2. Materials and methods2.1. Materi

2. Materials and methods
2.1. Materials and blend preparation
Roasted beans of different species, origins and roasting conditions were used:C. canephora cv. Robusta from Angola and Cameroon (light–medium roasted coffee beans—170 °C, 15 min), andC. Arabica from Brazil, Honduras and Timor Lorosae (medium roasted coffee beans—200 °C, 40 min). The humidity during roasting considered 70 L of water for 500 kg of samples. Roasted beans from different countries
and geographical areas were chosen considering their different composition mostly of phenolic trigonelline and caffeine contents (AlonsoSalces, Serra, Reniero, & Héberger, 2009; Bicho, Leitão, Ramalho, & Lidon, 2011; Bicho, Oliveira, Lidon, Ramalho, & Leitão, 2011; Bicho, Leitão, Ramalho, Alvarenga, & Lidon, 2013a, b). Accordingly, the functional coffee blend consisted of a mixture of 94% roasted coffee powder (C. canephora cv.RobustaandC. arabica,70/30,w/w), 3% cocoa powder
(from Ivory Coast), 2% coffee silverskin (fromC. canephora cv.Robusta from Angola) and 1% golden coffee (i.e., green coffee minimally processed). The coffee silverskin, employed as a raw material, is a byproduct from industrial pericarp removal from roasted beans. The blend was milled (milling machine adapted: 1.5% American Society for Testing and Materials 35/N0.500 mm; 51.7% American Society for Testing and Materials 60/0.500–0.250 mm and 46.8% American Society for Testing and Materials 80/b0.250) and stored in sealed packages, with degassing valves and capsules until use in the experiments. Storage was limited to, at the most, 4 days.

2.2. Preparation of espresso coffee samples
Using a coffee grinder, coffee blend was milled and 6.5 g was used to obtain a volume of 25 mL in 25 s of extraction by espresso coffee machines Briel Riviera (ES45, Portugal) and DQOOL (Brasilia, Italy).
The brewed coffee was immediately cooled to room temperature in an ice bath and stored at 20 °C. The operating conditions of the espresso machines were set, combining water pressure 15 bar (Briel)/19 bar
(DQOOL) and temperature (90 °C).

2.3. Chemical analysis of coffee blend and espresso beverages
HPLC analysis of caffeine, trigonelline and theobromine in coffee blends and beverages were performed according to procedures previously reported (Bicho, Leitão, Ramalho, & Lidon, 2011a)withminor
modifications. Ground roasted coffee blend (200 mg, fresh weight) and magnesium oxide (1 g) were homogenized in water (20 mL) and placed in a water bath, at 90 °C for 20 min, with continuous stirring.
After cooling, the volume was restored to the original level and the mixture wasfiltered (Whatman no. 1 paper filter) without washing the solid residue. An aliquot of 2 mL from the mixture was added to
10 mL of distilled water andfiltered through a 0.45μm PVDFfilter. The coffee blend extracts solution and beverages (20μL aliquots) were analyzed through a reversed-phase column Waters Spherisorb
ODS2 (4.6 × 250 mm, 5μm) in a HPLC (Beckman System Gold, Beckman Coulter, USA) equipped with a solvent system (Beckman Coulter, model 126) and a diode array detector (Beckman Coulter, model 168), using the 32 Karat Software (version 7.0, Beckman Coulter). Detection was performed at 254 nm, and the retention times for trigonelline, theobromine and caffeine wereca. 2.6, 6.9 and 19 min, respectively, using aflow rate of 1 mL min−1 and a mobile phase of 20 mM phosphate buffer and acetonitrile (90:10,v/v). Identification and quantification were carried out using specific standard curves with concentrations ranging between 20 and 1000 mg L−1 for caffeine and 10 and 500 mg L−1 for trigonelline
and theobromine. The determination of chlorogenic acids in the samples was accomplished based on the methodology previously described (Bicho, Leitão, Ramalho, & Lidon, 2011a; Moon, Yoo, & Shibamoto, 2009; Trugo & Macrae, 1984). Briefly, ground coffee samples (2 g) were extracted
four times with 10 mL of methanol/water (40/60, v/v), stirred for 30 min and centrifuged (15 min, 4000g,25°C),toafinal volume of 50 mL (golden and roasted coffee) and 25 mL (silverskin) with methanol/water solution. These extracts were clarified with Carrez solutions for 15 min,filtered by Whatman no. 1 and 0.45μm (PVDF) before injection. For coffee beverages, 20 mL was clarified with Carrez solutions and the solution of methanol/water was added until fulfil 50 mL. After 15 min extracts werefiltered by Whatman no. 1 and 0.45μm. Both types of extracts were analyzed through a reversed-phase column Waters Spherisorb ODS2 (4.6 × 250 mm, 5μm), using the HPLC system described above for caffeine determination, with aflow rate of 1mLmin−1 and a mobile phase consisting of a buffer solution of tripotassium citrate, 10 mM, pH 2.5 (solvent A) and methanol (solvent B), using the following gradient program: 0–5 min (20% B), 5–10 min (20–25% B), 10–15 min (25–30% B), 15–20 min (30–40% B), 20–25 min (40–45% B), 25–30 min (45–50% B), 30–35 min (50–60% B), 35–40 min (60–70% B), and 40–45 min (70–20% B) followed by an equilibration during 10 min before the next injection (20μL). Detection was performed at 325 nm (for quantification of FQAs) and 330 nm (for quantification of CQAs and di-CQAs). Identification and quantification were carried out using 5-O-caffeoylquinic acid (5-CQA) standard and its isomers, 3-O-caffeoylquinic acid (3-CQA) and 4-O-caffeoylquinic acid (4-CQA) and followingTrugo and Macrae (1984). Table 1provides the main validation data for analytical HPLC methods.
2.4. Total phenol content and antioxidant activity
The sample (1 g fresh weight) was extracted with 10 mL acetone/ water solvent (70:30,v/v) according toGu, House, Wu, Ou, & Prior (2006). After the addition of solvent, the sample was stirred for 2 h,
centrifuged (2000g, 15 min., 4 °C) and the supernatant was filtered (Whatman no. 3, protected from light). Total phenolic content in the extracts from each coffee blend was determined using the Folin–Ciocalteau method as described by Singleton & Rossi (1965). The extracts were properly diluted in order
to obtain an absorbance in the range of the prepared calibration curve. Then, the Folin–Ciocalteau reagent, the saturated Na2CO3solution and deionized water were added sequentially. After standing for 1 h at room temperature in darkness, the absorbance was measured at 750 nm using a UV 1203 spectrophotometer (Shimadzu, Tokyo, Japan). The results were expressed as mg of gallic acid equivalents per g for blends, per mL for beverage or cup (25 mL) of coffee espresso. Determination of the antioxidant capacity was carried out as previously detailed byGiao et al. (2007). The vitamin C equivalent antioxidant capacity method was used for the antioxidant analysis. This is a reaction
of free radicals with blue–green 2,2-azinobis (3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt using ascorbic acid as the standard with the resulting antioxidant activity being equivalent to a specific ascorbic acid concentration. This method is based on decolorization of radical cation of 2,2-azinobis (3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt, measured as percent reduction of absorbance at 734 nm. The stock solution of the radical cation of 2,2-azinobis (3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt (ABTS) was prepared via addition, at 1:1 (v/v), of 7 mM 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt to a solution of 2.45 mM K2S2O8. The developing reaction took place in the dark, for 16 h. This solution was adjusted with ultra-pure water to absorbanceca. 0.700 ± 0.020 at 734 nm, measured with a spectrophotometer. For the analysis
1 mL of diluted 2,2-azinobis (3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt solution was mixed with 20μL of sample and was assayed for inhibition percentage, after 6 min of reaction; triplicates of each sample were averaged to generate each datum point (which implies a total of twelve replicates per sample). Thefinal result was expressed as equivalent concentration of ascorbic acid (g L−1), using a calibration curve previously prepared. The determination of the scavenging effect on 2,2-diphenil-1-picrylhydrazyl free radical by the coffee blends and espresso coffee was carried out followingBrand-Williams, Cuvelier, & Berset (1995) with some modifications. The 2,2-diphenil-1-picrylhydrazyl (DPPH)
stock solution was prepared by addition of 3.9 mg of 2,2-diphenil-1-picrylhydrazyl to 100 mL of 70% acetone and then was diluted, at 1:1 (v/v). In order to obtain an absorbance of 0.565 ± 0.025 at 515 nm,
measured against the blank of acetone at 70% in a spectrophotometer, the above mentioned extract solution was diluted. A 100μL aliquot of the sample was assayed for inhibition percentage (between 20% and 80%, in order to ensure a linear response of the analytical method), after 30 min of reaction with 3.9 mL of diluted 2,2-diphenil-1-picrylhydrazyl in darkness; duplicates of each sample were averaged to generate each datum point (which implies a total of four replicates per sample). Thefinal result was expressed as an equivalent concentration of ascorbic acid (g L−1), using a calibration curve previously prepared. The results were expressed as mg of vitamin C equivalent antioxidant capacity per g for blends, per mL for beverage or cup (25 mL) of espresso coffee.

Hasil (Bahasa Indonesia) 2:

2. Bahan dan metode
2.1. Bahan dan campuran persiapan
Roasted biji dari spesies yang berbeda, asal-usul dan kondisi memanggang digunakan: C. canephora cv. Robusta dari Angola dan Kamerun (medium-light roasted biji-170 kopi ° C, 15 menit), andc. Arabika dari Brazil, Honduras dan Timor Lorosae (menengah biji-200 kopi panggang ° C, 40 menit). Kelembaban saat memanggang dianggap 70 L air untuk 500 kg sampel. Kacang panggang dari berbagai negara
dan wilayah geografis yang dipilih tersebut mengingat komposisi yang berbeda sebagian besar fenolik trigonelina dan isi kafein (AlonsoSalces, Serra, Reniero, & Héberger, 2009; Bicho, Leitão, Ramalho, & Lidon, 2011; Bicho, Oliveira, Lidon, Ramalho, & Leitão, 2011; Bicho, Leitão, Ramalho, Alvarenga, & Lidon, 2013a, b). Oleh karena itu, campuran kopi fungsional terdiri dari campuran 94% bubuk kopi panggang (C. canephora cv.RobustaandC. Arabika, 70/30, w / w), 3% cocoa powder
(dari Pantai Gading), 2% kopi silverskin ( fromC. canephora cv.Robusta dari Angola) dan 1% kopi emas (yaitu, kopi hijau minimal diproses). Kopi silverskin, digunakan sebagai bahan baku, adalah produk sampingan dari penghapusan pericarp industri dari kacang panggang. Campuran digiling (mesin penggilingan disesuaikan: 1,5% American Society for Testing dan Material 35 / N0.500 mm; 51,7% American Society for Testing dan Material 60 / 0,500-0,250 mm dan 46,8% American Society for Testing dan Material 80 / B0 0,250) dan disimpan dalam kemasan tertutup, dengan degassing katup dan kapsul sampai digunakan dalam percobaan. Penyimpanan terbatas, paling banyak, 4 hari. 2.2. Persiapan sampel kopi espresso Menggunakan penggiling kopi, kopi campuran digiling dan 6,5 g digunakan untuk memperoleh volume 25 mL pada 25 s ekstraksi dengan espresso mesin kopi Briel Riviera (ES45, Portugal) dan DQOOL (Brasilia, Italia). Kopi diseduh segera didinginkan sampai suhu kamar dalam penangas es dan disimpan pada 20 ° C. Kondisi operasi mesin espresso yang ditetapkan, menggabungkan tekanan air 15 bar (Briel) / 19 bar (DQOOL) dan suhu (90 ° C). 2.3. Analisis kimia kopi campuran dan minuman espresso analisis HPLC kafein, trigonelina dan theobromine dalam campuran kopi dan minuman dilakukan sesuai dengan prosedur yang dilaporkan sebelumnya (Bicho, Leitão, Ramalho, & Lidon, 2011a) withminor modifikasi. Tanah panggang campuran kopi (200 mg, berat segar) dan magnesium oksida (1 g) dihomogenisasi dalam air (20 ml) dan ditempatkan dalam bak air, pada 90 ° C selama 20 menit, sambil diaduk terus menerus. Setelah pendinginan, volume dipulihkan ke tingkat asli dan campuran wasfiltered (Whatman no. 1 kertas saring) tanpa mencuci residu padat. Sebuah alikuot dari 2 mL dari campuran ditambahkan ke 10 mL air suling andfiltered melalui PVDFfilter 0.45μm. Kopi solusi dan minuman ekstrak campuran (20μL aliquots) dianalisis melalui kolom fase terbalik Waters Spherisorb ODS2 (4,6 × 250 mm, 5μm) dalam HPLC (Beckman Sistem Emas, Beckman Coulter, USA) yang dilengkapi dengan sistem pelarut (Beckman Coulter, Model 126) dan detektor diode array (Beckman Coulter, Model 168), dengan menggunakan 32 Karat Software (versi 7.0, Beckman Coulter). Deteksi dilakukan pada 254 nm, dan waktu retensi untuk trigonelina, theobromine dan kafein wereca. 2.6, 6.9 dan 19 menit, masing-masing, dengan menggunakan tingkat Aflow dari 1 mL min-1 dan fase gerak dari 20 mM bufer fosfat dan asetonitril (90: 10, v / v). Identifikasi dan kuantifikasi dilakukan dengan menggunakan kurva standar tertentu dengan konsentrasi berkisar antara 20 dan 1000 mg L-1 untuk kafein dan 10 dan 500 mg L-1 untuk trigonelina dan theobromine. Penentuan asam chlorogenic di sampel dilakukan berdasarkan metodologi dijelaskan sebelumnya (Bicho, Leitão, Ramalho, & Lidon, 2011a; Bulan, Yoo, & Shibamoto, 2009; Trugo & Macrae, 1984). Secara singkat, sampel kopi darat (2 g) diekstraksi empat kali dengan 10 mL metanol / air (40/60, v / v), diaduk selama 30 menit dan disentrifugasi (15 menit, 4000g, 25 ° C), Volume toafinal dari 50 mL (emas dan kopi panggang) dan 25 mL (silverskin) dengan larutan metanol / air. Ekstrak tersebut diklarifikasi dengan solusi Carrez selama 15 menit, disaring oleh Whatman no. 1 dan 0.45μm (PVDF) sebelum injeksi. Untuk minuman kopi, 20 mL diklarifikasi dengan solusi Carrez dan solusi metanol / air ditambahkan hingga memenuhi 50 mL. Setelah 15 menit ekstrak werefiltered oleh Whatman no. 1 dan 0.45μm. Kedua jenis ekstrak dianalisis melalui kolom fase terbalik Waters Spherisorb ODS2 (4,6 × 250 mm, 5μm), menggunakan sistem HPLC dijelaskan di atas untuk penentuan kafein, dengan Aflow tingkat 1mLmin-1 dan fase gerak yang terdiri dari larutan penyangga dari tripotassium sitrat, 10 mM, pH 2,5 (solven A) dan metanol (pelarut B), dengan menggunakan program gradien berikut: 0-5 menit (20% B), 5-10 menit (20-25% B), 10- 15 menit (25-30% B), 15-20 menit (30-40% B), 20-25 menit (40-45% B), 25-30 menit (45-50% B), 30-35 min (50-60% B), 35-40 menit (60-70% B), dan 40-45 menit (70-20% B) diikuti oleh imbang selama 10 menit sebelum injeksi berikutnya (20μL). Deteksi dilakukan pada 325 nm (untuk kuantifikasi FQAs) dan 330 nm (untuk kuantifikasi CQAs dan di-CQAs). Identifikasi dan kuantifikasi dilakukan dengan menggunakan 5-O-caffeoylquinic asam (5-CQA) standar dan isomer nya, asam 3-O-caffeoylquinic (3-CQA) dan asam 4-O-caffeoylquinic (4-CQA) dan followingTrugo dan Macrae (1984). Tabel 1provides data validasi utama untuk metode HPLC analisis. 2.4. Total kandungan fenol dan aktivitas antioksidan Sampel (1 g berat basah) diekstraksi dengan 10 mL pelarut aseton / air (70: 30, v / v) menurut Togu, House, Wu, Ou, & Sebelum (2006). Setelah penambahan pelarut, sampel diaduk selama 2 jam, disentrifugasi (2000g, 15 menit., 4 ° C) dan supernatan disaring (Whatman no. 3, terlindung dari cahaya). Total kandungan phenolic dalam ekstrak dari masing-masing campuran kopi ditentukan dengan menggunakan metode Folin-Ciocalteau seperti yang dijelaskan oleh Singleton & Rossi (1965). Ekstrak benar diencerkan dalam rangka untuk mendapatkan absorbansi pada kisaran kurva kalibrasi disiapkan. Kemudian, reagen Folin-Ciocalteau, yang Na2CO3solution jenuh dan air deionisasi ditambahkan secara berurutan. Setelah berdiri selama 1 jam pada suhu kamar dalam kegelapan, absorbansi diukur pada 750 nm menggunakan spektrofotometer UV 1203 (Shimadzu, Tokyo, Jepang). Hasilnya dinyatakan sebagai mg setara asam galat per g untuk campuran, per mL untuk minuman atau cangkir (25 mL) kopi espresso. Penentuan kapasitas antioksidan dilakukan seperti sebelumnya rinci byGiao et al. (2007). Vitamin C Metode kapasitas antioksidan setara digunakan untuk analisis antioksidan. Ini adalah reaksi radikal bebas dengan biru-hijau 2,2-azinobis (asam 3-ethylbenzo thiazoline-6-sulfonat) garam diamonium menggunakan asam askorbat sebagai standar dengan aktivitas antioksidan yang dihasilkan menjadi setara dengan konsentrasi asam askorbat yang spesifik. Metode ini didasarkan pada dekolorisasi kation radikal 2,2-azinobis (asam 3-ethylbenzo thiazoline-6-sulfonat) garam diamonium, diukur sebagai pengurangan persen absorbansi pada 734 nm. Solusi stok kation radikal 2,2-azinobis (asam 3-ethylbenzo thiazoline-6-sulfonat) garam diamonium (ABTS) disiapkan melalui Selain itu, pada 1: 1 (v / v), dari 7 mM 2, 2-azinobis (asam 3-ethylbenzothiazoline-6-sulfonat) diamonium garam ke dalam larutan 2,45 mM K2S2O8. Reaksi berkembang berlangsung dalam gelap, selama 16 jam. Solusi ini disesuaikan dengan air ultra-murni untuk absorbanceca. 0.700 ± 0.020 di 734 nm, yang diukur dengan spektrofotometer. Untuk analisis 1 mL 2,2-azinobis (asam 3-ethylbenzo thiazoline-6-sulfonat) larutan garam diamonium diencerkan dicampur dengan 20μL sampel dan diuji untuk persentase penghambatan, setelah 6 menit reaksi; triplicates dari masing-masing sampel yang rata-rata untuk menghasilkan setiap titik datum (yang berarti total dua belas ulangan per sampel). Hasil Thefinal dinyatakan sebagai konsentrasi setara dengan asam askorbat (g L-1), dengan menggunakan kurva kalibrasi disiapkan sebelumnya. Penentuan efek scavenging pada 2,2-diphenil-1-pikrilhidrazil radikal bebas oleh campuran kopi dan kopi espresso dilakukan followingBrand-Williams, Cuvelier, & Berset (1995) dengan beberapa modifikasi. The 2,2-diphenil-1-pikrilhidrazil (DPPH) larutan stok disiapkan dengan penambahan 3,9 mg 2,2-diphenil-1-pikrilhidrazil sampai 100 ml 70% aseton dan kemudian diencerkan, pada 1: 1 (v / v). Untuk mendapatkan absorbansi 0,565 ± 0,025 pada 515 nm, diukur terhadap kosong aseton pada 70% pada spektrofotometer, larutan ekstrak tersebut di atas terdilusi. Sebuah alikuot 100μL sampel diuji untuk persentase inhibisi (antara 20% dan 80%, untuk memastikan respon linear metode analisis), setelah 30 menit reaksi dengan 3,9 mL diencerkan 2,2-diphenil-1- pikrilhidrazil dalam kegelapan; duplikat dari setiap sampel yang rata-rata untuk menghasilkan setiap titik datum (yang berarti total empat ulangan per sampel). Hasil Thefinal dinyatakan sebagai konsentrasi setara dengan asam askorbat (g L-1), dengan menggunakan kurva kalibrasi disiapkan sebelumnya. Hasilnya dinyatakan sebagai mg kapasitas antioksidan setara vitamin C per g untuk campuran, per mL untuk minuman atau cangkir (25 mL) kopi espresso.