# Potential Prebiotic and Anti-Obesity Effects of _Codium fragile_ Extract
## 2 Materials and Methods
### Stimulation of Probiotics Growth  
Two probioticic strains, _Lactobacillus plantarum_ subsp. _plantarum_ (_Lp_) and _Lactobacillus helevticus_ (_Lh_) were cultured in MRS broth, centrifuged and diluted in PBS to an optical density (OD\({}_{600}\)) of approximately 0.5. A 0.2% amount of the culture of each probioticic strain was added in MRS broth containing 1% CFE. Glucose was used as a control. Fermentations were monitored for 48 h at 37 \({}^{\circ}\)C, sampling every 8 h to determine the acidity and viable cell count of _Lp_ and _Lh_. The acidity was determined by measuring the amount of 0.1 N NaOH required to adjust the pH to 8.2. The viable cell counts of _Lp_ and _Lh_ were determined by counting the colony forming units (CFU) after plating on MRS agar and incubating under aerobic condition at 37 \({}^{\circ}\)C for 48 h.  
To determine SCFAs concentration, fresh feces samples were obtained from healthy seven-week-old Balb/c male mice that had not been treated with antibiotics. Feces were homogenized with 0.1 M anaerobic phosphate-buffered saline (pH 7.0) using a Bead Ruptor Homogenizer (Omni International, NW Kennesaw, GA, USA) to make 10% (\(w/v\)) slurries. Samples with or without 1% CFE were fermented in an anaerobic chamber at 37 \({}^{\circ}\)C for 24 h. After fermentation, SCFAs were determined using HPLC analysis. Two shim-pack SCR-102H (300 \(\times\) 8.0 mm, Shimadzu, Kyoto, Japan) were used in series together with a shim-pack guard column SCR-102H (50 \(\times\) 6.0 mm, Shimadzu, Kyoto, Japan) on the HPLC system (Shimadzu, Kyoto, Japan). The injection volume was 20 \(\mu\)L with a flow rate of 0.8 mL/min at 40 \({}^{\circ}\)C. The mobile phase was 4 mM \(\rho\)-toluenesulfonic acid in water.  
Additionally, total phenolic content (TPC), total flavonoid content (TFC), and radical scavenging activity (as determined by a DPPH assay) were determined before and after fermentation. TPC was determined according to the Folin-Ciocalteu procedure [37]. Briefly, samples (200 \(\mu\)L) were mixed with 2% Na\({}_{2}\)CO\({}_{3}\) (2.0 mL) and Folin-Ciocalteu's reagent (200 \(\mu\)L). After 15 min, the absorbance values at 750 nm were measured using a microplate reader. Gallic acid was used as a standard and TPC was expressed as mg of gallic acid equivalents (GAE)/g of the fermented broth. TFC was estimated using the protocol established by [PERSON] et al. [38]. Briefly, 150 \(\mu\)L of each fermented broth sample was mixed with 280 \(\mu\)L of de-ionized water, 10 \(\mu\)L of 10% aluminum chloride, and 10 \(\mu\)L of 1 M potassium acetate. After incubation for 30 min, the absorbance at 420 nm was measured using a microplate reader. The TFC content was presented as mg of quercetin equivalents (QAE)/g of the fermented broth. The radical scavenging activity of the fermented broth was determined using a DPPH assay [39]. Briefly, the fermented broth was prepared with different concentrations diluted with methanol. A mixture of 20 \(\mu\)L sample and 200 \(\mu\)L of 0.1 mM DPPH-methanol solution was taken and incubated in the dark for 30 min. The absorbance at 517 nm was measured. The fermented broth was replaced with methanol to serve as the negative control, and ascorbic acid was used as a positive control.