Spent Coffee grounds as a source of bioactive compounds

Spent Coffee grounds as a source of bioactive compounds Carmen Monente, Jimena Bravo, Ana I. Vitas, Leire Arbillaga, María Paz de Peña and Concepción Cid Dept Nutrition, Food Science, and Physiology Dept. Microbiology and Parasitology Dept. Pharmacology and Toxicology School of Pharmacy, University of Navarra (Pamplona, Spain) E-mail: mpdepena@unav.es 3rd Conference ISEKI Food 4 Athens, May 2014

Millions of coffee cups are consumed every day around the World (ICO, 2013) Coffee may reduce the risk of : Cancer (liver, kidney, oral, breast and colorectal) (Nkondjock, 2012) Neurodegenerative diseases: Parkinson (Ascherio et al., 2001) Alzheimer (Barranco Quintana et al., 2007) Amyotrophic Lateral Sclerosis (Beghi et al., 2011) Type 2 diabetes (van Dam & Hu, 2005; Matusheski et al., 2012) Cardiovascular diseases (Bonita et al., 2007) Coffee is one of the main source of antioxidants in diet (Pulido et al., 2003; Pellegrini et al., 2003; Svilaas et al., 2004)

Coffee antioxidant compounds Natural Formed during roasting Phenolic compounds Chlorogenic acids Chemical based assays: Antiradical activity (Daglia et al., 2004) In vitro assays: Protection against oxidative stress (Pavlica & Gebhardt, 2005; Cho et al., 2009) In vivo assays: Protection against oxidative DNA damage (Bichler et al., 2007; Hoelzl et al., 2010) Caffeine Chemical based assays: Scavenger of hydroxyl radicals (Daglia et al., 2004) In vitro assays: Prevention of LDL oxidation (Lee et al., 2000) Protection against oxidative DNA damage (Faustmann et al., 2009) Maillard Reaction Products (MRPs) Volatiles Non volatiles: Melanoidins Chemical based assays: Antiradical activity (Nicoli et al., 1997; Daglia et al., 2000; Del Castillo et al., 2002; Morales & Jimenez Perez, 2004) Metal chelating agents (Borrelli et al., 2002; Delgado Andrade et al., 2005) In vitro assays: Protection against oxidative stress (Goya et al., 2007) Genoprotective effect (Del Pino García et al., 2012) In vivo assays: Increase plasma total antioxidant capacity (Somoza et al., 2003) Protection of LDL oxidation (Dittrich et al., 2009)

Coffee compounds extraction Solubles Non solubles Chlorogenic acids Caffeine Soluble Melanoidins Hydrophilic volatile compunds Lipids Melanoidins Polisacharides Extraction method (Bell et al., 2000; Andueza et al., 2003a; Bravo et al.,2012; Ludwig et al., 2007) Grinding (Spiro & Selwod, 1984; Lingle, 1996; Barberá, 2000; Andueza et al., 2003a; Illy & Viani, 2005) Coffee/water ratio (Andueza et al., 2007) Water pressure and Temperature (Andueza et al., 2002, 2003b, 2007)

Coffee Brew Antioxidant capacity (Pérez Martínez et al., 2010; Bravo et al., 2012) Per g roasted coffee Per ml coffee brew > > > Pérez-Martínez et al., 2010 J. Agric. Food Chem. 58, 2958-2965 Spent coffee grounds: Source of natural antioxidants?

Millions of coffee cups are consumed every day around the World (ICO, 2013) SOLUBLE COFFEE Tons of spent coffee grounds INDUSTRY Compost Animal feed (Givens & Barber, 1986) Biofuel, biodiesel or bioethanol production (Silva et al., 1998; Kondamudi et al., 2008) Adsorbent and activated carbon (Namane et al., 2005; Franca et al., 2009) Antioxidants extraction (Yen et al., 2005; Ramalakshmi et al., 2009; Mussatto et al., 2011; Murthy & Madhava Naidu, 2012 ) COFFEE SHOPS AND DOMESTIC Compost (Cruz et al., 2014) Espresso coffee (Zuorro et al., 2013) Spent coffee grounds: Source of natural antioxidants?

OBJECTIVES To explore the possibility to obtain extracts of spent coffee grounds with antioxidant capacity and to evaluate their potential functional properties 1. To develop an extraction methodology of spent coffee with antioxidantcapacity. 2. To evaluate the antioxidant capacity and the main bioactive compounds in spent coffee extracts. 3. To evaluate the potential functional properties of spent coffee extracts: 1. Antioxidant and genoprotective effects in human cells. 2. Antimutagenic activity 3. Antimicrobial activity

1. Extraction methodology (Bravo et al., 2013) Arabica coffee (Guatemala) Spent coffee grounds Spent coffee:water 1:17 Extraction system Solid-liquid 1:10 (10 min) Filter coffeemaker (6 min) Soxhlet (1h) Soxhlet (2h) Solvents Water (W) (100%) Water:EtOH (W:E) (80:20,60:40,40:60,20:80) EtOH (E) (100%) Water:MeOH (W:M) (70:30,30:70) MeOH (M) (100%) Succesive extractions Water ph 4.5 7.0 9.5 Defatting Lyofilization Antioxidant capacity Folin Ciocalteu (Singleton and Rossi, 1965) ABTS (Re et al., 1999) DPPH (Brand-Williams et al., 1995) Browned compounds (Abs. 420 nm) Food Res. Int. 2013, 50, 610-616

μmoltrolox/g dm 1. Extraction methodology (Bravo et al., 2013) ABTS 160 140 120 100 80 60 40 20 0 c d Solid-liquid 1:10 (10 min) Filter coffeemaker (6 min) Soxhlet (1h) Soxhlet (2h) Solid-liquid Filter 1 Soxhlet Soxhlet coffeemaker 1h 3h µmol Trolox / g dm 160 140 120 100 80 60 40 20 0 a b μmoltrolox/g dm 180 160 140 120 100 80 60 40 20 0 Extraction Extraction Extraction Extraction Extraction 1 2 3 4 5 e f f d e de W W:Et W:Et W:Et W:Et 1 Et W:Me W:Me Me 100 80:20 60:40 40:60 20:80 100 70:30 30:70 100 µmoltrolox/g dm 220 200 180 160 140 120 100 80 60 40 20 0 a a b ph 4.5 ph 17.0 ph 9.5 Solvents Water (W) (100%) Water:EtOH (W:E) (80:20,60:40,40:60,20:80) EtOH (E) (100%) Water:MeOH (W:M) (70:30,30:70) MeOH (M) (100%) c c b Water ph 4.5 7.0 9.5 Food Res. Int. 2013, 50, 610-616

2. Antioxidants in spent coffee grounds extracts (Bravo et al., 2012) Arabica coffee (Guatemala) Robusta coffee (Vietnam) Spent Coffee Coffee brews Aqueous extraction (Bravo et al., 2013) Spent coffee extracts HPLC: SPE (Bicchi et al., 1995) HPLC method (Farah et al., 2005) Caffeoylquinic acids: 3-CQA, 4-CQA, 5-CQA Dicaffeoylquinic acids: 3,4-diCQA, 3,5-diCQA, 4,5-diCQA Caffeine 5-CQA Caffeine Antioxidant capacity Folin Ciocalteau (Singleton and Rossi, 1965) ABTS (Re et al., 1999) DPPH (Brand-Williams et al., 1995) Fremy s salt & TEMPO (Roesch et al., 2003; Caemmerer and Kroh, 2006) 3-CQA 4-CQA 3,4-diCQA 3,5-diCQA 4,5-diCQA 325 nm 276 nm Browned Compounds (Abs. 420 nm) J. Agric. Food Chem.. 2012, 60, 12565-12573

2. Antioxidants in spent coffee grounds extracts (Bravo et al., 2012) Arabica coffee (Guatemala) Robusta coffee (Vietnam) Spent Coffee Coffee brews Aqueous extraction (Bravo et al., 2013) Spent coffee extracts µmol Trolox/g ABTS 700 Arabica 600 500 d 400 c 300 d b a 200 c b 100 a 0 Filter Espresso Plunger Mocha Filter Espresso Plunger Mocha spent coffee coffee brew µmol Trolox/g ABTS 700 Robusta d 600 c 500 400 b 300 a 200 c d b 100 a 0 Filter Espresso Plunger Mocha Filter Espresso Plunger Mocha spent coffee coffee brew J. Agric. Food Chem.. 2012, 60, 12565-12573

2. Antioxidants in spent coffee grounds extracts (Bravo et al., 2012) Arabica coffee (Guatemala) Robusta coffee (Vietnam) caffeoylquinic acid Spent Coffee Aqueous extraction (Bravo et al., 2013) dicaffeoylquinic acid CGA 30 25 Coffee brews Arabica Spent coffee extracts 30 25 CGA Robusta Total CQAs (3-CQA, 4-CQA, 5-CQA) Total dicqas (3,4-diCQA, 3,5-diCQA, 4,5- dicqa) 20 20 mg/g 15 10 nd mg/g 15 10 nd 5 5 0 Filter Espresso Plunger Mocha Filter Espresso Plunger Mocha spent coffee coffee brew 0 Filter Espresso Plunger Mocha Filter Espresso Plunger Mocha spent coffee coffee brew J. Agric. Food Chem.. 2012, 60, 12565-12573

3. Potential Functional properties of spent coffee extracts Arabica coffee (Guatemala) Robusta coffee (Vietnam) Spent coffee Aqueous extraction (Bravo et al., 2013) Spent coffee extracts Spent coffee Aqueous extraction (Bravo et al., 2013) Spent coffee extracts 1. Antioxidant (ROS) and genoprotective (Comet assay) effects in human cells 2. Antimutagenic activity 3. Antimicrobial activity Food Chem. Toxicol. 2013, 60, 397-403

3. Potential Functional properties of spent coffee extracts Antimutagenic activity Salmonella mutagenicity test (Ames test) S. Typhimurium His, TA98 strain Toxicity assay: non toxic concentration selection Antimutagenicity test: Without metabolic activation ( S9): sample+phosphate buffer+culture+mutagen (NPD) With metabolic activation (+S9): sample+rat liver homogenate (S9mix)+culture+mutagen (NPD) 48h 37ºC Revertant colonies Spent coffee: Up to 92% (+S9) Arabica Filter Robusta Espresso Monente et al. Under revision

3. Potential Functional properties of spent coffee extracts Antimicrobial activity Arabica coffee (Guatemala) Robusta coffee (Vietnam) Spent coffee Aqueous extraction (Bravo et al., 2013) Spent coffee extracts Spent coffee Aqueous extraction (Bravo et al., 2013) Spent coffee extracts 1. Agar-well diffusion method 2. Minimum inhibitory concentration (MIC) 3. Growth inhibition curves Gram + Staphylococcus aureus Listeria monocytogenes Bacillus subtilis Fungal strains Candida albicans Aspergillus niger Gram - Escherichia coli Pseudomonas aeruginosa Salmonella Choleraesuis Monente et al. Under revision

3. Potential Functional properties of spent coffee extracts Antimicrobial activity 1. Agar well diffusion method Gram + Inhibition zones (mm) Arabica Filter Robusta espresso Organisms Spent Spent Coffee brew coffee coffee Coffee brew S. aureus 17.3 ± 0.6 ab 15.3 ± 0.6 a 15.3 ± 1.2 a 19.7 ± 1.2 b L. monocytogenes 20.0 ± 3.6 a 19.0 ± 2.7 a 18.0 ± 2.0 a 22.3 ± 3.1 a B. subtilis 8.8 ± 0.3 a 9.7 ± 0.6 a ND 11.9 ± 0.2 a E. coli ND ND ND 10.3 ± 1.2 S. Choleraesuis ND ND ND 10.7 ± 0.6 Ps. aeruginosa ND ND ND 10.3 ± 0.6 C. albicans 15.3 ± 1.2 ab 13.7 ± 0.6 a 15.7 ± 1.5 ab 17.7 ± 0.6 b A. niger ND ND ND ND 2. Minimum inhibitory concentration (MIC) Gram + MIC (mg/ml) Arabica filter Robusta espresso Organisms Spent Spent Coffee brew coffee coffee Coffee brew S. aureus 5 7.5 10 8.1 L. monocytogenes 20 30 20 16.3 B. subtilis 40 60 40 32.5 E. coli 80 60 160 32.5 S. choleraesuis 40 30 40 16.3 Ps. aeruginosa 40 60 80 16.3 C. albicans 40 60 40 32.5 Monente et al. Under revision

3. Potential Functional properties of spent coffee extracts Antimicrobial activity 3. Growth inhibition curves Control Arabica spent coffee Robusta spent coffee Monente et al. Under revision

CONCLUSIONS Spent coffee grounds are a good source of natural bioactive compounds (antioxidants) and it can be considered as a potential preservative to extent food shelf life or to applied to develop functional foods. But further research are needed: 1. To optimize technological extraction conditions, mainly at industry level 2. To study the addition of spent coffee grounds to other foods or products to develop functional foods (also in terms of sensorial properties) or other nutraceutical, cosmetic or pharmaceutical products 3. to study the bioavailability of bioactive compounds 4. To know antioxidant activity in animal and human studies

ACKNOWLEDGMENTS Coffee Research Team Toxicology lab Microbiology lab University of Navarra Financed by: Spanish Minister of Economy and Competitiveness (AGL2009-12052) Asociation of Friends of the University of Navarra (Grants) Unión Tostadora S.A.