*2.2. Characterization*

2.2.1. Physicochemical Analysis (Physical, Morphology, Elemental Composition, and Microbial Content)

EBNs with half-cup and striped-shaped physical characteristics were measured by a ruler in triplicate. The morphology characteristics were obtained by fixing EBN powder to a stub with carbon tape, coating with gold, and placing on the sample holder. Next, the sample holder was fixed on a rotatable disc inside the machine and observed using a scanning electron microscope (SEM) (FEI Quanta 400F). Meanwhile, energy dispersive

X-ray spectroscopy (EDX) was used to investigate the elemental constituents; the atomic and weight percentage of the elements were determined under low vacuum at an accelerating voltage of 15 kV and a current of 60–90 mA. The microbiological analysis for both EBN types was performed according to standard methods specified by the Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) and Official Methods of Analysis of Association of Analytical Chemists (AOAC), 16th Edition (1995). The FDA/BAM analytical methods were used to identify aerobic plate count (APC) (Chapter 3), yeasts and molds (Chapter 18), *Staphylococcus aureus* (*S. aureus*) (Chapter 12), and *Salmonella* (Chapter 5) [23], while AOAC: 991.14 (1995) was used to identify coliforms and *Escherichia coli* (*E. coli*) in the EBNs [24].

The APC was used to indicate the level of microorganisms in products. Briefly, the EBN sample (10 mL) was mixed with diluent (90 mL) to obtain <sup>10</sup>−2, <sup>10</sup>−3, and 10−<sup>4</sup> decimal dilutions. The inoculated diluents sample (1.0 mL) was mixed with plate count agar in a Petri dish and incubated aerobically at 37 ◦C for 48 h. Meanwhile, the yeas<sup>t</sup> and mold count in the EBN samples were determined out using a media supplemented by 100 mg/L chloramphenicol to inhibit bacterial growth. Peptone water (0.1%) was added to the EBN sample (25–50 g) and homogenized for 2 min. The solution (0.1 mL) was pipetted onto solidified dichloran rose bengal chloramphenicol (DRBC) agar and subjected to incubation in a dark place at 25 ◦C for 5 days. Coliforms and *E. coli* in EBN samples were determined using 3 M Petri film (3M Co., Saint Paul, MN, USA). The film was placed on a flat surface and inoculated with 1 mL of test suspension onto the center of the film base. The plate was incubated at 37 ◦C for 24 h. The red and blue colonies referred to coliforms and *E. coli,* respectively.

The presence of *S. aureus* in EBNs was examined by transferring 1 mL of EBN sample onto a Baird-Parker agar plate. The inoculum was spread evenly using a sterile bent glass streaking rod, and the plate was then incubated at 35–37 ◦C for 45–48 h. Detection of *Salmonella* in EBN was carried out by mixing a 25 g sample and 225 mL sterile lactose broth in a sterile blending container. The mixture was transferred into a sterile, wide-mouth, screw-cap jar and left at room temperature for 60 ± 5 min, with the jar securely capped. Then, the mixture was mixed well, and the jar cap was slightly loosened before incubation at 35 ◦C for 24 ± 2 h. Rappaport-Vassiliadis (RV) medium (1:100) and tetrathionate (TT) broth (1:10) were added, and the mixture was subsequently incubated as follows: RV: 24 ± 2 h at 42 ◦C; TT: 24 ± 2 h at 35 ◦C. About 10 μL of incubated TT broth was then used to streak 3 mm loopful on bismuth sulfite (BS) agar, xylose lysine deoxycholate (XLD) agar, and Hektoen enteric (HE) agar. The loopful step was repeated using RV medium (10 μL) on the same agar plates before being incubated for 24 ± 2 h at 35 ◦C. The results for APC, yeasts and molds, coliforms, and E. coli were expressed as log10 colony-forming unit per gram (CFU/g) EBN. Meanwhile, the result for *S. aureus* was reported as the most probable number per gram (MPN/g) of EBN, and *Salmonella* species were reported as either detected or not detected in 25 g of EBN.

#### 2.2.2. Chemical Analysis (Crude Protein and Amino Acid)

The crude protein in both EBNs was determined by the Kjeldahl method, using 6.25 as a conversion factor [25]. About 500 mg of EBN, 1 tablet of a catalytic amount of CuSO4/K2SO4, and 12 mL of sulfuric acid were added into the digestion tube to initiate the digestion process, until clear green or blue solution was obtained. The solution was cooled for 10–20 min before the addition of 75 mL of distilled water. The analysis was continued with the distillation process through the addition of 25 mL boric acid and 10 drops of bromocresol green as an indicator. Next, the cooled digestion tubes were placed in the digestion unit, and 50 mL of sodium hydroxide solution was added to samples for 5 min in a distillation unit. The distillate was titrated with hydrochloric acid until the changed color was obtained and was calculated in accordance with [26].

The amino acid composition was analyzed based on [27] using high-performance liquid chromatography (HPLC). About 0.3 g of EBN sample was hydrolyzed with 5 mL of 6 N

HCl at 110 ◦C for 24 h. The samples were cooled to room temperature and filtered through filter paper into a 100 mL volumetric flask. The 400 μL of internal standard (50 μmol/mL α-aminobutyric acid (AABA) in 0.1 M HCl) was added into the same flask and topped up to 100 mL with distilled water. The aliquot was filtered by using a 0.20 mm polytetrafluoroethylene microfilter. As for derivatization, 10 μL filtered hydrolysate samples or standard and 70 μL borate buffer solution were mixed well into a 1.5 mL glass vial. Then, 20 μL AccQ Flour reagen<sup>t</sup> (3 mg/mL in acetonitrile) was added to the mixture and vortexed for a few seconds. The 10 μL of samples and standards was injected into the HPLC (Waters 2475, Waters Co., Milford, MA, USA), with a 1 mL/min flow rate. Amino acid analysis was performed using AccQ Tag (3.9 × 150 mm) column with mobile phase A (Eluent A—200 mL AccQ Tag to 2 L of Milli-Q water) and mobile phase B (Eluent B—60% acetonitrile). The linear gradient condition was set according to the following times: 100% A and 0% B (start), 98% A and 2% B (0.5 min), 91% A and 9% B (15 min), 87% A and 13% B (19 min), 65% A and 35% B (32 min), 65% A and 35% B (34 min), 0% A and 100% B (35 min), 0% A and 100% B (38 min), 100% A and 0% B (39 min) and 100% A and 0% B (50 min). Amino acid content was read at 250 nm using a fluorescence detector (λ excitation and λ emission).
