Pectin

The Cow fecal sample was used as an isolation source. It was cultivated in ISP2 medium containing yeast extract (4 g), malt extract (10 g), dextrose (4 g), agar (2 g), and 2.5 mL of 1% clotrimazole. The isolated Streptomyces strains were inoculated in 0.4 g K₂HPO₄, 0.008 g MgSO₄, 0.2 g (NH₄)₂SO₄, 0.008 g FeCl₃, 0.1 g yeast extract, and 0.5 g pectin (101,845,988 Sigma) in 100 mL distilled water and incubated at 30 °C for 24 h. I/KI indicator was used to select the pectin-degrading colonies following the formation of a clear halo around the colonies⁴⁵ . The isolated strain was identified by the Iranian Biological Resource Center (IBRC). The universal primer pairs, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) as a forward primer and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) as a reverse primer, were used for amplification of 16S rRNA gene and then the sequencing was performed by Cosmogenetech, Korea. The sequence was submitted to NCBI, and the neighbor-joining method in MEGA X software was used to make the evolutionary history. The 16S rDNA sequence of Lactococcus garvieae was used as an outgroup (KF849269).

All isolates from different geographic locations that were initially cultivated on YAD media were tested for their ability to grow on selected alternative carbon sources: Pectin, a structural polysaccharide contained in the cell walls of terrestrial plants, and galacturonic acid, an oxidized form of D‐galactose, the primary component of Pectin. Cultivation of Sporobolomyces isolates was attempted using sterilized tap water media with the following amendments: (1) 3% Pectin (from apple, Sigma‐Aldrich, St. Louis, MO, USA) + 1.5% Agar + 0.3% NaHCO₃ (PA); (2) 3% Pectin; (3) 1% Galacturonic Acid + 0.1% KH₂PO₄ + 0.4% NaHCO₃ (GA), and (4) 0.5% Galacturonic Acid + 0.05% KH₂PO₄ + 0.2% NaHCO₃ + 1.5% Agar (GAA). Tests were processed under aerobic conditions on the benchtop, micro‐oxic conditions in a candle jar, and under anaerobic conditions in a COY hood with the same atmosphere described above.
For experiments testing the ability to grow under anaerobic conditions, we utilized the following 6 media: YAD, desiccated 1.5% agar medium with liquid yeast extract added, YAD with 30 μL of methanol in the lid, PA, GAA, and YAD with Baker's yeast precultivated on the plate.

All materials such as copper(II) nitrate trihydrate, 2, 6-pyridine dicarboxylic acid, chitosan (10 mg/mL acetic acid: water), pectin (pectin from citrus peel, impurities ≤10% moisture), and solvents with high purity were obtained from Merck and Sigma-Aldrich. The cultures such as Mueller Hinton agar and Mueller Hinton broth were obtained from Sigma-Aldrich. The bacterial and fungal strains were obtained from American Type Culture Collection.

Ingredients used for jelly candy production included: 0.5 g of the hydrolysate (3.36% of dry weight ingredients), 0.75 g agar (Merck, Dublin, Ireland), 0.75 g pectin (Merck, Dublin, Ireland), 0.75 g of guar gum (Sigma Aldrich, Dublin, Ireland), 0.2 mL of liquid sucrose (Dr Oetker, Dublin 12, Ireland), 0.5 mL of liquid food coloring (pink) (Dr Oetker, Dublin 12, Ireland), 0.5 mL orange extract (Dr Oetker, Dublin 12, Ireland), 3.5 sheets (approximately 12 g) of sheet bovine derived gelatin (Dr Oetker, Dublin 12, Ireland), 57 mL of ddH₂O. The jelly candies (jellies) were manufactured as described previously [17 (link),52 ]. A cold-set approach was used. Once achieved, the mixture was poured into polyethylene molds (2 × 1.5 cm) and refrigerated for 24 h at between 2 and 8 °C. The formulation used for the jelly candies was as follows: 6 g of Porphyridium sp. hydrolysate, 570 mL of ddH₂O, 0.75 g agar, 0.75 g pectin, 0.75 g guar gum, 5 g liquid sucrose, 1 g ascorbic acid, 1 mL food coloring (red), 1 mL of lemon extract essence, and 12 g of bovine gelatin (~3.5 sheets). All the Porphyridium sp. jellies produced were removed from the molds and stored in a freezer at −80 °C until the time of further analysis. The bioactive hydrolysate was added at a concentration of 3.36% (w/v) to the formulation. Jellies were rolled in cornstarch to prevent sticking following removal from molds.