*2.1. Materials*

Flour from five green banana cultivars, namely Grande Naine, Pisang Awak, Finger Rose, FHIA-01 and Du Roi, was kindly provided by the Agricultural Research Counsel (ARC) Tropical and Subtropical Crops, Nelspruit, Mbombela in South African. All reagents were analytical grade, Trolox, Folin–Ciocalteu reagent, gallic acid and quercetin were purchased from Sigma-Aldrich Pty. Ltd. (Johannesburg, South Africa). The resistant starch assay kit and the amylose/amylopectin kit were purchased from Megazyme Ltd. (Johannesburg, South Africa).

#### *2.2. Preparation of Banana Starch*

A water-alkaline extraction method was used to prepare banana starch, as described by Jiang et al. [16], with a few changes. Briefly, GBF (100 g) was macerated in distilled water (1 L) for 20 min at a low speed, then sieved through 100-mesh screens. The collected milk was centrifuged at 4000× *g* for 10 min to remove soluble fiber, and then 1 L NaOH solution (0.2%, *w*/*v*) was added to the sediment. The starch sediment was mixed with water and stirred for 5 min before resting for 2 h. Thereafter, the sediment was again suspended in water and allowed to settle. This was repeated until the wash water reached a neutral pH. The resultant material was then dried at 45 ◦C for 24 h. The desiccated starch was pulverized and passed through a 100 μm sieve.

#### *2.3. Proximate Composition of Green Banana Flour*

The moisture content was assayed using a vacuum oven dryer at 60 ◦C for 16 h using 2–3 g of sample, according to Rodriguez-Jimenez et al. [17]. A furnace was used to measure ash content using a method described by [9]. Soxhlet extraction was used for total fat content [14]. For protein content, the Kjeldahl method was followed, as demonstrated by Kumar et al. [9]. The carbohydrate percentage was calculated using the formula below.

Carbohydrate (g) = 100 − (protein (g) + moisture content (g) + lipid (g) + ash (g))

#### *2.4. Mineral Composition of Green Banana Flour*

Mineral analysis was conducted following a method by Jakavula et al. [18]. Briefly, the sample was digested using ultra-pure HNO3 on a microwave-accelerated reaction system (CEM, Matthews, NC, USA). This was conducted at high temperature and pressure for the extraction of acid-extractable elements with the sample material. After that, deionized water was added (50 mL), followed by analyses of the sample by ICP-OES (Thermo Scientific, Basingstoke, UK).

#### *2.5. Characterization of Functional Properties of Green Banana Flour*

#### 2.5.1. Water Absorption Capacity (WAC)

The WAC of GBF was determined using the method described by Kumar et al. [9], with some modifications. Precisely, 0.5 g flour sample was weighed into 50 mL centrifuge tubes followed by the addition of 5 mL distilled water. The suspensions were vortexed and rested for 1 h at room temperature (26 ± 2 ◦C). Thereafter, they were centrifuged at 3000 rpm for 30 min at 25 ◦C. The WAC was expressed as mL of water absorbed per gram of flour.

#### 2.5.2. Water Solubility Index and Swelling Power

The water solubility index and swelling power were determined following the method detailed by Kumar et al. [9]. Green banana flour (0.2 g) was mixed with distilled water (5 mL) for 30 s using a vortex. After that, the mixture was heated at 50 ◦C, 70 ◦C and 90 ◦C for 20 min, followed by cooling and centrifugation at 3000 rpm for 10 min. The supernatant was evaporated at 105 ◦C for 16 h in an oven. The solubility index was calculated as the ratio of the mass of dried supernatant to the mass of the flour expressed in percentage (g/100 g DW). After centrifugation, the filtrate was also weighed to obtain the swelling power.

#### *2.6. Microstructure Analysis of Green Banana Flour*

#### 2.6.1. Scanning Electron Microscopy (SEM)

A scanning electron microscope (SEM-EDX) (JEOL, JSM 7500F) was used to study the microstructures of the banana starch granules. The GBF starch samples were placed on aluminum cylinders that had a double-sided tape followed by coating with carbon. The acceleration voltage was 10.00 kV, as previously described by Maziya et al. [19]. An electron beam with the resolution set at a particle size of 20–200 μm was used to view the microstructure of the samples.

#### 2.6.2. X-Ray Diffraction (XRD)

The XRD analysis of the GBF samples was determined using Philips X'Pert XRD equipment (Malvern PANalytical, Almelo, The Netherlands). The power source was set at 40 kV and 40 mA power with a scanning interval of 5◦/min. The scanning range was 2θ = 5◦ to 90◦ [19].

#### *2.7. Molecular Structure Analysis of Green Banana Cultivars*

#### 2.7.1. Fourier Transform Infrared (ATR-FTIR) Spectroscopy

The ATR-FTIR spectra of GBF samples were measured using a 4000 FTIR spectrophotometer (JASCO, South Africa). The functional groups of the isolated compound were detected by ATR (JASCO, South Africa) with a diamond crystal plate with a scan rate of 16 runs per scan at a resolution of 4 cm<sup>−</sup><sup>1</sup> in wavenumbers from 500 to 4000 cm<sup>−</sup><sup>1</sup> [20].

2.7.2. Determination of Rapidly Digestible, Slow Digestible, Resistant, and Total Starch Contents of Green Banana Flour

The determination of rapidly digestible, slow digestible, resistant, and total starch contents of green banana starch was carried out using a Megazyme Resistant Starch Assay Kit (Megazyme Ltd., Johannesburg, SA). Briefly, the method involved incubating the GBF sample (80 mg) in a mixture of enzymes (pancreatic α-amylase and amyloglucosidase) in maleate buffer (pH 6.0) (K-RNTDF; AOAC Method 2017.16) [21].

#### 2.7.3. Amylose and Amylopectin

A commercial amylopectin/amylose kit (Megazyme Ltd., Johannesburg, South Africa) was used to quantify amylose content. The principle of the method involves the separation of amylopectin and amylose. Thereafter, amylopectin is precipitated with concanavalin-A (Con A), followed by centrifugation to eliminate it Jiang et al. [16].

#### *2.8. Total Phenols, Flavonoids Content and Antioxidant Properties*

#### 2.8.1. Total Phenolic Content (TPC)

The TPC was determined using the Folin–Ciocalteu assay method according to the procedure outlined by Blainski et al. [22]. Briefly, one gram of GBF was mixed with a 25 mL mixture of methanol and water (*v*/*<sup>v</sup>*, 20:5, respectively) followed by incubation at 37 ◦C for 4 h with shaking. The mixture was then centrifuged (4000× *g* for 10 min). Thereafter, the supernatant was mixed with 500 μL deionized water in a test tube with 30 μL standard/extracts and 50 μL Folin–Ciocalteu reagen<sup>t</sup> (Sigma-Aldrich, Johannesburg, South Africa). This was followed by the addition of 245 μL deionized water and 200 μL of Na2CO3. The sample mixture was then incubated at 27 ◦C for 30 min, and a microplate reader was used to measure the absorbance (750 nm). Gallic acid was used as standard, and the results were expressed as mg gallic acid equivalent (GAE)/g dry weight using the standard curve (R<sup>2</sup> = 0.9982).

#### 2.8.2. Total Flavonoid Content (TFC)

The TFC was determined following the method described by Jabri-Karoui et al. [23]. Briefly, where Quercetin (Sigma-Aldrich, Johannesburg, South Africa) was used as a standard. An aliquot of 30 μL of each extract or standard {Quercetin (Sigma-Aldrich, Johannesburg, South Africa)} was mixed with 20 μL of 10% AlCl3 and 20 μL of 2.5% NaNO3. After 5 min, 100 μL of NaOH solution was added to the mixture. From the mixture using a micropipette, 200 μL was pipetted into a microplate (96 well). A microplate reader was used to measure the absorbance (450 nm). The TPC was expressed as mg QE/mg dry weight using the standard curve (R<sup>2</sup> = 0.9991).
