Journal of Food Composition and Analysis 48 (2016) 8–12Contents lists available at ScienceDirect Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca Acrylamide in coffee: Estimation of exposure from vending machines Marta Mesías* , Francisco J. Morales Institute of Food Science, Technology and Nutrition, ICTAN-CSIC, José Antonio Novais 10, 28040 Madrid, Spain A R T I C L E I N F O A B S T R A C T Article history: Received 27 November 2015 Received in revised form 29 January 2016 Accepted 3 February 2016 Available online xxx The present study aimed to evaluate the levels of acrylamide in coffee brews acquired from coffee vending machines. Acrylamide content ranged from 7.7 to 40.0 mg/L (mean: 20.6 mg/L). These levels are similar to those found in coffee brews prepared by other coffee machines with different extraction procedures. The dietary exposure of the Spanish population to acrylamide through coffee drinks was estimated at 0.037 mg/kg body weight/day (range 0.018–0.056 mg/kg body weight/day). The maximum acrylamide exposure is lower than the value of 0.2 mg/kg body weight/day, defined as the lowest no observed adverse effect level (NOAEL) for a non-carcinogenic end-point. Therefore it may be suggested that exposure to acrylamide through vending machine coffees does not imply a high risk to public health. These data are useful to the different stakeholders involved in risk assessment, and in general to risk communicators, policy makers and the industry. Chemical compounds described in this article: acrylamide (PubChem CID: 6579); potassium hexacyanoferrate(II) trihydrate (PubChem CID: 161067); zinc acetate dehydrate (PubChem CID: 2724192); methanol (PubChem CID: 887); formic acid (PubChem CID: 284). ã 2016 Elsevier Inc. All rights reserved. Keywords: Acrylamide Coffee Exposure Processing contaminants Food analysis Food composition Food safety 1. Introduction Acrylamide is generated in several foods during processing as a consequence of the Maillard reaction and specifically as the result of the reaction between asparagine and reducing sugars as the main precursors (Stadler and Scholz, 2004). Recently the European Food Safety Authority (EFSA) has confirmed that the presence of acrylamide in food is a public health concern, requiring continued efforts to reduce its exposure (EFSA, 2015). According to the EFSA, coffee together with processed potatoes and cereal based food, are the main sources of exposure to acrylamide in the diet of adults (EFSA, 2014). Coffee is generally roasted at temperatures in the range of 220–250 C, triggering the Maillard reaction. This reaction is responsible for the changes in the sensory properties but also for the formation of acrylamide from its precursors naturally present in the coffee bean. The extent of acrylamide formation in coffee depends on initial concentrations of precursors and their ratio in the green bean. The roasting and storage conditions also affect the formation. For instance, Robusta beans contain higher levels of asparagine than Arabica, which results in higher levels of acrylamide after roasting (Lantz et al., 2006). Regarding the roasting conditions, acrylamide increases in the first stages of roasting and then declines with time * Corresponding author. Fax: +34 91 549 3627. E-mail address:
[email protected] (M. Mesías). http://dx.doi.org/10.1016/j.jfca.2016.02.005 0889-1575/ ã 2016 Elsevier Inc. All rights reserved. and temperature. This is probably due to the consumption of the precursors meaning that the loss of acrylamide from physical and chemical degradation predominates against its formation (Taeymans et al., 2004). Longer periods of storage also lead to a loss of acrylamide levels in roasted coffee (Hoenicke and Gatermann, 2005). In order to regulate and control the risk associated with acrylamide intake, the European Commission (EC) established recommendations on the monitoring of acrylamide levels in foods (EC, 2010) and adopted indicative values to control acrylamide content in certain foodstuffs. References values for coffee according to the latest EC Recommendation (2013/647/EU) are 450 mg/kg for roasted coffee and 900 mg/kg for instant coffee (EC, 2013). Concentrations in a range of 197–256 mg/kg for roasted coffee and 229–1123 mg/kg for instant coffee have been reported (EFSA, 2012). However, since coffee is not consumed as a powder, but as drink, it is important to estimate the real acrylamide exposure through the consumption of coffee. The main factors that affect acrylamide contents in the beverage are the coffee variety, ripeness of the coffee bean, roasting conditions, storage and beverage preparation (Soares et al., 2015). Acrylamide is extracted efficiently due to its high polarity and water solubility (Andrzejewski et al., 2004). Nevertheless, the level of extraction is also affected by factors such as water temperature, time of contact with ground coffee and pressure applied, all of which are conditioned by the type of coffee machine used. Levels of acrylamide from 1.7 to 75.0 mg/L have been found in coffee brews 2010. contact times and water temperature (Petracco. The machines belonged to twelve of the most relevant companies that provide vending services in Spain. In agreement with Alves et al. All measurements were made in triplicate. Mainz. dry weight and browning.1.3. Volume of coffee beverage was measured. roasting conditions.5 arbitrary units. The aim of this study was to determine levels of acrylamide in coffee acquired from coffee vending machines. The limit of quantification was set at 5 mg/L.6. coffee beverages were acquired from the most representative brands of coffee vending machines in the Spanish market. MA). Results and discussion 2. 2010). Mesías. An aliquot of the clear supernatant (1 mL) was loaded onto the cartridge at a flow rate of 2 mL/min and first drops were discarded and the rest collected. The pH values of the coffee brews ranged from 5. 5 mm. Afterwards.4 mL of water in polypropylene centrifuge tubes. 2004. Data were expressed as mean standard deviation (SD). France). 2. The vending machines were located in public areas with a high footfall of people. Syringe filter units (0. 2 mL of each sample was accurately weighed and then dried to a constant weight in an oven at 105 C for 24 h according to the Association of Official Analytical Chemists (AOAC) method (1995). Alves et al..7. Chemicals Acrylamide standard (99%).45-mm filter into an amber LC– MS vial. Statistical analysis Statistical analysis was performed using Statgraphics Centurion XV (Herndon. Thermo Scientific. Germany). Supernatant was diluted appropriately with water in order to obtain an absorbance reading less than 1.8 units of absorbance (data not shown). Cartridges were preconditioned with 1 mL of methanol and 1 mL of water. the dietary exposure to acrylamide by the Spanish population through coffee machines was estimated. Coffee brews were characterized by pH.3 to 5. Milford. [13C3]-Acrylamide (isotopic purity 99%) was from Cambridge Isotope Labs (Andover. Five-millilitre aliquots of each coffee brew samples were mixed with 4. 2. MA). types of coffee bean and roasting and different coffee/water ratios for extraction. Winooski. to estimate the exposure in real life conditions where the consumer does not have any impact on the preparation of the drink. Determination of dry weight 3. 2005. Louis. cellulose) were purchased from Análisis Vínicos (Tomelloso.. In the present study. (2015) with minor modifications. Fresh coffee samples were stored in 100-mL double-sealed containers and refrigerated at 4 C for a maximum of three days until analysis. Sirot et al. potassium hexacyanoferrate(II) trihydrate (98%).5–101%) and methanol (HPLC grade) (99. 2. Samples were cleaned up with Oasis-HLB cartridges (Waters Corp.4. pH was measured in coffee brews by using a pH meter at ambient temperature (CG-837 pH meter. Millipore. hospitals and universities. Finally. VA). Schott. Germany) at 30 C with formic acid in water eluant (0. Spain).3. This research has been intentionally limited to commercially available automatic coffee vending machines placed in busy public areas. Coffee samples Forty-one espresso coffee brew samples were acquired from different coffee vending machines in the Autonomous Community of Madrid (Spain).. Bremen. 2005). All other chemicals. it is more realistic to evaluate the exposure to acrylamide by analyzing the beverage rather than the ground coffee before preparation. These machines differ largely according to the technology 2. Morales / Journal of Food Composition and Analysis 48 (2016) 8–12 prepared with different extraction methods (Andrzejewski et al. Spain). Determination of browning Browning intensity was measured at 405 nm using a SynergyTM HT-multimode microplate spectrophotometer (BioTek Instruments. Formic acid (98%) was from Panreac (Barcelona. Determination of acrylamide by liquid chromatography– electrospray ionization–tandem mass spectrometry (LC–ESI-MS–MS) Acrylamide was determined by the method described by Mesías et al. Sample (10 mL) was separated on a Hypercarb column (50 2. All machines were different. VT) (Silva’n et al.7. (2010). MA). These beverages make a significant contribution to the coffee intake outside the home. dry weight from 1. Materials and methods 2. F. brewing procedure.05 confidence level. Solution was filtered through a 0. Mixture was spiked with 100 mL of a 5 mg/mL [13C3]-acrylamide methanolic solution as internal standard and later homogenized using an Ultra Turrax (IKA. such as train and bus stations. Results were expressed as mg/L of beverage. The accuracy of the results has been recently demonstrated in an interlaboratory comparison study launched by the Food Analysis Performance Assessment Scheme (FAPAS) program (2015). 2. zinc acetate dehydrate (99. Germany) for 10 min. 1 mL) were from Waters (Milford. Germany). MO). train and bus stations. CA) with positive electrospray ionization was used. Characterization of the coffee brews Dry weight was determined gravimetrically. Tuttlingen. involving different brewing procedures. Results were expressed as absorbance units/mL coffee.. 2. Santa Clara. coffee/water ratio. 3.45 mm. including hospitals. Molsheim. Measurement of pH 9 was clarified with 20 mL each of Carrez-I (15 g potassium hexacyanoferrate(II) trihydrate/100 mL water) and Carrez-II (30 g zinc acetate dihydrate/100 mL water) solutions and centrifuged (9000 g for 10 min) at 4 C. Reversed-phase Oasis-HLB cartridges (30 mg. FAPAS Proficiency Test 3056 corresponding to crispbread test material was analyzed.9%) were purchased from Sigma–Aldrich (St. fragmentation voltage was set at 76 V and the collision energy at 8 V.. yielding a z-score of 0.1. Şenyuva and Gökmen.1 mm. respectively. solvents and reagents were of analytical grade. The transitions m/z 72 ! m/z 55 and m/z 75 ! m/z 58 were monitored for acrylamide and [13C3]-acrylamide. The characteristics of coffee brews depend on variables such as green bean composition. Staufen. Milli-Q water used was produced using an Elix3 Millipore water purification system coupled to a Milli-Q module (model Advantage10.2. Relationships between the different parameters analyzed were evaluated by computing Pearson linear correlation coefficients at the p < 0.2% to 8.2 mL/100 mL) at a flow rate of 0. 1 mL of coffee brew .5.7% and browning from 1. etc. An Agilent 1200 liquid chromatograph coupled to an Agilent Triple Quadrupole MS detector (Agilent Technologies.4 mL/min.J.0 to 5. Precision (reproducibility) was less than 10% and recovery between 96 and 110%. universities. 2012).M. each sample was treated with 250 mL of each Carrez I and Carrez II solutions and centrifuged (9000 g for 10 min) at 4 C (Hettich Zentrifugen. grinding. Dotted line represents limit of quantification (LOQ). infusion and pressure (Soares et al.056 mg/kg bw/day (mean: 0.005 mg/kg bw/day) and Latvia (0. 1).10 M. and has to be further confirmed.05 Results are expressed as mean standard deviation (SD) (n = 41). All the samples contained acrylamide levels above the limit of quantification (LOQ = 5 mg/L). United Kingdom (0. This broad distribution in the acrylamide content is probably a consequence of the type of roasted coffee and the different conditions used to brew the coffee. ground coffee is kept in contact with water for a period of time.029 mg/kg bw/day) whereas Fig.01 Median Minimum Maximum P90a 20. in consequence.. Lower estimates have been reported for adults from Ireland (0.6 0. Finally.3. the pressure method is used to prepare espresso coffee.7 0.8 40. as expected.04 1. due to the high coffee/water ratio used to prepare espresso coffee. 3.04 0.7 0. 2013). However. Values are mean standard deviation (SD) (n = 2). The average for the acrylamide level in the forty-one samples was 1.3 0. hot water flows through the ground coffee during a short period of time and.5 g of ground roasted coffee per 40 mL of water (Andueza et al.037 mg/kg bw/day). Assuming that coffee beverages were prepared from 7.02 3. Morales / Journal of Food Composition and Analysis 48 (2016) 8–12 Table 1 Acrylamide content in coffee brew samples and estimations of acrylamide exposure.. F.6 7. This variability represents the broad scenario of coffee consumption from automatic vending services. Although in the present study some samples showed an acrylamide content lower than 10 mg/L.9 mg/L). In infusion preparations. 1.9 1. a P90: 90th percentile. no significant correlations were observed.4 Acrylamide exposure mg/person/day 2. higher than those observed in filter and French press cafetiere coffees. If we consider that the average coffee consumption for the Spanish population is 599 cups/person/year as reported by the Spanish Coffee Federation (SCF.7 to 40. 2001).6 mg/L (Table 1. 2010). the extraction increasing with time of contact and water temperature. Table 2 summarizes the acrylamide levels measured in coffee beverages prepared by different extraction methods as described in the literature. However. the acrylamide intake from coffee would range from 0. 2003) and considering that the extractability of acrylamide ranged from 92 to 99% (Alves et al.3 0. extraction is lower than in the decoction method (Petracco. since no information about the coffee brew preparation conditions is available.9 0. However this statement is based on an approximate calculation. hot water is percolated under high pressure through ground coffee for a very short time.6 mg/cup (Table 1).3–3.9 mg/cupb 1. The procedures to prepare a coffee brew are usually based on three different processes: decoction.6 mg/person/day (range: 1.004 mg/kg bw/day).018 to 0. 3.and whisker plot and distribution graph for the acrylamide content in coffee brews acquired from coffee vending machines.0 mg/L. intensity of roasting and extraction conditions.06 3. 2013). the coffee brews possessed different characteristics. Assuming a default value of 70 kg of body weight (bw) for an adult according to EFSA Scientific Committee (EFSA SC. which may result in an incomplete extraction of acrylamide.4 mg/cup. In the decoction. corresponding to 1. Volumes ranged from 40 to 100 mL/cup. the content of acrylamide in the ground coffee from which beverages were prepared could range from 41. Because of this. Mean SD Acrylamide content mg/L 20. taking into account the range of concentrations observed among the different samples. 2012).4 27.Fig. design. Figure represents the distribution of acrylamide content in 41 different coffee brews. c bw: body weight. 2015).6 6. which led to a total acrylamide content ranging from 0. In this case. the final acrylamide content may be higher than in other coffee brews.J. most of the coffee brews presented acrylamide values within the range reported for other espresso coffees and.0 mg/L and 39.5 to 232 mg/kg.9 mg/person/day).0 2. with a mean value of 20. Acrylamide levels in coffee brews Acrylamide levels in the coffee brews acquired from coffee vending machines ranged from 7. These results are similar to the daily intake of acrylamide from coffee estimated for adults from The Netherlands (0. Estimation of acrylamide exposure from coffee brews Exposure to acrylamide from drinking coffee was estimated.048 mg/kg bw/day). dry weight.7 mg/kg bwc/day 0.. coffee origin. Possible relationships between pH. This range is close to that reported by EFSA (2010) in roasted coffee (197–256 mg/kg) and no samples exceeded the indicative value of 450 mg/kg according to the recommendations of the European Commission (EC. Box. Mesías.0 2. then the mean daily acrylamide intake would be 2. Initially levels in the drinks were normalized to the volume of the beverage dispensed by each automatic machine. Data distribution showed a high variability in the acrylamide content among samples and two outliers were identified (36. .4 2.64 cups of coffee/person/day.2. b Volumes ranged from 40 to 100 mL/cup.6 0. browning and acrylamide content were analyzed.8 to 2. applied. (2010) Soares et al. higher exposures have been found for Denmark (0.0–0. Morales / Journal of Food Composition and Analysis 48 (2016) 8–12 11 Table 2 Acrylamide levels (mg/L) in coffee brews. GC–MS: gas chromatography-mass spectrometry. since coffee brew is considered one of the most important contributors to acrylamide exposure in the diet of adults. To avoid possible risk it is advisable to reduce acrylamide content in coffee and.3 11. respectively. (2010) Granby and Fagt (2004) Sirot et al. has been described (Granby and Fagt. where the total acrylamide intake was estimated to be 0.2 1.000–0.0 0.171 0.456 14–44. The dietary exposure of the Spanish population to acrylamide through coffee beverages does not imply a high risk to human health. considering certain preventive actions and mitigation strategies. The most approximate evaluation has been published by Delgado-Andrade et al. 2008). Extraction method not specified. Therefore. these exposures depend on the consumption habits of the population since coffee consumption is widely variable between different countries and regions. Therefore it can be deduced that the exposure of humans to acrylamide from coffee vending machines is similar to that of other coffee preparations. Table 3 shows a summary of the acrylamide exposure through the consumption of both roasted coffee beverages and instant coffee in adults and adolescents from several countries.2–42. 2015). Conclusions The differences observed in the acrylamide content of coffee brew may result from the different roasted coffees used and the different conditions of preparation in the coffee vending machines. However. 2011). France (0.18 mg/kg bw/day). as revised by Arisseto and Vicente (2015). it may be suggested that exposure to acrylamide through coffee consumption could imply a risk to public health. However. 4. defining the margin of exposure (MOE) as the value calculated by dividing the dose causing cancer in animal studies by exposure of humans to acrylamide (FAO/WHO. Coffee brews Method determination Acrylamide levels Reference Turkish coffee Espresso coffee LC–MS/MSa GC–MSb GC–MS LC–MS/MS LC–MS/MS GC–MS/MS LC–MS/MS LC–MS/MS 29.2 Adolescents Exposure (mg/kg bwa /day) Contribution (%) 0. coffee would account for 7. 2011) and the value of 2.31 mg/kg bw/day) and 4865 when the comparison is with the BMDL10 for Harderian gland tumors in male mice (0.J. (2006) Andrzejewski et al.565 Up to 55.M. (2004) Granby and Fagt (2004) Mojska et al.534 mg/kg bw/day.116 0. defined as the lowest no observed adverse effect level (NOAEL) for a non-carcinogenic end-point (FAO/WHO.4 0.000–0.4–36.0–9. although these conclusions should be revised for heavy coffee consumers (more than 5 cups/day). Consumption of coffee brew acquired from vending services contributes to the acrylamide exposure in humans in a way similar to other coffee brew preparations. 2004. it may be concluded that coffee consumption does not involve a high risk.0 3–15 5–14 2–16 37 3 Şenyuva and Gökmen (2005) Alves et al.000–0. In order to calculate the potential risk for human health through the consumption of acrylamide from coffee beverages. n.0 0. Population groups Roasted coffee Instant coffee Average consumers High consumers Average consumers High consumers Adults Exposure (mg/kg bwa /day) Contribution (%) 0. according to the recent review published by Arisseto and Vicente (2015). not available. Table 3 Summary of acrylamide exposure and contribution to the total acrylamide intake through the consumption of roasted coffee beverages and instant coffee. If these estimations are taken into account. the maximum acrylamide exposure estimated in our study is much less than both the value of 0. the results found in the present study lie within the range of exposure to acrylamide from roasted coffee for average coffee consumers (0.5 0.5% of the total acrylamide intake.168 mg/kg bw/day) and Sweden (0.010–0. As observed.9 0. To our knowledge..059–0.012 0.a. 2010). consequently the dietary exposure to acrylamide. The EFSA Scientific Committee considers that.5 0. In this respect.106 mg/kg bw/ day).007 0.166 n.0 33.a. it is necessary to define potential strategies to reduce the content of acrylamide in coffee. An average consumption of coffee from 210 mL/day in the Polish adult population to as much as 1100 and 900 mL/day for Danish men and women (35–44 years age group).003–0. Obviously. a MOE value of 8378 was obtained when comparing the mean exposure with the established BMDL10 (benchmark dose lower confidence limit 10%) for mammary tumors in female rats (0.5–41.0–17.171 mg/kg bw/day). there are no studies on the exposure of the Spanish population to acrylamide.171 mg/kg bw/ day) (Arisseto and Toledo. Mojska et al. F. 2010). Mesías. an MOE of 10.0–75. This result falls within the range of contribution of roasted coffee to the total acrylamide intake for both adults and adolescents who are average coffee consumers (Arisseto and Vicente.000 or higher is of low concern from a public health point of view (EFSA.003–0. (2012) Filter coffee French press cafetiere Brewed coffeec a b c LC–MS/MS: liquid chromatography-tandem mass spectrometry. Since the calculated MOE values are lower than 10000.000–0.2 mg/kg bw/day. 2015).b a b bw: body weight.7–14. for substances that are both genotoxic and carcinogenic. 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