# Volatile, Microbial, and Sensory Profiles and Consumer Acceptance of Coffee Cascara Kombuchas
## 2 Materials and Methods
### Kombucha Consortium, Black Tea, and Coffee Cascara Kombucha  
The kombucha consortium was part of the collection of the Microbiology Institute of the Federal University of Rio de Janeiro, Brazil. Previously cultivated in green tea,the consortium was fermented three times in black tea or coffee cascara tea infusion before experimental use to stabilize the microbial consortium in these matrixes [24]. All kombucha beverages were prepared according to the classical kombucha protocol described by [PERSON] [25]. _Infusions_--Black tea (BT) and coffee cascara (CC) infusions were prepared at 3% (weight/volume--\(w/v\)), pouring water at 95 \({}^{\circ}\)C over the raw material and letting it steep for 10 min. _Black tea kombucha_--Black tea kombucha (BT K) was prepared by mixing 10% (\(v/v\)) of black tea infusion, 10% (\(w/v\)) sugar (sucrose), and 2.5% (\(v/v\)) of the SCOBY. The mixture was allowed to ferment at 23 \({}^{\circ}\)C. Samples were collected before fermentation (day 0) and after 3, 6, and 9 days of fermentation. An extra sample with pH 2.8 \(\pm\) 0.05 was collected after 14 days of fermentation to be used as a starter for the coffee cascara kombucha production. _Coffee cascara kombucha_--Coffee cascara kombuchas (CC K) were prepared using 80% (\(v/v\)) of the coffee cascara infusion, 10% (\(v/v\)) of the black tea kombucha starter, 10% (\(w/v\)) sugar, and 2.5% (\(w/v\)) SCOBY. The mixture was allowed to ferment at 23 \({}^{\circ}\)C. Samples were collected before and after 3, 6, and 9 days of fermentation (d0, d3, d6, and d9, respectively). _2.3. pH, Total Titratable Acidity, Total Soluble Solids Determination, and Sugar Analysis_  
Triplicate samples were prepared for analysis. pH was measured using a pH meter (Kasvi K39-0014 PA, Sao Jose dos Pinhais, Parana, Brazil). Total titratable acidity was determined via titration with 0.1 N NaOH and phenolphthalein as an indicator, according to Adolfo Lutz Institute [26]. Results were expressed in mEq/L. Total soluble solids were evaluated using a handheld refracometer (Pocket Refractometer Pal-1, ATAGO, Tokyo, Japan). Results were expressed in \({}^{\circ}\)Brix. Sucrose was analyzed using a High Performance Liquid Chromatography Refractive Index Detector (RID) system (mod.# 2414, Waters, Milford, MA, USA), according to [PERSON] et al. [27], using a Hi-Plex column H 8 \(\upmu\)m (300 \(\times\) 7.7 mm) (Agilent, Santa Clara, CA, USA) at 30 \({}^{\circ}\)C with 20 \(\upmu\)L of injection volume and H\({}_{2}\)SO\({}_{4}\) 0.005 mol/L as mobile phase at 0.4 mL/min. For glucose and fructose, the column temperature was 60 \({}^{\circ}\)C, and the mobile phase flow was 0.6 mL/min. Calibration curves of external standards were used for the identification and quantification of all sugars (Supplementary Material Figure S1). _2.4. Analyses of Volatile Organic Compounds_  
Extraction of the volatile organic compounds from the infusions and kombuchas was performed via headspace solid-phase microextraction (HS-SPME), using a 50/30 \(\upmu\)m divinylbenzene/carboxen/polydimethylsiloxane fiber (DVB/CAR/PDMS, Supelco(r)). The analyses were performed in a gas chromatographer (Agilent, 6890 Little Falls, DE, USA) coupled to a mass spectrometer (Agilent 5975) (GC-MS) and equipped with an auto analyser Combi-PAL (CTC Analytics, Zwingen, Switzerland), according to the methodology described by [PERSON] et al. [28]. Before use, the fiber was conditioned according to the manufacturer's recommendations. Two milliliters of each sample were placed in a 20 mL SPME vial, which was immediately sealed with silicone septa and conditioned for 5 min at 50 \({}^{\circ}\)C under continuous agitation.