beverages Article Analysis of Phytosterols Content in Italian-Standard Espresso Coffee Franks Kamgang Nzekoue 1 , Laura Alessandroni 1 , Giovanni Caprioli 1,* , Gulzhan Khamitova 1 , Luciano Navarini 2 , Massimo Ricciutelli 1, Gianni Sagratini 1, Alba Nácher Sempere 3 and Sauro Vittori 1,4,* 1 School of Pharmacy, University of Camerino, Via Sant’Agostino 1, 62032 Camerino, Italy; [email protected] (F.K.N.); [email protected] (L.A.); [email protected] (G.K.); [email protected] (M.R.); [email protected] (G.S.) 2 Illycaffè S.p.A., Via Flavia 110, 34147 Trieste, Italy; [email protected] 3 Institut Educació Secundària Pare Vitoria, Avinguda d’Elx, 15, 03801 Alcoi, Spain; [email protected] 4 International Hub for Coffee Research and Innovation, Via Emilio Betti 1, 62020 Belforte del Chienti, Italy * Correspondence: [email protected] (G.C.); [email protected] (S.V.); Tel.: +39-0737402238 (G.C.) Abstract: This study aims to assess for the first time the content of phytosterols (PS) in espresso coffee (EC) to deepen the knowledge about the phytochemicals and health potentials of coffee brews. PS were extracted by hot saponification from 14 EC samples produced with coffee originating from 13 coffee-producing countries. PS were identified and quantified by high-performance liquid chromatography (HPLC) after derivatization. Among the detected PS, β-sitosterol (4.1–18.2 mg/L) was the most abundant followed by stigmasterol (1.1–4.9 mg/L), campesterol (0.9–4.7 mg/L), and Citation: Nzekoue, F.K.; cycloartenol (0.3–2.0 mg/L). Total PS fraction ranged from 6.5 mg/L to 30.0 mg/L with an average Alessandroni, L.; Caprioli, G.; level of 15.7 ± 5.8 mg/L. Therefore, a standard cup of EC (25 mL) could provide 0.4 ± 0.1 mg of PS. Khamitova, G.; Navarini, L.; Ricciutelli, M.; Sagratini, G.; Sempere, Keywords: espresso coffee; phytosterols; β-sitosterol; HPLC; cholesterol lowering A.N.; Vittori, S. Analysis of Phytosterols Content in Italian-Standard Espresso Coffee. Beverages 2021, 7, 61. https:// 1. Introduction doi.org/10.3390/beverages7030061 Espresso coffee (EC) is one of the most common coffee hot brews in the world, obtained Academic Editor: Edgar Chambers IV from the percolation of hot water under pressure through compacted cakes of roasted ground coffee, where the energy of the water pressure is spent within the cake [1]. Received: 2 August 2021 EC consumption is continually increasing internationally, being used not only as Accepted: 24 August 2021 coffee brew but also in the preparation of other highly consumed coffee beverages such Published: 27 August 2021 as cappuccino, Frappuccino, or flat white. According to the latest statistics from the International Coffee Organization (ICO), about 10 billion kg of coffee have been sold in Publisher’s Note: MDPI stays neutral 2020, so around 2.7 billion cups of coffee a day are consumed worldwide [2]. with regard to jurisdictional claims in Nowadays, coffee brewed from coffee capsule systems is slowly replacing traditional published maps and institutional affil- brewing methods. Coffee capsules are small recipients containing previously roasted and iations. ground coffee beans that are used in specially designed systems. These systems greatly increased and facilitated the consumption of homemade EC in the world [3]. EC brewing has been studied in various scientific experiments using different devices (e.g., espresso and capsule machines, etc.) and methodologies to optimize the beverage Copyright: © 2021 by the authors. preparation process [4–9]. Indeed, coffee is a popular beverage with a unique sensory Licensee MDPI, Basel, Switzerland. profile and also a complex source of hundreds of bioactive compounds, starting from This article is an open access article green beans up to the final cup of coffee. The most studied bioactive compounds in coffee distributed under the terms and brews include caffeine, trigonelline, diterpenes, and polyphenols such as chlorogenic acids conditions of the Creative Commons (Figure1)[10–15]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Beverages 2021, 7, 61. https://doi.org/10.3390/beverages7030061 https://www.mdpi.com/journal/beverages Beverages 2021, 7, 61 2 of 12 Beverages 2021, 7, 61 2 of 12 coffee brews include caffeine, trigonelline, diterpenes, and polyphenols such as chloro- genic acids (Figure 1) [10–15]. Figure 1. IllustrativeFigure summary 1. Illustrative of the summary main classes of the of bioactivemain classes compounds of bioactive present compounds and studied present in coffee and studied with an in example for each class. Phytosterolscoffee with an represent example the for only each class class. never Phytosterols studied in represent coffee brews. the only class never studied in coffee brews. Among all the bioactive compounds investigated in coffee-based products, few studies Amonghave all exploredthe bioactive the presence compounds and levelsinvestigated of phytosterols in coffee-based (PS) in coffee products, beans few and oil [16,17]. studies haveHowever, explored to the our presence knowledge, and levels PS content of phytosterols has never been(PS) in assessed coffee beans in a famous and oil coffee brew [16,17]. However,such as to EC. our PS, knowledge, which include PS content plant sterols/stanols, has never been are assessed common in bioactive a famous compounds coffee brewin such plants as exhibitingEC. PS, which anticancer, include hepatoprotective, plant sterols/stanols, and are blood common LDL cholesterol-loweringbioactive compoundsproperties in plants [exhibiting18,19]. The anticancer latter is opposed, hepatoprotective, to the effects and of diterpenes,blood LDL anothercholester- class of com- ol-loweringpounds properties found [18,19]. in coffee, The latter which is are opposed responsible to the for effects the controversial of diterpenes, association another between class of compoundscoffee drinking, found in blood coffee, cholesterol which ar increment,e responsible and for cardiovascular the controversial diseases associa- [20]. However, tion betweenthe coffee newest drinking, scientific blood outcomes cholesterol demonstrate increment, that regular and cardiovascular coffee consumption diseases is associated [20]. However,with the a reduced newest riskscientific of type outcomes 2 diabetes demonstrate mellitus, liver that disease, regular CVD, coffee and consump- some types of can- tion is associatedcer [21 ,with22]. Thus,a reduced considering risk of type the health 2 diabetes benefits mellitus, of PS, li itver is important disease, CVD, to assess and the human some typesintake of cancer of PS [21,22]. through Thus, coffee considering consumption the health and thus, benefits the potentialof PS, it is biological important benefits to of EC. assess the humanPS intake profiles of havePS through been characterized coffee consumption in various and foodsthus, the of thepotential human bio- diet, such as logical benefitsvegetable of EC.oil, cereals, fruits, legumes, and vegetables [23–25]. More recently, Nzekoue PS profileset al. have (2020) been studies characterized revealed thatin various coffee spentfoods groundof the human could bediet, a very such good as and eco- vegetable oil,friendly cereals, source fruits, of legumes, PS [26]. Thus,and ve wegetables hypothesized [23–25]. thatMore a recently, notable coffee Nzekoue brew et such as EC, al. (2020) studiesvery common revealed allthat over coffee the spent world, ground could could contain be PS.a very Therefore, good and this eco-friendly study aims to assess source of PSthe [26]. concentration Thus, we hypothesized of phytosterols that in oura notable everyday coffee cup brew of EC. such To as achieve EC, very this objective, common all14 over coffee thesamples world, originatedcould contain from PS. 13 differentTherefore, countries this study were aims collected to assess for EC the preparation. concentrationPS extractionof phytosterols and analysisin our everyday from obtained cup ofEC EC. were To achieve performed this throughobjective, optimized 14 and coffee samplesvalidated originated ultrasound-assisted from 13 different extraction countries and were HPLC collected methods. for EC preparation. PS extraction and analysis from obtained EC were performed through optimized and 2. Materials and Methods validated ultrasound-assisted extraction and HPLC methods. 2.1. Reagents and Standards 2. Materials andAnalytical Methods standards of Campesterol (C28H48O, CAS No. 474–62-4) were provided by β 2.1. ReagentsCarbosynth and Standards (Berkshire, United Kingdom), while -sitosterol (C29H50O, CAS No. 83–46-5), stigmasterol (C29H48O, CAS No. 83–48-7), cycloartenol (C30H50O, CAS No. 469–38-5), Analytical standards of Campesterol (C28H48O, CAS No. 474–62-4) were provided by and hexacosanol (1-hexacosanol) as internal standard (I.S, C26H54O, CAS No. 79983–71-4) Carbosynth (Berkshire, United Kingdom), while β-sitosterol (C29H50O, CAS No. 83–46-5), were supplied by Sigma-Aldrich (Milano, Italy). Dansyl chloride (C12H12ClNO2S, CAS No. 506–52-5) and 4-dimethylaminopyridine (DMAP) were provided by Sigma-Aldrich (Milano, Italy). All solvents used were HPLC or analytical grade. The deionized water Beverages 2021, 7, 61 3 of 12 (58 M cm resistivity) was obtained from the Milli-Q SP Reagent Water System (Milli- pore, Bedford, MA, USA), ethanol was supplied by Fisher Scientific (Loughborough, UK), hexane was supplied by Carlo Erba (Milan, Italy), while HPLC-grade acetonitrile and dichloromethane