*2.4. Characterisation of EPSRT7*

2.4.1. Monosaccharide Composition

The molecular weight of EPSRT7 was obtained via gel filtration chromatography with a Sephadex G-100 column (1.6 × 50 cm) eluting with 0.2 mol−<sup>1</sup> NaCl solution at a flow rate of 1 mL/min. Standard reference dextrans (5–80 KDa) were used [38].

The monosaccharide composition was determined with Bruker gas chromatography (EVOC GC–TQ) combined with mass spectrometry using the procedure described by Huang-Lin et al. [39]. For this, EPSRT7 hydrolysis was conducted with 0.5 M trifluoroacetic acid (TFA) at 120 ◦C for 2 h. Subsequently, the samples were treated with N2. Galactose, glucose, arabinose, fructose, and xylose were used as the standard. To determine the presence of amino acids and glucuronic acid, an Agilent Technologies 1100 series 6410B (HPLC/MSMS) and an ACE Excel 3 C18-Amide column were used as the stationary phase with a mobile phase of 0.1% formic acid in water. This was conducted at 40 ◦C (TQ, Waldbronn, Germany) with a flow rate of 0.2 mL/min.

#### 2.4.2. Attenuated Total Reflectance/FT-Infrared Spectroscopy (ATR/FTIR)

The IR spectra of the EPSRT7 were recorded using a BX– 187 FTIR spectrometer (Perkin Elmer, Waltham, MA, USA) with an ATR attachment (Pike Technologies, Cottonwood, AZ, USA). The spectra were carried out from accumulating 32 scans at a 4 cm−<sup>1</sup> resolution over a region from 400 to 4000 cm−<sup>1</sup> [40].

#### 2.4.3. Thermogravimetric (TGA) and Differential Scanning Calorimetric (DSC) Analysis

TGA was performed with a TGA Q500 (TA Instruments, New Castle, DE, USA). The EPSRT7 (1–3 mg) was placed into a platinum crucible and subjected to temperatures ranging from 20 to 800 ◦C at a heating rate of 10 ◦C/min under atmospheric pressure. DSC measurements were conducted using a DSC Q100 (TA Instruments, New Castle, DE, USA). The EPSRT7 (0.5–2 mg) was placed in an aluminium pan with its lid removed. The pans were heated from 20 to 600 ◦C at a rate of 10 ◦C/min. Data were analysed using TA Universal Analysis software [41].

#### *2.5. Emulsifying Activity Assesment*

The emulsifying activity of the EPSRT7 was measured at different pH levels (7.2, 5.1, 3.1) and at various concentrations (0.5, 1, and 2 mg/mL), using the method described by Meneghine et al. [42]. The experiment involved mixing 1.5 mL of an oil phase (olive oil, sunflower oil, sesame oil, coconut oil, diesel oil, hexane, or toluene) with an aqueous phase of 1.5 mL. For the aqueous phase, the commercial emulsifiers of Tween 20, sodium dodecyl sulphate (SDS), Triton X-100, and EPSRT7 were compared. The tubes were stirred for 2 min at 2400 rpm using a vortex. Emulsification indices E24, E48, and E168 were measured after 24, 48, and 168 h, respectively. Formula (2) was used to calculate the emulsification indices:

$$\text{EE } [\%] = \text{HEL/HT} \times 100 \tag{2}$$

where HEL (mm) represents the height of the emulsion layer, and HT (mm) refers to the total height.
