Pectin from citrus peel

The dried Tremella fuciformis was obtained from Gutian, Fujian, China. They were used to extract Tremella fuciformis polysaccharides in our laboratory, named L-TPS. For comparison, another 3 types of commercial polysaccharides (TMS, TPS-120, and TPS-160) were purchased from Shandong Freda Biological Co., Ltd. (Same raw material was used to extract these commercial polysaccharides. They were extracted and deproteinized with the same method and then subjected to degradation by enzyme and alkali solutions to obtain polysaccharides with different molecular masses). The sunflower oil was purchased from the Aldi supermarket in Ireland. The pectin from citrus peel and carboxymethylcellulose sodium salt were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The soluble soybean polysaccharide was provided by Zhengzhou Tianshun Food Additives Co., Ltd. (Zhengzhou, China). All other chemicals and solvents were of analytical grade.

The following chemicals were used: 3,5-dinitrosalicylic acid (DNSA) (98%, Thermo Fisher Scientific, Maharahstra, India), sodium sulphite (Lach-ner, Brno, Chez Republic), sodium hydroxide (Lach-ner, Brno, Chez Republic), phenol (99 + %, Thermo Fisher Scientific, Maharahstra, India), potassium sodium tartrate (Sigma-Aldrich, Buchs, Switzerland), Folin–Ciocalteu reagent (Sigma-Aldrich, Buchs, Switzerland), anhydrous sodium carbonate (Lach-ner, Brno, Chez Republic), gallic acid (anhydrous) for synthesis (Merck, Darmstadt, Germany), cellulase (from Aspergillus niger) (Sigma-Aldrich, Tokyo, Japan), pectinase (from Aspergillus niger) (Sigma-Aldrich, Buchs, Switzerland), xylanase (from Theryomyces, expressed in Aspergillus oryzae) (Sigma-Aldrich, Søborg, Denmark), beechwood xylan (Biosynth, Berkshir, England, UK), pectin from citrus peel (74.0%, Sigma-Aldrich, Buchs, Switzerland), sodium carboxymethyl cellulose (Sigma-Aldrich, Buchs, Switzerland), xylose (99%, Sigma-Aldrich, Buchs, Switzerland, D-( + )-glucose (99.5%, Sigma-Aldrich, Buchs, Switzerland), D-( + )-galacturonic acid monohydrate (97.0%, Sigma Aldrich, Buchs, Switzerland), C₉-C₂₅ alkanes, deuterated chloroform for NMR spectroscopy (CDCl₃-d with 0.03% v/v TMS, 99.80%, Eurisotop, Saint-Aubin, France).

pH and temperature modification of pectin was carried out as previously described[28 (link)]. Briefly, pectin from citrus peel (Sigma Aldrich, CA, USA) was dissolved in distilled water at a concentration of 1.5% at 60°C and the pH was adjusted to approx. 10.0 with NaOH. The solution was then cooled to room temperature while adjusting the pH to 3.0. Insoluble material was pelleted, and the supernatant was stored overnight at room temperature. The pH was next adjusted to 6.3, and the MCP was precipitated with 9 volumes of absolute ethanol and frozen at −20°C. The precipitate was filtered, washed with acetone on Whatman filters and then dried in vacuo. The doses of MCP (1 μg/μl) were chosen on the basis of previous studies [28 (link)] and pilot experiments performed in our laboratory.

Polygalacturonic acid, pectin from citrus peel, and pectin from apple were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium polygalacturonate was purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). D-Galacturonic acid sodium salt (purity, about 95%) was obtained from Sigma-Aldrich. All other chemicals and reagents were of analytical grade. The genome of Echinicola rosea JL3085^T harbored multi-gene polysaccharide utilization loci (PUL) systems involved in the degradation of pectin. This study used Escherichia coli DH5α and E. coli BL21 (DE3) for plasmid construction and as the hosts for gene expression, respectively.

Pectins are heteropolysaccharides which can harbor a high chemical complexity, comprising up to 17 different sugar units and containing more than 20 different linkages (35 (link)). On the other hand, pectins are soluble in water, which allows easy access of most gut microbes, and are present in the majority of plant food sources common in diets (35 (link)). Taking these factors together, we classified pectin as an intermediate-specificity dietary fiber. Pectin from citrus peel with galacturonic acid content of ≥74.0% with ≥6.7% methoxy groups (CAS number 9000-69-5) was obtained from Sigma-Aldrich (St. Louis, MO).

Native yellow pea flour was provided by Prof. Jiajia Rao, from North Dakota State University. Pectin from citrus peel (galacturonic acid ≥ 74.0% dried basis) was purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). ACTIVA RM transglutaminase (T-gase) preparation was purchased from Ajinomoto North America., Inc. (Chicago, IL, USA). Raw sea scallops were bought from a local grocery store (Stop & Shop, Amherst, MA, USA) and stored in a freezer (−20 °C) until used. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Fisher Scientific (Waltham, MA, USA). The Bradford reagent used for the protein determination was obtained from the Bio-Rad company (Hercules, CA, USA).

For pectinase activity measurement, each reaction mix contained 200 µL sodium acetate buffer 500 mM pH 5.5, 200 µL pectin solution [pectin from citrus peel (Sigma) 0.5%, pH 5.5] and 25 µL of the suitable supernatant sample. The reaction mix was incubated during 30 min at 37 °C. Reaction was stopped by addition of 640 µL of dinitrosalicylic acid solution (dinitrosalicylic acid 1%, sodium potassium tartrate 30% and NaOH 1.6%) and incubation at 95 °C for 5 min. At these conditions, dinitrosalicylic acid reacts with the reducing sugar released from pectin, producing a complex with maximal absorbance at 540 nm. Thus, the reaction was then cooled in ice by 5 min, and centrifuged to obtain the supernatant. Absorbance of the supernatant was measured at 540 nm, and absorbance data were interpolated in a suitable calibration plot. The pectinolytic activity (U/mL) was calculated as the enzyme necessary to release 1 µmol of reducing sugars for minute. Specific activity (U/mg) was obtained normalizing the activity by protein concentration, determined by the Bradford’s method [28 (link)].
To determinate the effect of temperature on pectinolytic activity, the same assay described above was performed, but at different temperatures. For details of temperatures used, see the respective Figure.