Cellulose dialysis tube

The proteins were incubated with phenolic compounds according to the procedure described by Rawel et al. [23 (link)] but with slight modifications. The protein–phenolic solutions were prepared by mixing ethanolic solution of pure phenolic compounds (GA, FA, CGA, Q, A, and CAT—0.3 mmol/5 mL) with 1 g of albumins or globulins suspended in 95 mL distilled water. The pH of reaction mixtures was adjusted to 9, and the samples were incubated for 24 h at 4 °C with continuous mixing. After incubation, the samples were transferred into the cellulose dialysis tube (Sigma–Aldrich), dialyzed for 24 h at 4 °C against distilled water, and lyophilized.
The incubation of proteins with phenolics from plant materials was conducted as follows: 1 g albumin and globulin were suspended in 50 mL distilled water, and the solutions were transferred into a dialysis tube and placed in plastic containers filled with 250 mL GT or GC extracts and the pH was adjusted to 9 with NaOH. After incubation, the protein samples were transferred to another container and dialyzed for 24 h at 4 °C against distilled water. Control samples were prepared under the same conditions but without the addition of phenolics.

The extraction of polysaccharides from P. cruentum was performed using the method proposed by Abdala-Díaz et al. (2010) [26 (link)] with minor modifications. Once the culture of P. cruentum reached the early stationary phase, centrifugation was performed at 4500 rpm for 10 min, and the supernatant was recovered for polysaccharides extraction. The polysaccharides were extracted by selective precipitation with 2% Cetylpyridinium bromide hydrate (w/v) (Cetavlon) (Sigma-Aldrich Ref: 285315). The precipitated sulfated polysaccharides were dissolved with 4 M NaCl (Sigma-Aldrich), centrifuged at 5000 rpm for 10 min and then flocculated with 96% (v/v) ethanol (Sigma-Aldrich). The final pellet was recovered and introduced into a dialysis membrane (Cellulose dialysis tube, Sigma-Aldrich) for overnight dialysis at low osmotic pressure (1M NaCl) and agitation at 4 °C. After dialysis, the entire membrane content was recovered and washed with 96% EtOH. Final centrifugation (4500 rpm, 2 min, at room temperature) was applied to recover the polysaccharides. The polysaccharides were then frozen at −80 °C in absolute ethanol for 24 h and lyophilized (Cryodos Lyophilizer, Telstar). The sulfated polysaccharide-enriched extract will hereinafter be referred to as “PcSPs”.