*2.10. In Vitro Digestion*

In order to evaluate the bioaccessibility of the bioactives and probiotics from powders, the KPB sample was selected in order to evaluate the flavonoids and probiotic viability in a simulated in vitro digestion model. The samples were selected based on the phytochemical profile and viable counts of *L. casei* 431®. The simulated model was prepared using a modified method described by Kim et al. [30]. In brief, the method involved successive steps of dissolving the powder in 10 mL of simulated saliva (Tris-HCl buffer, pH 7.7) containing 1 mg/mL α-amylase, followed by gastric digestion for 2 h and then intestinal digestion for another 2 h. An amount of 0.5 g of powder was dissolved in 10 mL of simulated saliva, which was then added to 20 mL of simulated gastric juice (SGS) composed of pepsin (1 mg/mL in 0.1 N HCl) at pH 2.0 and 1N HCl.

After 2 h of gastric digestion at 37 ◦C, with continuous stirring at 150 rpm, a volume of 15 mL of the digested sample was transferred to simulated intestinal juice (SIS) consisting of pancreatin with sodium bicarbonate at pH 7.0, followed by incubating the samples for 2 h at 37 ◦C with continuous stirring at 150 rpm on an SI-300R orbital shaker (Medline Scientific, UK). The TFC of the sample was measured in the initial and final phases of each digestion stage (after the oral phase, before and after 2 h of gastric digestion, and before and after 2 h of intestinal digestion).

The bioavailability of flavonoids refers to the amounts of compounds released from the powder after gastrointestinal digestion that could become available for absorption into the systemic circulation [31]. Bioaccessibility was calculated using Equation (2):

$$Biioaccibility\left(\%\right) = \frac{TFC\_1}{TFC\_0} \times 100\tag{2}$$

where *TFC*1 is the total flavonoid content (mg/g DW) in samples digested after gastrointestinal digestion, and *TFC*0 is the total flavonoid content (mg/g DW) in powder before gastrointestinal digestion.

For the survival rate of probiotics, the inoculated powder was dissolved in 10 mL of simulated saliva, and the aforementioned protocol describing the in vitro digestion of flavonoids was applied. In the initial step and after 2 h of gastric digestion or at 2 h of intestinal digestion, samples were analyzed in terms of the viability of probiotic cells. The survival rate of probiotic cells was calculated using Equation (3):

$$\text{Survival rate } \left( \% \right) = \frac{\text{Log } \left( N\_1 \right)}{\text{Log } \left( N\_0 \right)} \times 100 \tag{3}$$

where *N*1 is the total number of viable cells after each stage of simulated digestion, and *N*0 is the initial total number of viable cells before exposure at each stage of digestion.

### *2.11. CIEL\*a\*b\* Analysis of Freeze-Dried Powders*

For the color analysis of powders, a CR 410 Chroma Meter (Konica Minolta, Tokyo, Japan) colorimeter was used to determine the color coordinates: L\* (illumination, brightness, 0 black, 100 white), a\* (positive value red, negative value green), and b\* (positive value yellow, negative value blue).

### *2.12. Powder Structure and Morphology*

The powder morphology was observed by scanning electron microscopy (SEM). In order to assess the structural and morphological characteristics, each powder was placed on a sample holder and fixed with double-sided tape. Then, it was sputter-coated with gold and observed in a Quanta FEG 250 SEM (FEI, United States of America).
