Guar gum

A 1.5% guar gum gel (w/v) solution was obtained by dissolving 0.75 g of guar gum (Sigma-Aldrich, St. Louis, MO, USA) in 50 mL of distilled sterile water. Then, the solution was sterilized by autoclave, and nisin was incorporated within the guar gum gel (biogel) in a proportion of 1:1, to obtain a 0.75% biogel (w/v) [25 (link)].

A stock solution of nisin (1000 µg/mL) was obtained by dissolving 1 g of nisin powder (2.5% purity, 1000 IU/mg, Sigma-Aldrich, St. Louis, MO, USA) in 25 mL of HCl (0.02 M) (Merck, Darmstadt, Germany). This solution was sterilized by filtration through a 0.22 µm cellulose acetate membrane filter (Millipore, Burlington, MA, USA) and stored at 4 °C [27 (link)].
Brain Heart Infusion (BHI) or Trypticase Soy Broth (TSB) with guar-gum gel at 0.75% (w/v) were prepared by dissolving 3.75 g of guar-gum (Sigma-Aldrich, St. Louis, MO, USA) and 18.5 g of BHI powder (VWR Chemicals, Leuven, Belgium) or 15 g of TSB powder (VWR Chemicals, Leuven, Belgium), respectively, in 500 mL of sterile distilled water, and heat sterilized by autoclave [27 (link)].
Clindamycin is an alternative antibiotic currently used in clinical practice associated with mild DFIs and was used in the present study at 1/2 MIC as a control for comparing the effects of nisin-biogel at sub-MICs on S. aureus DFI isolates virulence factors expression [28 (link)]. A stock solution of clindamycin was obtained by dissolving 6.6 mg of clindamycin powder (Sigma-Aldrich, St. Louis, MO, USA) in 10 mL of sterile water and filtered using a 0.22 μm cellulose acetate membrane filter. This stock solution was kept frozen at −80 °C and diluted with sterile water to the final concentration of 0.0165 µg/mL when required.

Nisin Z and guar gum were prepared as described [31 (link)]. Stock solutions from ultrapure Nisin Z (≥95% purity, NISIN Z) (Handary, Brussels, Belgium) were prepared in milli-Q purified water (Sigma-Aldrich, Darmstadt, Germany), filtered using a 0.2 µm Millipore filter (VWR, Leuven, Belgium), and stored at 4 °C.
A guar gum gel of 1.5% (w/v) was prepared by dissolving 0.75 g of guar gum (Sigma-Aldrich, Darmstadt, Germany) in 50 mL of sterile distilled water and heat sterilized by autoclave. An EDTA disodium salt (Sigma-Aldrich, Darmstadt, Germany) stock solution of 64 mg/mL was prepared by dissolving 6.4 g of EDTA in 100 mL of sterile distilled water and filtered.
A set of dilutions of Nisin Z were prepared, corresponding to the following concentrations: 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 15, 25, 50, 100, 200, 400, 800, 1000, and 1250 µg/mL. The set of dilutions of Nisin Z were incorporated within the gel in a proportion of 1:1, followed by vortex homogenization, obtaining a final gel of 0.75% (w/v), as previously described by us [31 (link)]. EDTA was added to a final concentration of 0.4% (4000 µg/mL) (see results).

Mannobiose (M₂), mannotriose (M₃) and mannotetraose (M₄) standards were purchased from Megazyme (Bray, Ireland). Locust bean gum (LBG), solka floc, glucose, mannose, guar gum, p-nitrophenyl-α-d-galactopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-mannopyranoside, p-nitrophenol (pNP) and other chemicals were sourced from Sigma-Aldrich, USA. Copra meal was obtained from Parker Biotech Private Ltd., Tamil Nadu, Chennai, India. Food-grade konjac gum (glucomannan) was obtained from New Foods, Bloomingdale, Illinois, USA. Fenugreek seed (Trigonella foenum-graecum) meal, Aloevera pulp, rice husk, wheat straw and wheat bran were purchased from local markets.

The pET28a-RmMan5A was constructed as described previously [30 (link)]. E. coli DH5α and BL21 (DE3) were used for plasmids propagating and gene expression, respectively. Vector pET28a (+) was obtained from Novagen (Madison, WI, USA). Chelating Sepharose (Ni-IDA) resin matrix was purchased from GE Life Sciences (Pittsburgh, PA, USA). LBG and guar gum were obtained from Sigma (St. Louis, MO, USA). Unless otherwise stated, all other chemicals were of analytical grade.

𝛼-Lactalbumin (LA), bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), poly-L-arginine hydrochloride (PA), silver nitrate (AgNO₃), sodium borohydrate (NaBH₄), and guar gum were obtained from Sigma-Aldrich, sodium citrate was from J. T. Baker, sodium tetraborate was from Fluka, Luria-Bertani (LB) broth and Dulbecco’s modified Eagle’s medium (DMEM) were from ThermoFisher Scientific, agar was from Difco, and synthetic diamond powders (Micron + MDA) were from Element Six.