Acidity and Antioxidant Activity of Cold Brew Coffee

Abstract

The acidity and antioxidant activity of cold brew coffee were investigated using light roast coffees from Brazil, two regions of Ethiopia, Columbia, Myanmar, and Mexico. The concentrations of three caffeoylquinic acid (CQA) isomers were also determined. Cold brew coffee chemistry was compared to that of hot brew coffee prepared with the same grind-to-coffee ratio. The pH values of the cold and hot brew samples were found to be comparable, ranging from 4.85 to 5.13. The hot brew coffees were found to have higher concentrations of total titratable acids, as well as higher antioxidant activity, than that of their cold brew counterparts. It was also noted that both the concentration of total titratable acids and antioxidant activity correlated poorly with total CQA concentration in hot brew coffee. This work suggests that the hot brew method tends to extract more non-deprotonated acids than the cold brew method. These acids may be responsible for the higher antioxidant activities observed in the hot brew coffee samples.

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Scientific RepoRts | (2018) 8:16030 | DOI:10.1038/s41598-018-34392-w
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Acidity and Antioxidant Activity of
Cold Brew Coee
Niny Z. Rao & Megan Fuller
The acidity and antioxidant activity of cold brew coee were investigated using light roast coees
from Brazil, two regions of Ethiopia, Columbia, Myanmar, and Mexico. The concentrations of three
caeoylquinic acid (CQA) isomers were also determined. Cold brew coee chemistry was compared to
that of hot brew coee prepared with the same grind-to-coee ratio. The pH values of the cold and hot
brew samples were found to be comparable, ranging from 4.85 to 5.13. The hot brew coees were found
to have higher concentrations of total titratable acids, as well as higher antioxidant activity, than that
of their cold brew counterparts. It was also noted that both the concentration of total titratable acids
and antioxidant activity correlated poorly with total CQA concentration in hot brew coee. This work
suggests that the hot brew method tends to extract more non-deprotonated acids than the cold brew
method. These acids may be responsible for the higher antioxidant activities observed in the hot brew
coee samples.
Cold brew coee is a popular phenomenon that has recently invigorated the coee industry, particularly in the
warm summer months1. e domestic cold brew coee market grew 580% from 2011 to 20162. Roast Magazine
reports a 460% increase in retail sales of refrigerated cold brew coee in the United States from 2015 to 2017, gen-
erating $38 million in 2017 alone3. Cold brew coee is made through a low-temperature, long-contact brewing
method. Regional coee vendors, such as Starbucks and Dunkin Donuts have marketed the product as tasting
smoother and less bitter than traditional hot brewed coees4. Consumer interest has also been spurred by a range
of online health and lifestyle blogs publishing recipes and specic health claims for cold brew coee. A recent arti-
cle in Healthy Living Made Simple, a bimonthly publication with 4 million readers, states that “coee brewed hot
is far more acidic than cold-brewed, according to a number of scientic studies, and some say cold-brewed coee
even has a sweeter taste because of its lower acidity”5. A blog post on Coee Brewing Methods makes several
claims regarding the decreased acidity, decreased caeine levels, and increased antioxidant content of cold brew
coee6. At the time of publication, there was very little published research on the chemistry of cold brew coee
and no published research on the health eects of cold brew coee.
In fact, the health benets and risks of traditional hot brew coee consumption remain controversial. Coee
has long been associated with indigestion, heartburn, and other gastrointestinal symptoms. Epidemiological
meta-analyses and patient-based experimentation have led to conicting outcomes regarding the relationship
between coee consumption and gastrointestinal disorders. Early work by omas et al.7 found that coee con-
sumption in 20 healthy individuals and 16 patients with reux esophagitis resulted in the decrease of lower eso-
phageal sphincter (LES) pressure. e reduction of LES pressure, found in both cohorts following consumption
of coee with pH values of 4.5 and 7.0, could lead to aggravated heartburn symptoms7. Because the decrease in
LES pressure occurred at both an acidic and neutral pH, acidity may not be the inciting factor in heartburn fol-
lowing coee consumption. Two studies by Wendl et al.8 and Pehl et al.9 observed gastro-oesophageal reux in
asymptomatic individuals (n = 16) and patients with gastro-oesophageal disease (GERD) (n = 17), respectively,
and found that both cohorts experienced decreased oesophageal reux aer consuming decaeinated coee, indi-
cating that caeine may responsible for coee-related heartburn symptoms8,9. A recent population-based study of
GERD patients (n = 317) and asymptomatic individuals (n = 182) found no association between GERD symptom
frequency or severity and coee consumption10. Kubo et al.’s work is in agreement with other meta-analyses that
use patient-reported symptoms. Shimamato et al.11 used a large-scale multivariate analysis (n = 8,013) to evaluate
coee consumption as a contributor to the occurrence of gastric ulcers, duodenal ulcers, reux esophagitis, and
non-erosive reux disease. Shimamato et al.11 found no signicant relationships between coee consumption
and these four major acid-related gastrointestinal disorders11. Given the disagreement found in the literature
Department of Chemistry and Biochemistry, Thomas Jeerson University, East Falls Campus, Philadelphia, PA, 19144,
USA. Correspondence and requests for materials should be addressed to N.Z.R. (email: niny.rao@jeerson.edu)
Received: 10 July 2018
Accepted: 15 October 2018
Published: xx xx xxxx
OPEN
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regarding the health impacts of traditional hot coee, it is understandable that the general public views coee as
a potential health risk despite signicant evidence to the contrary.
Beyond gastrointestinal symptoms, coee has been shown to correlate to multiple potential health benets.
A substantial umbrella review of numerous meta-analyses found no consistent evidence of harmful associations
between coee consumption and diverse health outcomes, with the exception of issues related to pregnancy and
risk of bone fractures in women12. is work by Poole et al.12 evaluated previous research relating coee con-
sumption to cardiovascular health (including cardiovascular disease, coronary heart disease, and stroke), and
found a reduction in health risks when three cups of coee per day were consumed13–16. Poole et al.12 also found
coee consumption to be associated with decreased risk of liver17,18, metabolic19,20, and neurologic diseases21,22.
e causal pathways for these chemoprotective associations between coee consumption and disease are not
well understood; however, recent studies of coee have shown the beverage to exhibit high antioxidant capacity
and anti-inammatory eects. Work by Bakuradze et al.23 showed compounds present in coee roast products -
notably 5-caeoylquinic acid, a type of chlorogenic acid, and caeic acid - demonstrated direct antioxidant
activity in HT-29 (human colon) cells23. e role of antioxidant compounds as radical-scavengers in the body is
well-researched24–26, but the relationship between coee consumption, antioxidant activity, and brewing methods
is largely uncharacterized. A recent review by Naveed et al.27 further highlighted the therapeutic roles of chloro-
genic acids in human health and called for further research in the area27. Work by Chu et al.28 found that roasted
coees contained higher antioxidant capacities and higher chlorogenic acid and phenolic concentrations than
green coee beans. Chu et al.'s work also found a strong correlation between neuroprotective ecacy of roasted
coee and total chlorogenic acid concentration28.
Despite the growing popularity of cold brew coee, very little research has been published on its chemi-
cal attributes, including pH and total antioxidant activity, and associated health eects. An exhaustive literature
search returned only four peer-reviewed studies related to cold brew coee29–32. None of these studies provided
enough information to either support or refute the health claims about cold brew coee made by commercial
coee vendors and cold brew enthusiasts.
Given the signicant growth of the cold brew coee market and the potential importance of coee’s bioactive
compounds to human health, this study quanties the pH, total titratable acidity, and total antioxidant capacity of
cold brew coee produced from grinds sourced from six dierent coee-growing regions. Further, this research
quanties 5- caeoylquinic (5-CQA), 4-caeoylquinic (4-CQA), and 3-caeoylquinic acid (3-CQA) in these cold
brew coees to better understand the relationship between CQA content and total antioxidant capacity of coee.
e total antioxidant capacity is a measure of radical scavenging capacity and was determined using a ABTS
((2,2′-Azino-bi(3-ethylbenzo-thiazonile-6-sulfonic acid) diammonium salt) radical cation decolourization assay.
All coees used in this study were light-to-medium roast, pre-ground beans purchased from a commercial ven-
dor. Traditional hot brew coees and cold brew coees were compared to determine what, if any, dierences exist
in the acidity and antioxidant capacity of the resulting beverages as a function of brewing temperature and time.
Results
Hot Brew Coee. e results from the hot brew coee analyses are shown in Tables1 and 2. e hot brew
coee samples analyzed in this study were found to have pH values ranging from 4.85 to 5.10. e Ethiopian-Ardi
samples were observed to be the most acidic with a pH of 4.85 ± 0.09, whereas the Brazilian samples were the least
acidic with a pH of 5.10 ± 0.02. Of the three CQA isomers analyzed, 5-CQA was found to have the highest con-
centration in all samples, in agreement with previous studies33–39. e Ethiopian-Ardi samples were also found
to have the highest 5-CQA and total CQA concentration (1721 ± 99 mg/L of coee and 3270 ± 90 mg/L of coee,
respectively). e Brazilian samples had the lowest 5-CQA and total CQA concentration (1261 ± 111 mg/L of
coee and 2503 ± 103 mg/L of coee, respectively). e 3-CQA and 4-CQA concentrations were the highest in
the Ethiopian-Ardi samples, while Myanmar samples contained the lowest concentration of these two isomers.
Previous work by Moon et al.35 suggested that lower CQA concentration is correlated with a higher pH35. A sim-
ilar trend was observed among the samples analyzed in this study, with a Pearson correlation coecient of -0.70.
ese results agree well with pH data presented by Moon et al.35 for light roast hot brew coees.
e total titratable acidity (TA) of the coees is expressed in mL of 0.10 N NaOH required to titrate 40 ml of
coee to a pH of 6 and a pH of 8. ere have been multiple attempts to understand the chemical characteristics of
coee that cause the perception of bitterness in coee. Bähre et al. has demonstrated that TA shows better corre-
lation to sourness than pH40. Maier et al. found that the sourness of coee correlates well with TA titrated to pH
6.041. Balzer suggested that phenolic acids deprotonate at pH values greater than 842. us, TA titrated to pH 8.0
5-CQA
(mg/L) 4-CQA
(mg/L) 3-CQA
(mg/L) Total CQA
(mg/L)
Brazilian 1261 ± 111 693 ± 45 550 ± 27 2503 ± 103
Ethiopian - Ardi 1721 ± 100 842 ± 22 707 ± 34 3270 ± 90
Ethiopian - Yirgz 1385 ± 285 635 ± 101 510 ± 78 2530 ± 261
Myanmar 1433 ± 341 595 ± 38 489 ± 30 2517 ± 277
Columbia 1429 ± 67 677 ± 22 562 ± 27 2669 ± 64
Mexico 1476 ± 111 721 ± 41 611 ± 38 2808 ± 105
Table 1. Hot Brew Coee Samples: concentration of 5-CQA, 4-CQA, 3-CQA, and total CQA concentration
(milligrams per liter of brewed coee) of hot brew coee samples (Mean ± 95% Condence Interval, n = 6).
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may be better end point for titration42. Although sourness is not the focus of this study, TA titrated to these two
endpoints may provide some insights about the acid contents in coee. An earlier study by Gloess et al.36 found
no correlation between pH and TA36. For hot brew coee samples, Columbia coee was found to have the highest
concentration of total titratable acids at both pH of 6 and pH of 8. Brazilian and Myanmar samples were observed
to have the lowest concentrations of total titratable acids at both pH of 6 and pH of 8. Data collected in this study
showed little correlation between the pH and TA titrated to pH 6 (Pearson correlation coecient = -0.15) and TA
titrated to pH of 8 (Pearson correlation coecient = -0.09) for hot brew coee, in support of ndings by Gloess
et al.36.
Ethiopian-Yirgz samples were observed to have the highest antioxidant activity and Brazilian samples were
observed to have the lowest antioxidant activity. In general, the results of this study for hot brew coee agree well
with the general body of knowledge regarding the chemical characterization of light-to-medium roast coees,
including CQA content34,35 and antioxidant activity43–46.
Cold Brew Coee. e results from the cold brew coee analyses are shown in Tables3 and 4. ere is little
published data to contextualize these results. However, comparison with the hot brew coee characteristics in
Table1 point to the existence of chemical dierences between cold and hot brew coees prepared from the same
coee beans and extracted at the same ratio of water volume to grind weight. ese data indicate that the tem-
perature of the water used in brewing inuences the release and diusion of compounds in the resulting coee
beverage.
e pH values of cold brew samples ranged from 4.96 to 5.13, with Ethiopian-Yirgz being the most acidic
(pH = 4.96 ± 0.08) and Myanmar being the least acidic (5.13 ± 0.03). Similar to the hot brew counterparts,
5-CQA was found to be the most abundant CQA isomer in cold brew coee. Brazilian samples were observed to
have the highest concentration of all three CQA isomers whereas Mexican samples had the lowest CQA isomer
concentrations. e correlation between pH and total CQA concentration in cold brew coee is somewhat weak
(Pearson correlation coecient = -0.52).
In terms of total titratable acids, Mexican samples had the lowest concentration of total titratable acids at both
pH of 6 and pH of 8. Columbia samples had the highest concentration of total titratable acids (TA) at pH of 6 and
Brazilian samples had the highest concentration of total titratable acids at pH of 8. Similar to the hot brew sam-
ples, no correlation between pH and TA were observed for the cold brew samples. Ethiopian-Ardi samples were
observed to have the highest antioxidant activity, Myanmar and Ethiopian-Yirgz samples had the lowest antioxi-
dant activity. In general, the cold brew extracts were found to have pH values comparable to those of the hot brew
extracts, but lower total acidity measures, lower total CQA concentrations, and lower total antioxidant activities.
Hot and Cold Brew Comparisons. Total acidity and pH. Measurements of pH quantify the concen-
tration of aqueous hydrogen ions at the time of analysis, providing a metric for the quantity of deprotonated
acid molecules in a sample. Total titratable acidity (TA) is a measure of all acidic protons in a sample, including
non-dissociated protons, that can be neutralized through the addition of a strong base.
pH
Total Acidity
pH = 6
(mL of 0.10 N NaOH)
Total Acidity
pH = 8
(mL of 0.10 N NaOH)
Antioxidant Activity
(mmol equivalence
Trolox/L coee)
Brazilian 5.10 ± 0.02 3.17 ± 0.20 6.53 ± 0.38 18.34 ± 2.34
Ethiopian - Ardi 4.85 ± 0.09 3.62 ± 0.31 7.08 ± 0.74 19.95 ± 1.62
Ethiopian - Yirgz 4.96 ± 0.02 3.83 ± 0.33 7.45 ± 0.59 20.72 ± 3.12
Myanmar 4.92 ± 0.03 3.18 ± 0.75 6.40 ± 0.79 19.72 ± 1.17
Columbia 4.99 ± 0.10 4.27 ± 0.21 7.85 ± 0.06 19.98 ± 2.74
Mexico 4.95 ± 0.04 3.58 ± 0.41 6.68 ± 0.62 20.18 ± 1.65
Table 2. Hot Brew Coee Samples: pH, total titratable acid concentration titrated to a pH of 6 and 8 (milliliters
of 0.10 N NaOH per 40 milliliters of brewed coee), and antioxidant activity (millimoles equivalence Trolox per
liter of brewed coee) of hot brew coee samples (Mean ± 95% Condence Interval, n = 6).
5-CQA
(mg/L) 4-CQA
(mg/L) 3-CQA
(mg/L) Total CQA
(mg/L)
Brazilian 1124 ± 63 564 ± 23 513 ± 19 2201 ± 53
Ethiopian - Ardi 1133 ± 36 552 ± 14 464 ± 10 2149 ± 30
Ethiopian - Yirgz 1031 ± 127 480 ± 46 384 ± 34 1895 ± 104
Myanmar 912 ± 126 429 ± 28 355 ± 20 1697 ± 94
Columbia 1018 ± 157 488 ± 51 406 ± 41 1912 ± 127
Mexico 857 ± 138 416 ± 44 344 ± 35 1616 ± 111
Table 3. Cold Brew Coee Samples: concentration of 5-CQA, 4-CQA, 3-CQA, and total CQA concentration
(milligrams per liter of brewed coee) of cold brew coee samples (Mean ± 95% Condence Interval, n = 8).
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Commercial vendors and coee enthusiasts oen suggest that cold brew and hot brew coees boast dierent
taste proles due to diering acidity levels; and that cold brew coee, being supposedly less acidic, may reduce
gastrointestinal symptoms sometimes associated with coee consumption6,47–50. is work found the pH meas-
urements for all coee samples tested to be comparable, ranging between 4.85 to 5.13. Varying the temperature
of the extraction water did not result in distinguishable pH values between hot and cold brew coees (Fig.1).
However, TA results indicate substantially dierent concentrations of total acidic compounds between hot and
cold brew coees. is research found hot coee extracts to have larger measures of titratable acidity, indicating
higher concentrations of extracted acids and/or additional acidic compounds not found in the cold brew coee
extracts (Fig.2). e Pearson correlation coecients for both hot and cold brew samples are less than 0.5. e lack
of a correlation in this data agrees with the ndings of Gloess et al.36 and suggests that pH is a poor measurement
for the complex acid chemistry in both hot and cold brew coee extracts.
In general, these results suggest that cold and hot brew coees are similar in their total concentrations of
deprotonated acid compounds, but dier in the concentration and possibly the complexity of protonated acids
at the pH of extraction. e total CQA concentration data, shown in Tables1 and 3, found hot brew extracts to
have higher total CQA concentrations (Fig.3). is is one source of the dierence in total titratable acidities
(TA). e compounds present in hot brew coee but absent from cold brew coee may be larger molecules
with temperature-dependent solubilities, and/or compounds with signicant intermolecular forces that result in
strong coee matrix-compound attraction.
Antioxidant activity and Total CQA Concentration. e family of chlorogenic acid compounds are known to
contribute signicantly to the antioxidant activity of coee. Work by Daglia et al.51 and Stadler et al.52 have found
the polyphenolic compounds in coee to have antioxidant and antiradical activity in radical-mediated mutagenic
pathways. Given the importance of this family of compounds, correlations between antioxidant activity and CQA
concentrations were analyzed.
Similar to CQA data and TA, the data collected in this study indicated that hot brew extracts have higher anti-
oxidant activity than their cold brew counterparts (Fig.3). Figure4 shows the relationship between antioxidant
activity and total CQA concentration for hot and cold brew coees. e cold brew samples were found to have
a Pearson correlation coecient of 0.82, indicating a relatively strong correlation between these two chemical
characteristics. However, the antioxidant capacity and total CQA concentration of hot brew coee were found to
have a Pearson correlation coecient of 0.22, indicating a much weaker relationship between antioxidant activity
pH
Total Acidity
pH = 6
(mL of 0.10 N NaOH)
Total Acidity
pH = 8
(mL of 0.10 N NaOH)
Antioxidant Activity
(mmol equivalence
Trolox/L coee)
Brazilian 5.04 ± 0.16 2.83 ± 0.21 5.88 ± 0.31 16.10 ± 3.02
Ethiopian - Ardi 5.01 ± 0.02 2.55 ± 0.18 5.25 ± 0.19 17.45 ± 2.05
Ethiopian - Yirgz 4.96 ± 0.08 2.58 ± 0.18 5.18 ± 0.14 13.36 ± 0.99
Myanmar 5.13 ± 0.03 2.52 ± 0.14 5.32 ± 0.21 13.36 ± 2.85
Columbia 5.00 ± 0.05 2.93 ± 0.18 5.52 ± 0.32 15.33 ± 1.92
Mexico 5.08 ± 0.04 2.13 ± 0.11 4.75 ± 0.27 13.92 ± 2.69
Table 4. Cold Brew Coee Samples: pH, total titratable acid concentration titrated to a pH of 6 and 8 (milliliters
of 0.10 N NaOH per 40 milliliters of brewed coee), and antioxidant activity (millimoles equivalence Trolox per
liter of brewed coee) of cold brew coee samples (Mean ± 95% Condence Interval, n = 6).
4.7
4.8
4.9
5.0
5.1
5.2
5.3
BrazilianEth. Ardi Eth. YirgzMyanmar Columbia Mexico
pH
HotCold
Figure 1. pH values of six coee samples brewed using both hot and cold brewing methods. e error bars
represent 95% condence level.
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and chlorogenic acid concentration. Given that hot coee extracts exhibited higher antioxidant activity than their
cold brew counterparts, hot water must extract additional bioactive compounds. Hot brew coees analyzed here
were found to have increased concentrations of CQA isomers, and likely had increased concentrations of other
chlorogenic acids. is may account for the dierence in antioxidant activity between hot and cold brews, but
there may be additional compounds responsible for this dierential. e strong correlation between antioxidant
activity and total CQA concentration in cold brew coee suggests that CQA isomers are important drivers of cold
brew coee antioxidant activity.
Discussion
Cold brew coee extracts were found to have lower concentrations of acidic compounds and may be less chem-
ically diverse than hot brew coee extracts prepared from the same beans. is can be seen in both total acidity
and antioxidant activity measurements. Hot coee brews were found to have higher titratable acid levels, indicat-
ing higher concentrations of acidic compounds than in cold brew extracts, and/or additional acidic compounds
not found in cold brew extracts. All cold brew coee samples analyzed in this study were found to have lower
titratable acid levels than their hot brew counterparts. Coee is composed of dozens of low molecular mass com-
pounds, including numerous carboxylic acids such as citric, malic, quinic, succinic, and gluconic acids40,53. While
all of these acids are readily soluble in water, their ability to detach from the coee matrix and diuse through
the intra- and intergranular pore spaces in room temperature water as is used in cold brew method is poorly
understood.
Hot brew coees had higher antioxidant capacities than their cold brew counterparts, indicating that addi-
tional radical-scavenging compounds and/or higher concentrations of such compounds were present in the hot
brew samples. For cold brew coee, a strong correlation was found between total CQA concentration and total
antioxidant activity, while a weak correlation was seen for hot brew coee. e total CQA concentration failed to
correlate with antioxidant activity in hot brew coee likely because those hot water extracts had a more diverse
and complex chemistry than the cold brew samples. It can be assumed that many of the compounds absent from
the cold brew coees were acidic molecules, as the total acidity levels in the hot coees were found to be greater.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
BrazilianEth. Ardi Eth. YirgzMyanmar Columbia Mexico
HotCold
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
BrazilianEth. Ardi Eth. YirgzMyanmarColumbiaMexico
HotCold
Figure 2. Total titratable acids of six coee samples brewed using both hot and cold brewing methods measure
at (le) pH of 6.0 and (right) pH of 8.0. e values are reported as milliliters of 0.1 NaOH per 40 milliliters of
brewed coee. e error bars represent 95% condence level.
0
5
10
15
20
25
30
BrazilianEth. Ardi Eth. YirgzMyanmar Co lumbia Mexico
Antioxidant Activity
HotCold
0
500
1000
1500
2000
2500
3000
3500
4000
BrazilianEth. Ardi Eth. YirgzMyanmar Co lumbia Mexico
HotCold
Figure 3. (le) 3-CGA concentration in milligrams per liter of brewed coee and (right) antioxidant activity in
mmol equivalent of Trolox per liter of brewed coee of the six coee samples brewed using both hot and cold
brewing methods. e error bars represent 95% condence level.
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is research nds that water temperature during aqueous extraction inuences the transport of acidic mol-
ecules from the coee matrix into the water phase substantially enough to alter the total titratable acidity and
antioxidant activity of the resulting coee beverage.
Conclusions and Future Work
is research reveals important fundamental dierences between hot and cold brew coee that may have impli-
cations for possible health impacts on drinkers. It is oen claimed by cold brew coee enthusiasts that cold
brew coee has lower acidity than its hot brew counterparts, and thus may be a better alternative for those who
suer from gastrointestinal symptoms. is study suggests that the hot brew method tends to extract additional
non-deprotonated acids in comparison to the cold brew method. ese acids may be responsible for the higher
antioxidant activities observed in hot brew coee samples. Additionally, the chemical composition of hot brew
coee may be more diverse and complex than that of cold brew coee. Additional research is needed to fully
understand any possible dierences in the health eects of coee as a function of brewing temperature and time.
e lower antioxidant capacity in cold brew coees may decrease the chemoprotective benets known to be asso-
ciated with hot brew coees.
To better understand the relationship between brewing temperature and chemical complexity of the resulting
coee, compound-specic analysis of the extracts is needed. ere are several classes of compounds present
in coee extracts that may be the cause of the dierences seen in hot and cold brew coee in this study. One
possible class of compounds that may inuence pH and antioxidant activity levels are melanoidins. Melanoidin
compounds are known to have antiradical properties and account for upwards of 25% of coee’s dry matter54,55,
however, they have not been characterized in cold brew coees.
Previous studies have reported extensively on the chemical composition of coee34–37,42,56,57. Future work to
identify and quantify compounds present in hot and cold brew coee would help to better elucidate the chemical
dierences between the two beverages. Further work could also be done to characterize the antioxidant activity
of specic compounds and classes of compounds to better understand the role of brewing temperature on total
antioxidant character of the resulting coee beverages.
Materials and Methods
Materials. Pre-ground, light roast Brazilian, Colombian, Ethiopian, Mexican, and Myanmar coees were pur-
chased from commercial vendors. Coee samples from two regions of Ethiopia (labeled as Ardi and Yirgz by the
vendor) were analyzed separately.
5-Caeoylquinic acid (5-CQA, CAS: 327-97-9), 4-CQA (CAS: 905-99-7), and 3-CQA (CAS: 906-33-2) were
purchased from Sigma-Aldrich (Milwaukee, WI). HPLC grade methanol was obtained from Fisher Scientic
(Nazareth, PA). Phosphoric acid (85% wt.) was obtained from Sigma-Aldrich (Milwaukee, WI) and diluted to
2.0 mM concentration using deionized (DI) water. Standard stock solutions of 2.5 mM Trolox (6-hydroxy-2,5,7,
8-tetramethylchroman-2-carboxylic acid) were prepared in ethanol weekly. Trolox and ethanol were purchased
from Sigma-Aldrich (Milwaukee, WI). ABTS˙+ (2,2′-azionbis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-
nium salt) radical cation solutions were prepared every 48 hours and stored in the dark at room temperature. e
ABTS˙+ solution was allowed to stand for 12 hours aer mixing to achieve maximal color formation. e potas-
sium persulfate and ABTS reagents used to generate the radical solution were both obtained from Sigma-Aldrich
(Milwaukee, WI). Standardized 0.1 N NaOH from Sigma-Aldrich (Milwaukee, WI) was used to nd the total
titratable acidity of each coee. Filtered municipal tap water was used to brew the coees. Analysis of this water,
conducted by Penn State University’s Agricultural Analytical Services Laboratory, found the water to have a total
hardness of 174 mg/L and a pH of 7.5.
0
5
10
15
20
25
010002000300
04
00
0
Antioxidant Activity
Cold Hot
PC = 0.82
PC = 0.22
Figure 4. Relationship between 3-CGA concentration (mg/L brewed coee) and antioxidant activity (mmol
equivalent Trolox/L brewed coee) for hot and cold brew coees from the six regional coee samples.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

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7
Scientific RepoRts | (2018) 8:16030 | DOI:10.1038/s41598-018-34392-w
Methods. Cold brew experiments. e cold brewing process was carried out at room temperature (ranging
from 21 °C to 25 °C over the experimental period) adapted from a home-brewing recipe published on e New
York Times’s Cooking website58. A sample of 35.0 g of coee was placed in 350 mL of carbon-ltered municipal
water in a 32-ounce Mason jar tted with a screw-top lid. e coee was allowed to brew for 7 hours as suggested
by our previous study32. e coee samples were then ltered using the Hario V60 paper lter before analysis.
Four samples were taken from each batch of ltered cold brew coee, and each experiment was performed in
duplicate (n = 8).
Hot brew experiments. Hot brew extraction was conducted using the same coee-to-water ratio as was used in
the cold brew method. e water was heated to boiling, then added to coee grounds in a traditional French press
carafe. e coee samples were brewed for 6 minutes before ltering using the Hario V60 paper lter. It is noted
that the samples at the time of ltering were dierent between hot and cold brew experiments. e experiments
were designed to simulate typical brewing environments for consumption. us, the ltering process was not
temperature controlled. ree samples were taken from each batch of ltered hot brew coee, and each experi-
ment was performed in duplicate (n = 6).
Sample Storage. Both cold brew and hot brew samples were freshly prepared for each experiment. All samples
were analyzed within 10 minutes of brewing.
HPLC Analysis. Standard solutions and coee extracts were analyzed using an adapted methodology reported
in GL Sciences Technical Note No. 6759. An Agilent 1200 Series high-performance liquid chromatography system
(HPLC) was tted with a Supelco 5 µm column (15 cm × 4.6 cm) (Supelco, Bellefonte, PA) run at 40.0 °C with a
mobile phase mixture of 75% mobile phase A and 25% mobile phase B (A: 95% 2.0 mM phosphoric acid and 5%
methanol; B: 95% methanol and 5% 2.0 mM phosphoric acid). e ow rate was 1.0 mL/min with an injection
volume of 10.0 µL. CQA isomers were detected using a diode array detector at 325 nm. 5-CQA was quantied
via standard calibration curves. 4-CQA and 3-CQA standards were used to determine the retention time of each
isomer. Quantitation of the other CQA isomers was accomplished using the area of 5-CQA standard combined
with the respective molar extinction coecients of other two isomers as reported previously33,34,38.
Total acidity and pH measurements. e pH of each brewed coee sample was measured with a Mettler Toledo
FiveEasyTM F20 benchtop pH/mV meter. A 40 mL aliquot of coee brew was titrated with 0.1 N NaOH at 22 °C to
a pH of 6.0 and a pH of 8.0.
Antioxidant activity measurements. Total antioxidant activity of hot and cold brew coees was determined using
an ABTS radical cation decolorization assay modied from Re et al. and Vignoli et al.60,61. To summarize the pro-
cedure, a stock solution of ABTS˙+ was made by mixing equal parts 7.0 mM ABTS and 2.45 mM potassium per-
sulfate to form the ABTS˙+ radical cation. e mixture was allowed to stand in the dark at room temperature for
14 to 16 hours to reach optimal absorbance at 734 nm. A dilute working solution of ABTS˙+ with an absorbance
between 0.80 and 0.90 at 734 nm was made by diluting the stock solution with DI water. Trolox standards were
tested by mixing 30 µL of 2.5 mM Trolox solution with 4.0 mL of diluted ABTS˙+ solution and allowing to stand
for 6 minutes. e resulting solution was analyzed by UV-Vis spectroscopy at 734 nm using a ermo Scientic
Evolution 201 spectrophotometer, and ABTS˙+ scavenging capacity was determined by absorbance dierence
between the working standard and the Trolox - ABTS˙+ sample.
Filtered coee samples were diluted 1:2 with DI water and centrifuged at 8000 rev/min for 2 minutes to further
remove any particulates from the sample. A 5.0 µL aliquot of coee was pipetted into 4.0 mL of the dilute ABTS˙+
and allowed to stand for 6 minutes. e resulting solution was analyzed by UV-Vis following the procedure for the
Trolox standards. e total antioxidant capacity of each coee sample was calculated as mmol Trolox equivalent
per liter of brewed coee.
Statistical analysis. ANOVA (Table S1) andtwo-tailed student’s t-test(Table S2) were employed to determine
similarities in antioxidant activities, pH values, total acidities, and equilibrium concentrations of CQA with con-
sideration to the origin of the coee and brewing method. e output of the statistical analysis is included in the
supplementary information.
Data Availability
All data generated or analyzed during this study are included in this published article (and its Supplementary
Information les).
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Acknowledgements
e authors would like to thank Maria Latorre Socas, Javika Shah, Kelly Fallon, Bhumi Patel, Cailyn Chow, Nicole
Misiorski, Alyssa Olewine, Danielle Adams, Madisyn Peoples, Amritpal Jagra, Paulina Czerwinska, and Lauren
Mehrfor their contribution in data collection. is work would not have been possible without their oversight of
sample collection, HPLC analysis, pH measurement, titration, and antioxidant measurement. e authors would
like to thank the generous funding support provided by the Eileen Martinson ’86 Fund for the Undergraduate
Capstone Experience at the omas Jeerson University East Falls Campus as well as the University Faculty
Research, Scholarship, and Practice-based Grant.Publication made possible in part by support from the omas
Jeerson University + Philadelphia University Open Access Fund.
Author Contributions
N.Z.R. conceived of the total acidity study and contributed to the experimental design and execution of the study.
M.F. conceived of the antioxidant study and contributed to the experimental design and execution of the study.
N.Z.R. and M.F. contributed equally to all versions of the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-34392-w.
Competing Interests: e authors declare no competing interests.
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