidelines of the American Association for Accreditation of Laboratory Animal Care (Bethesda, MD, USA) and was approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee. Pleural adhesion assays were performed with porcine small bowel, which was procured by a local vendor (Research 86, Boston, MA, USA), and studied with a protocol approved by the Brigham and Women’s Hospital Institutional Animal Care and Use Committee.
Pectin. The unstandardized citrus pectins used in this study were obtained from a commercial source (Cargill, Minneapolis, MN, USA). The characterization of the high methoxyl citrus pectin has been detailed elsewhere [27 (link)]. Briefly, the proportion of galacturonic acid residues in the methyl ester form determines the degree of methoxylation. The high-methoxyl pectins (HMP) demonstrated a greater than 50% degree of methoxylation. The pectin powder was stored in low humidity at 25 °C.
Pectin dissolution in water. The pectin powder was dissolved at 25 °C by a controlled increase in added water to avoid undissolved powder [28 (link)]. Sequential swelling, softening, and fluidization of the particles was followed by dissolution [29 (link)]. As described elsewhere [30 (link)], the complete dissolution of the pectin was produced using a high-shear 10,000 rpm rotor-stator mixer (L5M-A, Silverson, East Longmeadow, MA, USA). Reproducible viscosity was ensured using a digital tachometer and ammeter (DataLogger, Silverson). The dissolved pectin was poured into variable-sized molds for further studies.
Pressure-sensitive adhesive (PSA). The PSA was a proprietary multi-purpose acrylic adhesive made available through the cooperation of the 3M Corporate Research Materials Laboratory (St. Paul, MN, USA).
Nanocellulose fibers (NCF). The NCF powder, obtained from the University of Maine (Process Development Center, Orono, ME, USA), was dissolved at 25 °C by a controlled increase in water similar to previous reports. Briefly, NCF dissolution was obtained with progressive hydration, followed by a high-shear 10,000 rpm rotor-stator mixer (L5M-A, Silverson). The dissolved NCF was poured into standardized molds and cured for further studies.
Adhesion testing. Pectin–lung adhesion experiments were performed with a custom fixture designed for the TA-XT plus with 5 kg of the load cell (Stable Micro Systems). The fixture was composed of a 30 mm diameter flat-ended stainless-steel cylindrical probe and a flat stainless-steel fixture surface; both surfaces used adhesive mounts to fix the pectin film and tissue specimen. The probe descended at the selected probe velocity until encountering a trigger force of 1N. The probe compressed the pectin films and pleural sample at a selectable compression force (typically 1–5N) and development time. The probe was then withdrawn at 0.5 mm/s with constant force and distance recordings at 500 pps.
Transillumination stereo microscopy. The film interface was transilluminated with a 4000 lumen 6000 K LED light with custom diffusion filter to assure uniform illumination. Probe compression and withdrawal was recorded with a 16 Mega pixel camera (Hayear, Shenzhen, PRC) at 60 frames per second mounted to a Nikon SMZ 1000 stereo microscope (Nikon, Tokyo, Japan) as previously described [31 (link)]. The recorded MOV files were converted to MetaMorph (Molecular Devices, Downington, PA, USA) compatible STK files for morphometric analysis. Time-base correction was integrated into the recordings for calibration of the image stacks.
Scanning electron microscopy. After coating with 20–25 A gold in an argon atmosphere, the pectin films were imaged using a Philips XL30 ESEM scanning electron microscope (Philips, Eindhoven, the Netherlands) at 15 Kev and 21 μA. A eucentric sample holder was used for standardized automation.
Enzyme treatment. The tissues were treated with three commercially obtained enzymes previously used (Sigma-Aldrich, St. Louis, MO, USA). Hyaluronidase cleaved the 1/4 linkages between N-acetyl-D-glucosamine and D-glucuronate. The hyaluronidase solution and tissues were maintained at 37 °C during a 90-min incubation. After enzyme treatment, the tissues were washed with PBS three times.
Statistical analysis. The statistical analysis was based on measurements in at least three different samples. The unpaired Student’s t-test for samples of unequal variances was used to calculate statistical significance. The data was expressed as mean ± one standard deviation. The significance level for the sample distribution was defined as p < 0.01.

Citrus pectin films were developed following the procedure outlined in Çavdaroğlu et al. [20 (link)], with a slight modification. Kanpei citrus peel pectin (amount specified in Table 1) was mixed with 100 mL of distilled water. The mixture was homogenized at 5000 rpm for 2 min using a homogenizer (Daihan Scientific Co., Ltd., Republic of Korea) before being left to stir in a water bath at 60 °C for 30 min. Next, 30% glycerol (w/w) was added, and the solution was continuously stirred at 60 °C for another 30 min. Glycerol was added as a plasticizer due to its stability and compatibility with pectin hydrophilic biopolymer chains [21 (link)]. To remove air bubbles, the mixture was vacuum degassed using an ultrasonic device (JAC-3010, Kodo Technical Research Co., Ltd., Hwaseong, Republic of Korea). After that, films were cast on leveled plastic plates using 25 mL of the film-forming solution for each plate and dried at 45 °C in an oven dryer for 2 days. After drying, the films were carefully removed from the plates and set aside for further processing.
For the pre-dry PLA component, the PLA pellet was pre-dried in an oven dryer (OTEC-004-m, Republic of Korea) at 60 °C overnight, and then a predetermined amount of PLA, as outlined in Table 1, was dissolved in 100 mL of chloroform through 2 h of stirring until complete dissolution was achieved. The subsequent step involved the combination of PLA and dried pectin films: 25 mL of the PLA solution was applied to a glass casting plate, onto which the dried citrus pectin film was delicately placed. The solution was allowed to dry naturally at room temperature until both PLA and pectin films were thoroughly dried and harmoniously fused.

This study included four dietary carbohydrates provided by General Mills. Agave-derived powdered long-chain inulin consisted of fructose polymers (>90% dry weight) with minor amounts of fructose (<12% dry weight), glucose, and sucrose. Corn fiber consisted of a mixture of glucose polymers and insoluble non-digestible carbohydrates (>85% dry weight) derived from partially hydrolyzed starch-made glucose syrup with a minor monosaccharide content (<15% dry weight). Polydextrose consisted of synthetic highly branched and randomly bonded polymers of glucose (>90% dry weight) with minor amounts of bound sorbitol and citric acid (<2% dry weight). Sourced from by-products of the juice and citrus-oil processing industries, citrus peel-derived pectin corresponds to low-ester pectin standardized with sugars.