The Type and Concentration of Milk Increase the in Vitro Bioaccessibility of Coffee Chlorogenic Acids Davide Tagliazucchi,* ,†,§ Ahmed Helal, †,‡,§ Elena Verzelloni, † and Angela Conte † † Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42100 Reggio Emilia, Italy ‡ Department of Food and Dairy Sciences and Technology, Damanhour University, 22516 Damanhour, Egypt ABSTRACT: Coffee with different types and concentrations of milk was digested with pepsin (2 h) and pancreatin (2 h) to simulate gastropancreatic digestion. Chlorogenic acids (CGAs) were determined by high-performance liquid chromatography- electrospray ionization-tandem mass spectrometry in ultrafiltrate (cutoff 3 kDa) to evaluate their bioaccessibility. After digestion, bioaccessible CGAs decreased from 80.2 to 53.0 and 69.5 μmol/200 mL in coffee without milk and coffee-whole milk, respectively. When whole, semiskimmed, skimmed, or diluted milk were present, the increase in bioaccessibility was dependent on fat content (r = 0.99, p < 0.001). No relationship was observed between bioaccessibility and proteins, carbohydrates, and calcium content. The addition of milk to coffee caused an immediate decrease in the bioaccessibility due to CGAs binding to proteins. After digestion, 86-94% of bound CGAs remained in the high molecular weight fraction. Casein bound 5-caffeoylquinic acid with high affinity (K D of 37.9 ± 2.3 μmol/L; n = 0.88 ± 0.06). KEYWORDS: coffee, chlorogenic acids, digestion, milk, fat, bioaccessibility, protein, casein ■ INTRODUCTION Coffee is among the most widely consumed pharmacologically active beverages in the world. Epidemiological studies have associated coffee consumption with a reduced risk of several diseases, including cardiovascular disorders and type 2 diabetes. 1,2 Coffee contains over a thousand chemicals, comprising the bioactive compounds caffeine, diterpenes, chlorogenic acids (CGAs), and melanoidins that are formed during the roasting process. It has been suggested that CGAs are the principal responsible for the protective effect of coffee in cardiovascular diseases and diabetes, 1,2 although a role for high molecular weight melanoidins has been recently reported. 3 Several health benefits have been linked with the dietary introduction of CGAs in the human body, such as decrease in blood pressure, 4 and in the relative risk of cardiovascular disease, 5 chemoprotective and antigenotoxic activities, 6 and prevention of type 2 diabetes. 7 On the contrary, some other studies have shown that CGAs could induce genotoxic effects that may increase the risk of some types of cancer. 8 The genotoxic activity of CGAs is mediated by a pro-oxidant mechanism (involving H 2 O 2 production) and resulted in the induction of DNA damage in cultured cells. 9 The genotoxic effect was found at millimolar concentrations of CGAs, but at the submicromolar concentrations found in the body, the antioxidant and chemopreventive activities may prevail. 9 CGAs represent 4-12% of green coffee constituents in mass, but because of their thermal instability, they may be largely degraded during intense roasting. 10 CGAs are esters of hydroxycinnamic acid with quinic acid. The most representative are the isomers of caffeoylquinic acid (CQA), feruloylquinic acid (FQA), and p-coumaroylquinic acid (p-CoQA). 11,12 There are also low concentrations of dicaffeoylquinic acids (diCQA) and CQA lactones. 13 The bioavailability and metabolism of coffee CGAs in humans have been recently studied. 14-16 During the passage through the body, extensive metabolism of CGAs occurs. Stalmach et al. 14 found that the only unmetabolized compounds detected in human plasma were FQAs and traces of 5-CQA, after ingestion of a cup of coffee containing more than 400 μmol of total CGAs. The Stalmach's studies 14,15 carried out on healthy subjects and ileostomists highlights the role of the small intestine and colon in the bioavailability of dietary CGAs. It was found that the small intestine is the primary site for the hydrolysis of CQAs and FQAs with the release of caffeic and ferulic acids, which are subsequently metabolized (methylate, sulfate, and glucuronide derivatives) mainly in the liver. The colon is the site for the conversion of ferulic acid and caffeic acid to dihydroferulic and dihydrocaffeic acids, which are further sulfated. 17 In addition, caffeic acid may be metabolized by the colonic micro fl ora in 3-hydroxyphenylacetic, (3,4- dihydroxyphenyl)propionic, 2,4-dihydroxybenzoic, and trans- 3-hydroxycinnamic acids. 18 Free and sulfated forms of dihydroferulic acid and dihydrocaffeic acid are found in plasma, suggesting that the CGAs not absorbed at stomach and small intestine level are modified by the intestinal microflora prior to the absorption by large bowel. 19 In human plasma, Monteiro et al. 20 detected micromole amounts of unmetabolized CQAs and diCQAs after ingestion of coffee containing more than 3 mmol of total CGAs. Presently, many of the studies investigating the bioavailability of coffee CGAs have been focused on simple beverages, while only a few studies have been carried out to test the effect of formulations on their bioavailability. 21 Dupas et al. 22 showed that milk proteins (after 25% semiskimmed or skimmed milk addition to coffee) bound CGAs in vitro. Recently, a human Received: June 21, 2012 Revised: October 19, 2012 Accepted: October 23, 2012 Published: October 30, 2012 Article pubs.acs.org/JAFC © 2012 American Chemical Society 11056 dx.doi.org/10.1021/jf302694a | J. Agric. Food Chem. 2012, 60, 11056-11064