Locust bean gum

For the purposes of this work a Lactobacillus rhamnosus GG strain (E-96666, VTT, Espoo, Finland) of established probiotic functionality was used. Low ester content (<50%) amidated pectin (LM-101 AS, Genu^®, CPKelco, UK), low viscosity sodium alginate (LFR5/60, Protanal^®, 65–75% guluronic acid units, 25–35% mannuronic acid, units, 35–60 kDa, Drammen, Norway), high viscosity sodium alginate (RF6650, Protanal^®, 45–55% guluronic acid units, 45–55% mannuronic acid, units, ∼100 kDa, Drammen, Norway), locust bean gum (Sigma Aldrich, UK), kappa-carrageenan (Sigma Aldrich, UK) and bovine skin gelatin B (Sigma Aldrich, UK) were used as film forming agents. Whey protein concentrate (81 ± 2% whey protein, 9% lactose, Lacprodan^® DI-8090) was used as a co-structuring component, glycerol (97% purity, Sigma Aldrich, UK) was used as the plasticiser.

The selected strains of xylose utilizing LAB from Eri silkworm midgut were investigated for their abilities to utilize the selected polysaccharides as the sole carbon source. Briefly, individual LAB isolate was spiked on MRS agar containing 0.5% (w/v) of single carbon source of individual polysaccharides including starch (Fisher Scientific, Loughborough, Leicestershire, UK), pectin (Fisher Scientific, Loughborough, Leicestershire, UK), beechwood xylan (Megazyme International, Bray, Co. Wicklow, Ireland), locust bean gum (Sigma Aldrich, St. Louis, MO, USA) or carboxymethyl cellulose (Sigma Aldrich, St. Louis, MO, USA) supplemented with bromocresol purple (0.04%, w/v). After incubation at 37 °C for 24 h, the bacterial growth and the yellow halo-formed surrounding colonies were observed. Further duplicated experiment was also carried out, but the bromocresol purple was replaced with trypan blue (0.01%, w/v) for detection of extracellular activities of amylase, pectinase, xylanase, β-mannanase, and cellulase, respectively. The clear zone formed surrounding colonies were observed after incubation at 37 °C for 24 h.

For β-mannanase activity measurement, the crude enzyme solution was diluted to 500 μL using 0.2 M acetic acid–sodium acetate buffer (pH 4.8) and then mixed with 1.5 mL of 1% (w/v) locust bean gum (Sigma-Aldrich). The reaction system was incubated at 50 °C for 30 min and ended with the addition of 3 mL 3,5-dinitrosalicylic acid reagent. After boiling for 10 min and the addition of 20 mL distilled water, absorbance at 540 nm was determined. The concentration of released product was calculated according to the standard curve of mannose. One unit of β-mannanase activity was defined as the amount of enzyme required to release 1 μmol mannose from the substrate per minute under the above condition. Polygalacturonase activity was measured using the same method except that the substrate was replaced by 1% (w/v) polygalacturonic acid (Sigma-Aldrich). The culture supernatants were mixed with 5 × SDS-PAGE loading buffer (GenStar, China), boiled for 10 min, and centrifuged at 8,000 rpm for 1 min. Equal volumes (30 µl) of samples were loaded into 12.5% (w/v) SDS-PAGE gels for electrophoresis at 120 V for 1 h. Gels were stained with Coomassie Blue R-250 solution for 1 h and then washed with destaining solution (1:1:8 ethanol/acetic acid/water, v/v/v) until a clear background was obtained.

Flake chitin from crab shells, flake chitin from shrimp shells, ethylene glycol chitin, Avicel PH-101, carboxymethylcellulose, soluble starch, locust bean gum, beechwood xylan, and lignin were obtained from Sigma-Aldrich (St. Louis, MO, USA). A series of N-acetyl-β-d-COSs of (GlcNAc)₂ to (GlcNAc)₆, ivory nut mannan, and wheat arabinoxylan was purchased from Megazyme International Ireland Ltd. (Wicklow, Ireland). Flake chitosan from crab shells and GlcNAc were provided by USB Co. (Cleveland, OH, USA) and TCI Co., Ltd. (Tokyo, Japan), respectively.

Locust Bean Gum (LB), NaCl (S) and Ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma Aldrich/Merck and used without further purification. Sand (SiO₂) Fraction E (90–150 μm) was purchased from David Ball sand specialists. Fe₂O₃ (Hematite) was acquired from Mineral Waters Ltd and used as supplied.