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摘 要： 分别对添加了每日膳食营养素推荐供给量标准（RDA为90 mg）的50%，75%和100%维生素C的咖啡粉进行了维生素C的潜在载体的评估研究.旨在评价咖啡冲泡和贮藏3个月后产品中维生素C的稳定性，并探究消费者是否能感知普通咖啡和维生素C强化咖啡之间的差异.实验结果表明：添加50%RDA水平的咖啡在冲泡过程中损失16%的维生素C;但更高强化水平的咖啡未发现维生素C的损失.室温贮藏3个月后，添加100%RDA水平的强化咖啡未见维生素C降解，而在另两个低强化水平下检测到5%～6%维生素C的损失.每份添加50%或75%RDA维生素C的冲泡咖啡的口感与未强化咖啡不存在显著性差异.因此，添加75%RDA维生素C的强化咖啡粉可确保在不改变其感官特性的情况下，将添加的维生素完全保留在冲泡咖啡饮料中.咖啡粉可用于日常膳食维生素C强化的潜在载体.
关键词： L-抗坏血酸; 维生素C; 咖啡; 三角检验; 感官; 强化
1 Introduction
Vitamin C is the most widely used supplement worldwide[1-3] and is one of the most commonly used antioxidants for enriching beverages[4-5].Recommended dietary allowance （RDA） of vitamin C has been set as low as 45 mg?d-1 by the US.Food and Nutrition Board of the National Academy of Sciences-National Research Council for scurvy prevention[6].Although diseases caused by nutritional deficiencies are no longer widespread in the western world[2]，addition of nutrients to food has been directed towards overall health and well-being.
Currently，US RDA is 90 mg?d-1 and 75 mg?d-1 for men and women，respectively[7].Additionally，a tolerable upper limit intake of 2 000 mg?d-1 of vitamin C has been widely cited as safe[2]due to low risk of hypervitaminosis，as amounts not utilized by the body are excreted in the urine[8-9].Furthermore，if the recommended United States Department of Agriculture （USDA） guideline of eating at least five servings of fruits and vegetables is followed，daily vitamin C intake would be 210-280 mg[10].However，about 25% of women and 33% of men consume less than 2.5 servings of fruits and vegetables daily[10]，and thus do not meet or barely meet RDA of vitamin C.
Coffee may be an ideal beverage medium for vitamin C fortification.At least 83% of Americans drink coffee[11] with an average of one cup of coffee consumed on any given day by more than 50% of men and women age of 20 years old and over [12].Additionally，compared to other beverages consumed during breakfast，coffee （and coffee-based beverages） is consumed the highest （44%） followed by water （16%），juice （14%），soft drinks （11%），milk （8%），and tea （7%） [12].This study was conducted to evaluate potential for fortification of ground coffee with vitamin C. Coffee pods used for single-serve brewing applications are increasingly gaining popularity attributed to convenience that caters to the busy life-style of today’s society.Although this study evaluates the potential for fortifying coffee with vitamin C （in general），the application in ground coffee packaged in single-serve pods may ideally deliver vitamin C for people who want to save time （short coffee brewing process） or want a beverage that can be taken easily on-the-go.
The objectives of the present study were：（1） to determine the effect of the brewing process on retention of vitamin C added to ground coffee;（2） to evaluate vitamin C stability in ground coffee after 3 months storage;and （3） to evaluate whether consumers can detect a difference in sensory attributes of coffee with and without vitamin C.
2 Materials and methods
2.1 Chemicals
Food grade L-ascorbic acid （>99.56%，purification;DNP International Company，Inc.，Santa Fe Springs，CA，USA） was used as received.The mobile phase for HPLC analysis of vitamin C were of analytical reagent grade， including the following：sodium phosphate monobasic （Sigma-Aldrich，St.Louis，MO，USA），HPLC-grade water and methanol （Fisher Scientific，Fair Lawn，NJ，USA）.Deionized water was used for the preparation of all coffee samples.Ground coffee （ground 100% Arabica coffee，Luigi Lavazza S.P.A.，Torino，Italy） was purchased from a local store.
2.2 Preparation of coffee with vitamin C
A predetermined amount of vitamin C （50%，75% or 100 % RDA level;100% RDA is 90 mg per serving） and ground coffee （7 g coffee per serving） were placed inside a clear bag （4 servings per bag） with round wire closure （Fisher Scientific，Fair Lawn，NJ，USA）.These bags were placed in a small corrugated box and shaken using Vortex Genie-2 （Fisher Scientific，Fair Lawn，NJ，USA） 5 h prior to brewing to allow mixing of vitamin C and the ground coffee.A 7 g sample was taken from the bag and placed in the My K-Cup? reusable coffee filter basket and assembled for brewing （7 g coffee with vitamin C per 250 mL deionized water for brewing resulting in （236±1.3） mL brewed coffee） using the Mini Plus single-cup home brewing system [13]（Keurig Green Mountain，Waterbury，VT，USA）.The internal temperature of the water in the brewer was estimated to be 89 ℃ （www.keurig.com/support/k-cup-brewers）.Brewed samples were immediately cooled in ice bath.Samples were stirred using a magnetic stir bar，and a 2 mL aliquot sample at room temperature was filtered through a 0.45 μm PTFE（polytetrafluoroethylene） Whatman syringe filter （Sigma-Aldrich，St.Louis，MO，USA） prior to injection in HPLC. Quantitative analysis of vitamin C was performed by HPLC-UV with a Waters 2695 Series system （Waters Corporation，Milford，MA，USA） equipped with a Waters 2998 model diode array detector （operated at 245 nm） and an injection valve with a 20 ?L sample loop.Compounds were separated on a 4.6 mm×150 mm i.d.，5 ?m pore size reversed-phase Zorbax Eclipse Plus C18 column protected by a Zorbax Eclipse Plus C18 analytical guard column with 4.6 mm×12.5 mm，2.1 mm id.，5 ?m pore size （Agilent Technologies，Santa Clara，CA，USA）.Vitamin C was eluted with a gradient elution of mobile phases A [50 mmol?L-1 sodium phosphate monobasic /MeOH （90/10），pH=2.5] and B [50 mmol?L-1 sodium phosphate monobasic/MeOH （10/90），pH=2.5] at a flow rate of 1 mL?min-1.The elution started at 100% A to reach 70% B at the 18th minute，changed back to reach 100% A at the 20th minute，and kept isocratic for 6 min to equilibrate the column prior to injection of the next sample.
2.3 Preparation of vitamin C for storage stability
Samples of 30 g ground coffee were placed in a 7-layer flexible foil pouch （MIL-PRF，provided by Cadillac Products Packaging Co.，Paris，IL，USA）.Each pouch of ground coffee was fortified with predetermined levels of vitamin C （50%，75% or 100% RDA;based on 100% RDA is 90 mg vitamin C per 7 g of ground coffee）.Controls were non-fortified ground coffee.Pouches were purged for 10 s in a vacuum sealer with nitrogen，and sealed with Multivac A300/52 （Multivac Inc.，Kansas City，MO，USA）.Samples were stored at ambient temperature.Vitamin C storage stability in ground coffee after 3 months was evaluated after a brewing process using similar protocols and quantification methods outlined in section 2.2.
2.4 Determination of retention of vitamin C
Chromatograms were integrated with Waters Empower? Software （Waters Corporation，Milford，MA，USA） and were compared with the retention time of vitamin C standard （R2=0.997 3） prepared using plain brewed coffee （7 g ground coffee per 250 mL deionized water） as solvent as shown in figure 1.Each experiment had a minimum of three replicates from each bag.
Figure 1 A comparison of HPLC chromatograms of （a） coffee with no vitamin C，and （b） coffee with vitamin C.Zorbax Eclipse Plus C18 column with mobile phases A [50 mmol?L-1 sodium phosphate monobasic/MeOH （90/10），pH=2.5]，and B [50 mmol?L-1 sodium phosphate monobasic/MeOH （10/90），pH=2.5] at a flow rate of 1 mL?min-1. 2.5 Sensory evaluation of coffee fortified with vitamin C
Panelists were recruited for willingness to taste coffee served at room temperature.The panel included college students，staff，and faculty from The University of Tennessee （Knoxville，TN，USA）.Prior to tasting，all panelists signed an informed consent form in compliance with Research Involving Human Subjects.A total of three triangle tests were conducted to evaluate if panelists are able to detect differences between non-fortified coffee versus coffee fortified with vitamin C.Triangle tests were conducted on three formulations of coffee （50%，75%，or 100% RDA vitamin C per serving;100% RDA is 90 mg per serving） against regular plain coffee.Each of the three triangle tests consisted of 60 panelists.Participants included 19 male，41 female;22 male，38 female;or 21 male，39 female （triangle test of coffee with 50%，75%，or 100% RDA of vitamin C，respectively）.
Testing was conducted in a central location testing facility with individual booths under white fluorescent lighting between 8：00 and 11：00 a.m.Coffees were first prepared and brewed approximately 2.5 h before evaluation.Deionized water （Crystal Springs Water，San Luis Obispo，CA，USA） was used for coffee brewing.Each batch of brewed coffee was stirred occassionally using a glass rod while cooling to room temperature.Once coffee temperature reached 32 ℃ or lower，vitamin C （50%，75%，or 100% RDA） was added to the brewed coffee.Predetermined amounts of vitamin C were calculated based on a 7 g ground coffee per 250 mL brewed coffee serving.The samples were distributed in 50 mL clear glass beakers，each containing 30 mL sample.Each cup was identified with a random three-digit number.The order of presentation was balanced.All 3 samples of the triangle test were presented together at ambient temperature.Panelists were instructed to taste the samples from left to right，to identify the odd sample and to describe the difference.An expectorant cup with 20 mL distilled water （Crystal Springs Water，San Luis Obispo，CA，USA） at ambient temperature was provided for rinsing between samples.
2.6 Data analysis
Data are expressed as means ± standard deviation，analyzed using SPSS （version 16.0，SPSS，Chicago，IL，USA）.One-way analysis of variance was carried out to test any significant differences between the means.Differences were considered significant at p<0.05.FIZZ Acquisition （Version 2.47b，Fizz Biosystems，Counternon，France） was used for sensory data gathering and statistical analysis. 3 Results and discussion
3.1 Retention of vitamin C after coffee brewing
Vitamin C retention after coffee brewing is presented in table 1.Brewing of ground coffee fortified with 75% or 100% RDA （67.5 mg or 90 mg，respectively） of vitamin C per serving did not significantly reduce the vitamin content in the brewed coffee.However，brewing of coffee with 50% RDA （45 mg） resulted in a 16% degradation loss of vitamin C （down to 37.6 mg per serving）.
a Based on RDA 100%=90 mg of vitamin C in a serving;One single-cup serving of coffee obtained by using 250 mL water used to brew 7 g ground coffee （single-coffee-cup after brew=（236.0±1.3） mL）;* significantly reducing.
The stability of vitamin C during brewing of fortified ground coffee may be attributed to both the sample pH value and the short exposure time at a relatively high temperature.The non-fortified brewed coffee had a pH value of 5.05±0.06;whereas，the coffee with vitamin C （50%-100% RDA per serving） had a pH value of 4.93±0.13.Although ascorbic acid easily oxidizes at the pH value above 6，it is relatively stable at lower pH[14-15].The internal temperature of the hot water in the Keurig coffee maker was estimated at 89 ℃ based on the Keurig brewer support website[13].Immediately after brewing，the internal temperature of the brewed coffee was （78±3） ℃.The brewed coffee was cooled rapidly in an ice-bath prior to HPLC analysis.Thus，vitamin C in all samples was exposed to potentially unfavorable conditions for a short time.
The relatively good stability of vitamin C either at short exposure to high temperatures or at low pH has been demonstrated by other studies.For example，blanching of dehydrated sliced carrots at 98 ℃ for 1 min reduced vitamin C content for 15% [16].Orange juice （pH=3.4） had ascorbic acid content of 40 mg per 100 mL even after pasteurization at 80 ℃ for 30 s[17]，and no loss of vitamin C was determined in homogenized raw clementines even after a one year storage at -60 ℃ under nitrogen[18].Vitamin C naturally present in fresh broccoli florets （117.7 mg per 100 g） showed no loss when broccoli was steamed for 3.5 min[19].
3.2 Storage stability
When vitamin C （50%，75%，or 100% RDA） was added to ground coffee and stored at room temperature for 3 months，no losses of vitamin C were observed at the level of 100% RDA;and 5%-6% losses were detected at a lower level of fortification.Stability of the added vitamin C in ground coffee may be attributed to the preparation and type of packaging used for storage.Samples were nitrogen flushed and vacuum-sealed prior to sealing of the packaging to simulate commercial packaging of ground coffee.The use of laminated flexible foil pouches for packaging other foods，such as high pressurized orange juice，has similarly resulted in lower rates of ascorbic acid degradation [17]. Vitamin C storage stability is influenced by factors such as presence of oxygen，moisture，pH，and length of storage[20-23].For instance，when infant formula was fortified with vitamin C，packaged in a nitrogen-flushed hermetically sealed container and stored for 3，6，and 9 months at 25 ℃，an average of 6.5% loss in vitamin C was detected with respect to initial content [24].However，90 d storage at ambient temperature caused 22% decrease in vitamin C content in strawberry drink with initial pH=3.40[25].Vitamin C fortified powdered fruit drinks showed adequate stability with >80% vitamin C retention after one-year of storage at ambient temperature[26].Vitamin C added to deaerated peanut spreads was also shown to be relatively stable （<25% loss） after 3 months storage at ambient temperature [27].On the contrary，more significant vitamin C losses were observed in other vitamin C fortified food products.For example，in production of potato flakes，drum-drying reduced the vitamin C level to 44% and consequent 12 months storage at 25 ℃ further decreased the vitamin C content to <20% from the initial level[28].Canned pumpkin or pineapple cubes packed in a sucrose solution （pH value of 4.63 or 4.22，respectively） resulted in 25%-30% vitamin C loss after 6 months storage in room temperature[29].
3.3 Triangle test between unfortified and vitamin C fortified coffee
Low levels of vitamin C （50% or 75% RDA per serving） added to coffee were non-detectable by panelists （p=0.750 5 and p=0.244 4，respectively） when tasted against unfortified coffee.However，panelists were able to detect a difference between unfortified and fortified coffee at the 100% RDA level （p=0.021 9），checking flavor as the main difference.Specifically，all panelists who correctly identified the odd sample indicated a perceived flavor difference.Attributed reasons for differences include the following comments： coffee sample with vitamin C was “more acid （sour）”，“more acidic”，“vinegar-tasting”，“sour aftertaste”;whereas，coffee without vitamin C was described as “less acidic”，“less sour”，“not as tart”，and “didn’t have the slight vinegar-like flavor”.Furthermore，a few panelists commented a “more bitter” taste on coffee without vitamin C;whereas，vitamin C fortified coffee had “less bitter” attributes. The levels of vitamin C in these samples are higher than detection threshold levels （0.016-0.031 mmol?L-1;citric acid） for sour taste[30].However，perceived sourness depends on not only the carrier medium[31] but also individual differences of the subject’s sensitivity to the stimuli[30].Sour taste from ascorbic acid at vitamin C levels （100% RDA per serving;2.04 mmol?L-1） may not be the main contributor of the perceived difference because coffee’s characteristic taste includes both acid and bitter flavor profiles[32].However，results of the study suggest that panelists may potentially perceive a greater sour taste in their coffee when vitamin C is added at levels of 90 mg vitamin C per serving.
Panelists’ indication of no difference between unfortified and fortified coffee with low levels of vitamin C （45 mg and 67.5 mg per serving，respectively） is similar to what has been observed in other model beverages.For example，50 g Yerba mate （made of dried and minced leaves or twigs） fortified with low levels of vitamin C （15 mg） resulted in no flavor alteration after a 20 mL liquid extraction[33].Additionally，L?KER et al[34] showed that liquid milk fortified with vitamin C （30 mg per 100 g） resulted in no increase in acidity and no negative effect on the sensory quality of the milk.
Coffee beverages are served both hot and cold.A study on liking of coffee showed coffee acceptability increased at temperatures that are either high （>70 ℃） or low （40 ℃ or 55 ℃） with lowest acceptability at ambient temperature[35].In this study，coffee was served at room temperature so that panelists can focus if ascorbic acid imparts a flavor difference when added to coffee，rather than citing temperature as a source of variation between samples.
Product and purchase intents of panelists who were consumers of coffee are shown in table 2.Overall，the results showed that a higher percentage of panelists （>50%） indicated they would choose coffee that is vitamin-fortified than the non-fortified one after tasting at lower levels of added vitamin C （50% or 75% RDA）.Likewise，lower vitamin C levels （50% or 75% RDA per serving） in coffee resulted in at least 56% of panelists indicating that vitamins in coffee provide nutritional benefit.Lastly，about 72% of the panelists indicated that they might or would buy fortified coffee. Coffee consumption patterns of the panelists were evaluated.Ninety percent of the panelists consume coffee hot.Twenty-six percent of the panelists drink their coffee black;whereas，40% of the panelists add both cream and sugar to their coffee （others only cream 23%;or only sugar 11% of panelists）.Lastly，sixty-percent of the panelists drink coffee at least once a day.
Addition of low levels of vitamin C in widely consumed beverages may provide value added benefits due to higher likelihood of consumer acceptance.With the widespread consumption of coffee on a daily basis，its fortification with vitamin C has the potential to reach numerous segments of the population.
4 Conclusions
The busy lifestyles of today’s society require food products to not only be a good source of nutrients，but also be easier to take on-the-go in order to be incorporated in people’s diets.Vitamin C fortification in ground coffee beans may be advantageous because it is a popularly consumed beverage that reaches a multitude of people.As demonstrated in this study，vitamin C added at levels of 75% （67.5 mg） per 7 g ground coffee may be a suitable fortification level because it withstands the brewing process and does not alter flavor and appearance of the beverage.
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关键词： L-抗坏血酸; 维生素C; 咖啡; 三角检验; 感官; 强化
1 Introduction
Vitamin C is the most widely used supplement worldwide[1-3] and is one of the most commonly used antioxidants for enriching beverages[4-5].Recommended dietary allowance （RDA） of vitamin C has been set as low as 45 mg?d-1 by the US.Food and Nutrition Board of the National Academy of Sciences-National Research Council for scurvy prevention[6].Although diseases caused by nutritional deficiencies are no longer widespread in the western world[2]，addition of nutrients to food has been directed towards overall health and well-being.
Currently，US RDA is 90 mg?d-1 and 75 mg?d-1 for men and women，respectively[7].Additionally，a tolerable upper limit intake of 2 000 mg?d-1 of vitamin C has been widely cited as safe[2]due to low risk of hypervitaminosis，as amounts not utilized by the body are excreted in the urine[8-9].Furthermore，if the recommended United States Department of Agriculture （USDA） guideline of eating at least five servings of fruits and vegetables is followed，daily vitamin C intake would be 210-280 mg[10].However，about 25% of women and 33% of men consume less than 2.5 servings of fruits and vegetables daily[10]，and thus do not meet or barely meet RDA of vitamin C.
Coffee may be an ideal beverage medium for vitamin C fortification.At least 83% of Americans drink coffee[11] with an average of one cup of coffee consumed on any given day by more than 50% of men and women age of 20 years old and over [12].Additionally，compared to other beverages consumed during breakfast，coffee （and coffee-based beverages） is consumed the highest （44%） followed by water （16%），juice （14%），soft drinks （11%），milk （8%），and tea （7%） [12].This study was conducted to evaluate potential for fortification of ground coffee with vitamin C. Coffee pods used for single-serve brewing applications are increasingly gaining popularity attributed to convenience that caters to the busy life-style of today’s society.Although this study evaluates the potential for fortifying coffee with vitamin C （in general），the application in ground coffee packaged in single-serve pods may ideally deliver vitamin C for people who want to save time （short coffee brewing process） or want a beverage that can be taken easily on-the-go.
The objectives of the present study were：（1） to determine the effect of the brewing process on retention of vitamin C added to ground coffee;（2） to evaluate vitamin C stability in ground coffee after 3 months storage;and （3） to evaluate whether consumers can detect a difference in sensory attributes of coffee with and without vitamin C.
2 Materials and methods
2.1 Chemicals
Food grade L-ascorbic acid （>99.56%，purification;DNP International Company，Inc.，Santa Fe Springs，CA，USA） was used as received.The mobile phase for HPLC analysis of vitamin C were of analytical reagent grade， including the following：sodium phosphate monobasic （Sigma-Aldrich，St.Louis，MO，USA），HPLC-grade water and methanol （Fisher Scientific，Fair Lawn，NJ，USA）.Deionized water was used for the preparation of all coffee samples.Ground coffee （ground 100% Arabica coffee，Luigi Lavazza S.P.A.，Torino，Italy） was purchased from a local store.
2.2 Preparation of coffee with vitamin C
A predetermined amount of vitamin C （50%，75% or 100 % RDA level;100% RDA is 90 mg per serving） and ground coffee （7 g coffee per serving） were placed inside a clear bag （4 servings per bag） with round wire closure （Fisher Scientific，Fair Lawn，NJ，USA）.These bags were placed in a small corrugated box and shaken using Vortex Genie-2 （Fisher Scientific，Fair Lawn，NJ，USA） 5 h prior to brewing to allow mixing of vitamin C and the ground coffee.A 7 g sample was taken from the bag and placed in the My K-Cup? reusable coffee filter basket and assembled for brewing （7 g coffee with vitamin C per 250 mL deionized water for brewing resulting in （236±1.3） mL brewed coffee） using the Mini Plus single-cup home brewing system [13]（Keurig Green Mountain，Waterbury，VT，USA）.The internal temperature of the water in the brewer was estimated to be 89 ℃ （www.keurig.com/support/k-cup-brewers）.Brewed samples were immediately cooled in ice bath.Samples were stirred using a magnetic stir bar，and a 2 mL aliquot sample at room temperature was filtered through a 0.45 μm PTFE（polytetrafluoroethylene） Whatman syringe filter （Sigma-Aldrich，St.Louis，MO，USA） prior to injection in HPLC. Quantitative analysis of vitamin C was performed by HPLC-UV with a Waters 2695 Series system （Waters Corporation，Milford，MA，USA） equipped with a Waters 2998 model diode array detector （operated at 245 nm） and an injection valve with a 20 ?L sample loop.Compounds were separated on a 4.6 mm×150 mm i.d.，5 ?m pore size reversed-phase Zorbax Eclipse Plus C18 column protected by a Zorbax Eclipse Plus C18 analytical guard column with 4.6 mm×12.5 mm，2.1 mm id.，5 ?m pore size （Agilent Technologies，Santa Clara，CA，USA）.Vitamin C was eluted with a gradient elution of mobile phases A [50 mmol?L-1 sodium phosphate monobasic /MeOH （90/10），pH=2.5] and B [50 mmol?L-1 sodium phosphate monobasic/MeOH （10/90），pH=2.5] at a flow rate of 1 mL?min-1.The elution started at 100% A to reach 70% B at the 18th minute，changed back to reach 100% A at the 20th minute，and kept isocratic for 6 min to equilibrate the column prior to injection of the next sample.
2.3 Preparation of vitamin C for storage stability
Samples of 30 g ground coffee were placed in a 7-layer flexible foil pouch （MIL-PRF，provided by Cadillac Products Packaging Co.，Paris，IL，USA）.Each pouch of ground coffee was fortified with predetermined levels of vitamin C （50%，75% or 100% RDA;based on 100% RDA is 90 mg vitamin C per 7 g of ground coffee）.Controls were non-fortified ground coffee.Pouches were purged for 10 s in a vacuum sealer with nitrogen，and sealed with Multivac A300/52 （Multivac Inc.，Kansas City，MO，USA）.Samples were stored at ambient temperature.Vitamin C storage stability in ground coffee after 3 months was evaluated after a brewing process using similar protocols and quantification methods outlined in section 2.2.
2.4 Determination of retention of vitamin C
Chromatograms were integrated with Waters Empower? Software （Waters Corporation，Milford，MA，USA） and were compared with the retention time of vitamin C standard （R2=0.997 3） prepared using plain brewed coffee （7 g ground coffee per 250 mL deionized water） as solvent as shown in figure 1.Each experiment had a minimum of three replicates from each bag.
Figure 1 A comparison of HPLC chromatograms of （a） coffee with no vitamin C，and （b） coffee with vitamin C.Zorbax Eclipse Plus C18 column with mobile phases A [50 mmol?L-1 sodium phosphate monobasic/MeOH （90/10），pH=2.5]，and B [50 mmol?L-1 sodium phosphate monobasic/MeOH （10/90），pH=2.5] at a flow rate of 1 mL?min-1. 2.5 Sensory evaluation of coffee fortified with vitamin C
Panelists were recruited for willingness to taste coffee served at room temperature.The panel included college students，staff，and faculty from The University of Tennessee （Knoxville，TN，USA）.Prior to tasting，all panelists signed an informed consent form in compliance with Research Involving Human Subjects.A total of three triangle tests were conducted to evaluate if panelists are able to detect differences between non-fortified coffee versus coffee fortified with vitamin C.Triangle tests were conducted on three formulations of coffee （50%，75%，or 100% RDA vitamin C per serving;100% RDA is 90 mg per serving） against regular plain coffee.Each of the three triangle tests consisted of 60 panelists.Participants included 19 male，41 female;22 male，38 female;or 21 male，39 female （triangle test of coffee with 50%，75%，or 100% RDA of vitamin C，respectively）.
Testing was conducted in a central location testing facility with individual booths under white fluorescent lighting between 8：00 and 11：00 a.m.Coffees were first prepared and brewed approximately 2.5 h before evaluation.Deionized water （Crystal Springs Water，San Luis Obispo，CA，USA） was used for coffee brewing.Each batch of brewed coffee was stirred occassionally using a glass rod while cooling to room temperature.Once coffee temperature reached 32 ℃ or lower，vitamin C （50%，75%，or 100% RDA） was added to the brewed coffee.Predetermined amounts of vitamin C were calculated based on a 7 g ground coffee per 250 mL brewed coffee serving.The samples were distributed in 50 mL clear glass beakers，each containing 30 mL sample.Each cup was identified with a random three-digit number.The order of presentation was balanced.All 3 samples of the triangle test were presented together at ambient temperature.Panelists were instructed to taste the samples from left to right，to identify the odd sample and to describe the difference.An expectorant cup with 20 mL distilled water （Crystal Springs Water，San Luis Obispo，CA，USA） at ambient temperature was provided for rinsing between samples.
2.6 Data analysis
Data are expressed as means ± standard deviation，analyzed using SPSS （version 16.0，SPSS，Chicago，IL，USA）.One-way analysis of variance was carried out to test any significant differences between the means.Differences were considered significant at p<0.05.FIZZ Acquisition （Version 2.47b，Fizz Biosystems，Counternon，France） was used for sensory data gathering and statistical analysis. 3 Results and discussion
3.1 Retention of vitamin C after coffee brewing
Vitamin C retention after coffee brewing is presented in table 1.Brewing of ground coffee fortified with 75% or 100% RDA （67.5 mg or 90 mg，respectively） of vitamin C per serving did not significantly reduce the vitamin content in the brewed coffee.However，brewing of coffee with 50% RDA （45 mg） resulted in a 16% degradation loss of vitamin C （down to 37.6 mg per serving）.
a Based on RDA 100%=90 mg of vitamin C in a serving;One single-cup serving of coffee obtained by using 250 mL water used to brew 7 g ground coffee （single-coffee-cup after brew=（236.0±1.3） mL）;* significantly reducing.
The stability of vitamin C during brewing of fortified ground coffee may be attributed to both the sample pH value and the short exposure time at a relatively high temperature.The non-fortified brewed coffee had a pH value of 5.05±0.06;whereas，the coffee with vitamin C （50%-100% RDA per serving） had a pH value of 4.93±0.13.Although ascorbic acid easily oxidizes at the pH value above 6，it is relatively stable at lower pH[14-15].The internal temperature of the hot water in the Keurig coffee maker was estimated at 89 ℃ based on the Keurig brewer support website[13].Immediately after brewing，the internal temperature of the brewed coffee was （78±3） ℃.The brewed coffee was cooled rapidly in an ice-bath prior to HPLC analysis.Thus，vitamin C in all samples was exposed to potentially unfavorable conditions for a short time.
The relatively good stability of vitamin C either at short exposure to high temperatures or at low pH has been demonstrated by other studies.For example，blanching of dehydrated sliced carrots at 98 ℃ for 1 min reduced vitamin C content for 15% [16].Orange juice （pH=3.4） had ascorbic acid content of 40 mg per 100 mL even after pasteurization at 80 ℃ for 30 s[17]，and no loss of vitamin C was determined in homogenized raw clementines even after a one year storage at -60 ℃ under nitrogen[18].Vitamin C naturally present in fresh broccoli florets （117.7 mg per 100 g） showed no loss when broccoli was steamed for 3.5 min[19].
3.2 Storage stability
When vitamin C （50%，75%，or 100% RDA） was added to ground coffee and stored at room temperature for 3 months，no losses of vitamin C were observed at the level of 100% RDA;and 5%-6% losses were detected at a lower level of fortification.Stability of the added vitamin C in ground coffee may be attributed to the preparation and type of packaging used for storage.Samples were nitrogen flushed and vacuum-sealed prior to sealing of the packaging to simulate commercial packaging of ground coffee.The use of laminated flexible foil pouches for packaging other foods，such as high pressurized orange juice，has similarly resulted in lower rates of ascorbic acid degradation [17]. Vitamin C storage stability is influenced by factors such as presence of oxygen，moisture，pH，and length of storage[20-23].For instance，when infant formula was fortified with vitamin C，packaged in a nitrogen-flushed hermetically sealed container and stored for 3，6，and 9 months at 25 ℃，an average of 6.5% loss in vitamin C was detected with respect to initial content [24].However，90 d storage at ambient temperature caused 22% decrease in vitamin C content in strawberry drink with initial pH=3.40[25].Vitamin C fortified powdered fruit drinks showed adequate stability with >80% vitamin C retention after one-year of storage at ambient temperature[26].Vitamin C added to deaerated peanut spreads was also shown to be relatively stable （<25% loss） after 3 months storage at ambient temperature [27].On the contrary，more significant vitamin C losses were observed in other vitamin C fortified food products.For example，in production of potato flakes，drum-drying reduced the vitamin C level to 44% and consequent 12 months storage at 25 ℃ further decreased the vitamin C content to <20% from the initial level[28].Canned pumpkin or pineapple cubes packed in a sucrose solution （pH value of 4.63 or 4.22，respectively） resulted in 25%-30% vitamin C loss after 6 months storage in room temperature[29].
3.3 Triangle test between unfortified and vitamin C fortified coffee
Low levels of vitamin C （50% or 75% RDA per serving） added to coffee were non-detectable by panelists （p=0.750 5 and p=0.244 4，respectively） when tasted against unfortified coffee.However，panelists were able to detect a difference between unfortified and fortified coffee at the 100% RDA level （p=0.021 9），checking flavor as the main difference.Specifically，all panelists who correctly identified the odd sample indicated a perceived flavor difference.Attributed reasons for differences include the following comments： coffee sample with vitamin C was “more acid （sour）”，“more acidic”，“vinegar-tasting”，“sour aftertaste”;whereas，coffee without vitamin C was described as “less acidic”，“less sour”，“not as tart”，and “didn’t have the slight vinegar-like flavor”.Furthermore，a few panelists commented a “more bitter” taste on coffee without vitamin C;whereas，vitamin C fortified coffee had “less bitter” attributes. The levels of vitamin C in these samples are higher than detection threshold levels （0.016-0.031 mmol?L-1;citric acid） for sour taste[30].However，perceived sourness depends on not only the carrier medium[31] but also individual differences of the subject’s sensitivity to the stimuli[30].Sour taste from ascorbic acid at vitamin C levels （100% RDA per serving;2.04 mmol?L-1） may not be the main contributor of the perceived difference because coffee’s characteristic taste includes both acid and bitter flavor profiles[32].However，results of the study suggest that panelists may potentially perceive a greater sour taste in their coffee when vitamin C is added at levels of 90 mg vitamin C per serving.
Panelists’ indication of no difference between unfortified and fortified coffee with low levels of vitamin C （45 mg and 67.5 mg per serving，respectively） is similar to what has been observed in other model beverages.For example，50 g Yerba mate （made of dried and minced leaves or twigs） fortified with low levels of vitamin C （15 mg） resulted in no flavor alteration after a 20 mL liquid extraction[33].Additionally，L?KER et al[34] showed that liquid milk fortified with vitamin C （30 mg per 100 g） resulted in no increase in acidity and no negative effect on the sensory quality of the milk.
Coffee beverages are served both hot and cold.A study on liking of coffee showed coffee acceptability increased at temperatures that are either high （>70 ℃） or low （40 ℃ or 55 ℃） with lowest acceptability at ambient temperature[35].In this study，coffee was served at room temperature so that panelists can focus if ascorbic acid imparts a flavor difference when added to coffee，rather than citing temperature as a source of variation between samples.
Product and purchase intents of panelists who were consumers of coffee are shown in table 2.Overall，the results showed that a higher percentage of panelists （>50%） indicated they would choose coffee that is vitamin-fortified than the non-fortified one after tasting at lower levels of added vitamin C （50% or 75% RDA）.Likewise，lower vitamin C levels （50% or 75% RDA per serving） in coffee resulted in at least 56% of panelists indicating that vitamins in coffee provide nutritional benefit.Lastly，about 72% of the panelists indicated that they might or would buy fortified coffee. Coffee consumption patterns of the panelists were evaluated.Ninety percent of the panelists consume coffee hot.Twenty-six percent of the panelists drink their coffee black;whereas，40% of the panelists add both cream and sugar to their coffee （others only cream 23%;or only sugar 11% of panelists）.Lastly，sixty-percent of the panelists drink coffee at least once a day.
Addition of low levels of vitamin C in widely consumed beverages may provide value added benefits due to higher likelihood of consumer acceptance.With the widespread consumption of coffee on a daily basis，its fortification with vitamin C has the potential to reach numerous segments of the population.
4 Conclusions
The busy lifestyles of today’s society require food products to not only be a good source of nutrients，but also be easier to take on-the-go in order to be incorporated in people’s diets.Vitamin C fortification in ground coffee beans may be advantageous because it is a popularly consumed beverage that reaches a multitude of people.As demonstrated in this study，vitamin C added at levels of 75% （67.5 mg） per 7 g ground coffee may be a suitable fortification level because it withstands the brewing process and does not alter flavor and appearance of the beverage.
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