Millipore membrane

CPS and biomass isolation were conducted following Di Pippoa et al. [23 (link)] with a few modifications. Leptolyngbya sp. cells and their envelopes (capsular polysaccharides [CPS] were re-suspended in distilled water (1:10), incubated at 60 °C for 60 min and then centrifuged at 10,000 g for 20 min to remove the cells which were then washed three times with deionized water, freeze-dried and lyophilized. The supernatant fluid was ultra-filtrated using a tangential ultra-filtration cell (Millipore, Bedford, MA, USA) and Millipore membranes (30 kDa pore size) as described above (RPS isolation). The purified CPS was freeze-dried and lyophilized.

Viscosity of culture broths was measured using a cone/plate rheometer as described by Peña et al. [17 ]. The alginate molecular weight distribution was estimated by gel permeation chromatography (GPC) with a serial set of Ultrahydrogel columns (UG 500 and Linear Waters), using a HPLC system with a differential refractometer detector (Waters, 410). Elution was performed with 0.1 M NaNO3 at 35 °C at a flow rate of 0.9 ml min⁻¹. The detector signal was processed with a PC compatible software (Empower GPC, Waters). Calibration of the columns were performed via a standard calibration method using as molecular weight standards, the standard pullulans within a range of 58–2350 kDa. For this purpose, the standard kit P-82 (58–1600 kDa) and P2500 (2350 kDa) from Shodex.Co (Japan) were used. Alginate solutions were filtered through 0.22 μm Millipore membranes to remove microgels [9 (link)].

Nanoparticles were produced by an ionic crosslinking technique [10 (link)], using sodium tripolyphosphate (TPP) as the crosslinking agent, in 30% (w/w) concentration ratio of TPP to total chitosan amount, and mechanical stirring at approximately 350 rpm (Multistirrer 15 Magnetic Stirrer, Velp Scientific Inc., Bohemia, NY, USA). In general, for each 4 mL of chitosan solution at 2.5 mg/mL (in acetic acid 1%, v/v, pH 5.0, previously filtered through 0.45 μm Millipore™ membranes), 6 mL of TPP solution at 0.5 mg/mL (filtered through 0.22 μm) was added dropwise (30 s). Immediately after the addition of TPP, 1 mL of standard bromelain solution was added to produce chitosan–bromelain nanoparticles, or 1 mL of distilled water to produce bromelain-free chitosan nanoparticles (blank nanoparticles). After the addition of all components, the solution was kept under magnetic stirring for 40 min. To compare different specifications and origin, three chitosan types were used for nanoparticles production, i.e., low molecular weight chitosan (LMW), chitosan from shrimp shells (SHR), and chitosan oligosaccharide lactate (LAC).

In order to eliminate the water-insoluble fraction (WIF), which could consist of cellulose, hemicellulose, and lignin, the pectic substances extracted from the conventional procedure or subcritical water at the optimum conditions were purified by a centrifugation process. The dried pectic substances were dissolved in fresh Milli-Q water (1:200, w/v), prepared from a Milli-Q water purification system (Millipore Co., Milford, MA, USA), for 15 h and centrifuged at 9072 g during 20 min at room temperature in a LABOFUGE ll centrifuge (Ollmann and Co. KG, LABSCO, Friedberg, Germany) to remove WIF. The supernatants were filtered through 11 and 3 µm Millipore membranes (Millipore Co., Milford, MA, USA), respectively. The filtrates were oven-dried at 40 °C for 12 h to recover the “purified” pectin, and weighed. The obtained pectins were used for the characterization.

After analyzing the microbial count data, an effective acid mixture (i.e., 0.8% AA + 0.2% LA), 1% AA, 1% LA, and the control were selected, after which 16S rRNA sequencing was performed to detect naturally present microbes. The microbial count was analyzed once more to ensure that the results obtained showed a similar trend with Section 2.4. The disinfection process was performed as described in Section 2.3. The microbial suspension was prepared as described in Section 2.4. For total microbial DNA extraction, 40 mL of the suspension was drawn with a sterile syringe and filtered through two Millipore membranes (0.22 μm; 20 mL each; Billerica, MA, USA), after which DNA was extracted from the resulting membranes using Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. DNA quality and concentration were determined by agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. The agar type and culture conditions are described in Section 2.4. Each treatment was replicated six times. Verification of microbiological analysis was performed three times with six replicates, and the results are expressed as microbial reduction (log CFU g⁻¹).

Bacteriophages were isolated from sewage using enrichment cultures [16] . All phages used in this study were isolated using the same method. For example, to isolate IME-SA1, approximately 2 mL of filtered (Millipore membranes, pore diameter 0.45 µm) sewage was mixed with 2 mL of 3×LB and 100 µl overnight culture of S. aureus (isolated from clinical samples in the same local hospital as the sewage). The enrichment culture was incubated at 37°C for at least 14 hours with agitation to allow amplification of bacteriophages, and was then centrifuged (10 min, 10,000 g, 4°C). The supernatant was filtered (Millipore membranes, pore diameter 0.45 µm) to remove the residual bacterial cells and 200 µl of bacteriophage was mixed with 500 µl of S. aureus cells in the exponential growth phase (OD₆₀₀ = 0.2 to 0.5) and incubated at 37°C for 30 min. About 5 mL of top agar (LB with 0.7% agar) at 50°C was then added, and the mixture was poured onto an LB plate prewarmed at 37°C (double-layer method) [16] . The plates were then incubated at 37°C overnight to obtain bacteriophage plaques [17] .