Journal Pre-proofs

Bitterness Compounds in Coffee Brew Measured by Analytical Instruments

and Taste Sensing System

Hirofumi Fujimoto, Yusaku Narita, Kazuya Iwai, Taku Hanzawa, Tsukasa

Kobayashi, Misako Kakiuchi, Shingo Ariki, Xiao Wu, Kazunari Miyake,
Yusuke Tahara, Hidekazu Ikezaki, Taiji Fukunaga, Kiyoshi Toko

PII: S0308-8146(20)32090-2
DOI: https://doi.org/10.1016/j.foodchem.2020.128228
Reference: FOCH 128228

To appear in: Food Chemistry

Received Date: 20 January 2020

Revised Date: 24 September 2020
Accepted Date: 24 September 2020

Please cite this article as: Fujimoto, H., Narita, Y., Iwai, K., Hanzawa, T., Kobayashi, T., Kakiuchi, M., Ariki, S.,
Wu, X., Miyake, K., Tahara, Y., Ikezaki, H., Fukunaga, T., Toko, K., Bitterness Compounds in Coffee Brew
Measured by Analytical Instruments and Taste Sensing System, Food Chemistry (2020), doi: https://doi.org/
10.1016/j.foodchem.2020.128228

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 Bitterness Compounds in Coffee Brew Measured by Analytical Instruments

and Taste Sensing System

Hirofumi FUJIMOTOa, Yusaku NARITAa,*, Kazuya IWAIa, Taku HANZAWAa, Tsukasa

KOBAYASHIa, Misako KAKIUCHIa, Shingo ARIKIa, Xiao WUb, Kazunari MIYAKEc, Yusuke

TAHARAb, Hidekazu IKEZAKId, Taiji FUKUNAGAa, Kiyoshi TOKOb,e

aR&D Department, UCC Ueshima Coffee Co., Ltd., Hyogo, Japan

bResearch and Development Center for Five-Sense Devices, Kyushu University., Fukuoka, Japan
cGraduate School of Information Science and Electrical Engineering, Kyushu University, Fukuoka, Japan

dIntelligent Sensor Technology, Inc., Kanagawa, Japan

eInstitute for Advanced Study, Kyushu University, Fukuoka, Japan

AUTHOR E-MAIL ADDRESS:

Hirofumi Fujimoto < hirofumi-fujimoto@ucc.co.jp >

Yusaku Narita < yuusaku-narita@ucc.co.jp >

Kazuya Iwai < kazuya-iwai@ucc.co.jp >

Taku Hanzawa < taku-hanzawa@ucc.co.jp >

Tsukasa Kobayashi < tsukasa_-kobayashi@ucc.co.jp >

Misako Kakiuchi < misako-kakiuchi@ucc.co.jp >

Shingo Ariki < shingo-ariki@ucc.co.jp >

Xiao Wu < wu.xiao@nbelab.ed.kyushu-u.ac.jp >

Kazunari Miyake < kazunari.miyake.771@s.kyushu-u.ac.jp >

Yusuke Tahara < tahara.yusuke.494@m.kyushu-u.ac.jp >

1
Hidekazu Ikezaki < ikezaki.hidekazu@insent.co.jp >

Taiji Fukunaga < taiji-fukunaga@ucc.co.jp >

Kiyoshi Toko < toko@ed.kyushu-u.ac.jp >

*CORRESPONDING AUTHOR:

Tel.: +81-78-304-8805. Fax: +81-78-304-8806.

E-mail address: yuusaku-narita@ucc.co.jp.

Postal address: Innovation Center, UCC Ueshima Coffee Co., Ltd., 7-7-7 Minatojima-Nakamachi, Chuo-

ku, Kobe-Shi, Hyogo, 650-8577, Japan

2
Abstract

We investigated the bitter compounds in coffee brews using multivariate analysis of the data obtained

from analytical instrument and electronic taste sensor experiments. Coffee brews were prepared from

coffee beans roasted to four different degrees. Each brew was fractionated into four fractions by liquid–

liquid extraction. The relative amounts of 30 compounds in each fraction were analyzed by analytical

instruments, and the bitterness response value of each fraction was analyzed by a taste sensor. Candidate

bitter compounds in the coffee brews were identified with reference to their variable importance in

projection and by coefficient of projection to latent structure regression (PLS-R) analysis. PLS-R analysis

suggested that nicotinic acid, L-lactic acid, and nicotinamide contributed to the bitterness of the coffee

brews. In fact, the coffee brews with added nicotinic acid, L-lactic acid, and nicotinamide had an

increased bitterness response value compared to those without.

Keywords

Coffee, Taste sensor, Bitterness, Liquid–liquid extraction, Projection to latent structure regression

Chemical compounds

L-lactic acid (PubChem CID: 107689)

Nicotinic acid (PubChem CID: 938)

Nicotinamide (PubChem CID: 936)

3
1. Introduction

Brewed coffee is one of the most widely consumed and enjoyed bitter beverages in the world (Masi et

al., 2015), and research on coffee’s bitter compounds has been conducted for a long time. Chen (1979)

was the first to report two bitter compounds in coffee brew, caffeine and trigonelline which, respectively,

account for 10%–30% and 1% of the total bitterness. Since then, much research has been conducted on

the bitter compounds other than caffeine in the knowledge that decaffeinated coffee also has a bitter taste

and that the bitterness of coffee increases as the degree of roasting rises. Bitter tasting heterocyclic

compounds generated by the roasting process, such as furfuryl alcohol (Shibamoto et al., 1981), 5-

hydroxymethyl-2-furanaldehyde (Belitz, 1977), and 2,5-diketopiperazines (Ginz et al., 2000), attracted

attention, and more recently chlorogenic acid lactones (CGLs), i.e., intramolecular condensation

compounds of chlorogenic acids (CGAs), and 4-vinylcatechol oligomers (VCOs) have been recognized

as bitter compounds as well (Frank et al., 2006; Frank et al., 2007; Frank et al., 2008).

While many bitter components have been reported, they have not been fully documented because it is

assumed that there are over 800 compounds contained in roasted coffee, and this number includes only

the volatile compounds (Korhoňová et al., 2009)

Previously, compounds contributing to the taste of coffee have been measured using methods such as

gas chromatography–mass spectrometry (GC-MS) (Jackels et al, 2014), liquid chromatography–mass

spectrometry (LC-MS/MS) (Frank et al., 2007), and one-/two-dimensional nuclear magnetic resonance

(1D/2D NMR) spectroscopy (Wei et al., 2014). In recent years, taste analyses have increasingly

employed electronic taste sensor (Hayashi et al., 2008; Phat et al., 2016) as an alternative to standard

sensory test, as these objectively quantify the taste of foods and beverages. The taste sensor (an

“electronic tongue” with global selectivity), developed by Toko, yields results that correlate well with

human sensory evaluation of foods (Fukunaga et al., 1996; Toko, 2000). The taste sensor comprises

several different lipid/polymer membranes that can transform information about substances that humans

4
perceive as tastes into electrical signals. The sensor’s output has been shown to produce similar patterns

for groups of chemical substances with similar tastes.

Our aim in this study was to investigate bitter compounds in coffee brews using a taste sensor and

measurements from LC-MS/MS, liquid chromatography–photo diode array (LC-PDA), and liquid

chromatography–ultraviolet detector (LC-UV). First, coffee brews made from beans roasted to various

degrees were fractionated into four fractions according to polarity via liquid–liquid extraction using

organic solvents; since the membranes of taste sensors are composed of lipids, it was assumed that

responses would vary with polarity. Second, the bitterness response values of each fraction were

analyzed using a taste sensor, and the relative abundance of the compounds contained in each fraction

were quantified using LC-MS/MS, LC-PDA, and LC-UV. Then, we identified the bitter compounds

using projection to latent structure regression (PLS-R) analysis of the results. Finally, a Brazilian

arabica coffee brewed from beans roasted to achieve an L value (in the Hunter Lab color space) of 20

and with added L-lactic acid, nicotinic acid, and nicotinamide was analyzed to confirm whether or not

the previously identified compounds were recognized as bitterness compounds by the taste sensor.

2. Materials and methods

2.1. Materials and reagents

Green coffee beans (Coffea arabica from Brazil, natural process) were obtained from UCC Ueshima

Coffee Co., Ltd. (Kobe, Japan). The following chemicals were obtained commercially from Nacalai

Tesque, Inc. (Kyoto, Japan): 1-butanol, acetic acid, nicotinic acid, nicotinamide, acetic acid, citric acid,

L-malic acid, glycolic acid, L-lactic acid, quinic acid, bromothymol blue (BTB), disodium hydrogen

phosphate, and sodium hydroxide. We obtained formic acid and ultrapure water (LC/MS grade) from

FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and acetonitrile and perchloric acid from

Kanto Chemical Co., Inc. (Tokyo, Japan). The reference solution containing 30 mmol/L KCl and 0.3

5
mmol/L tartaric acid, and alcohol solution for rinsing the electrode of the taste sensor were from

Intelligent Sensor Technology Co., Ltd. (Kanagawa, Japan), and Strata C18-E cartridge (500 mg, 6

mL) was purchased from Phenomenex, Inc. (Torrance, CA, U.S.A.)

2.2. Roasting of coffee beans

Four batches of 500 g of green coffee beans were separately roasted in a ZR1 Plus coffee roaster

(Scolari Engineering, Milano, Italy) to achieve four final roast degrees (L values of 30, 25, 20, and 15;

the lower the L value, the darker the color from the roasting). The roasting air temperature was 350 °C

and the roasting times to achieve L values of 30, 25, 20, and 15 were 9 min 43 s, 10 min 56 s, 11 min

37 s, and 13 min 2 s, respectively, and the temperature reached before cooling to achieve these values

were 185 °C, 196 °C, 204 °C, and 220 °C, respectively. The L value of the roasted coffee beans was

analyzed using a ZE 6000 color meter (Nippon Denshoku Industries Co., Ltd., Tokyo, Japan). The

roasted coffee beans were then packed under vacuum conditions in aluminum packages and stored at

4 °C in the dark until testing.

2.3. Preparation of coffee brews

The four batches of roasted coffee beans were ground into medium fine grounds using a BM-570N

coffee cutter (Lucky Coffee Machine Co., Ltd., Hyogo, Japan). Then, 100 g of the ground coffee was

extracted with a paper filter and 1.5 L of hot water at 90 °C using a coffee brewer (BM-1200, Lucky

Coffee Machine Co., Ltd.). Each collected coffee brew was kept at 4 °C until fractionation.

2.4. Fractionation of coffee brews by liquid–liquid extraction

Each coffee brew was fractionated into four fractions by liquid–liquid extraction according to

methods previously described (Mumin et al., 2006; Bianco et al., 2013) with some modifications (Fig.

6
1). First, 500 mL of the coffee brew was added to 500 mL ethyl acetate and the mixture was stirred

with a magnetic stirrer at a constant rate of 600 rpm at room temperature for 1 h. The mixture separated

into a hydrophobic (upper/ethyl acetate) layer and a hydrophilic (lower/aqueous) layer, and each layer

was collected. The collected aqueous layer was fractionated twice more with ethyl acetate in the same

way. The collected ethyl acetate and aqueous layers were dried with an evaporator (EYELA N-1110V,

Tokyo Rikakikai Co., Ltd, Tokyo, Japan) and named fraction_A and fraction_B, respectively.

Fraction_A was added to a mixture consisting of 100 mL ultrapure water and 100 mL chloroform

which was stirred with a magnetic stirrer at a constant rate of 800 rpm at room temperature for 1 h. The

mixture separated into two layers, a hydrophilic (aqueous) layer and a hydrophobic (chloroform) layer,

and each layer was collected. Fractionation of the aqueous layer was repeated twice more by the

addition of 100 mL chloroform each time. The chloroform layer and aqueous layer were named

fraction_1 and fraction_2, respectively (Fig. 1). Fraction_B was added to 1,000 mL of a mixture of 498

mL ultrapure water, 2 mL formic acid, and 500 mL 1-butanol and stirred with a magnetic stirrer at a

constant rate of 500 rpm at room temperature for 1 h. The mixture separated into two layers, a

hydrophilic (aqueous) layer and a hydrophobic (1-butanol) layer, and each layer was collected.

Fractionation of the aqueous layer was repeated twice more by the addition of 500 mL 1-butanol each

time. The 1-butanol layer and the aqueous layer were named fraction_3 and fraction_4, respectively

(Fig. 1). Thereafter, 500 mL of each coffee brew and fraction_1 to fraction_4 were lyophilized using a

freeze dryer (Okawara MFG. Co., Ltd., Shizuoka, Japan) and their dry weights measured before

dissolving each in 50 mL of 20% ethanol to prepare sample solutions which were stored at –20 °C until

analysis.

(Fig. 1)

7
2.5. Taste sensor analysis of the coffee brew fractions

Taste sensor analysis was performed according to methods previously reported (Ito et al., 2011; Wu

et al., 2016) with some modifications. Approximately 10 mL ultrapure water was added to 8 mL of the

sample solutions and the mixtures lyophilized using a freeze dryer. Then, 80 mL of ultrapure water

was added to the lyophilized samples and mixed. The mixture was filtered through Kiriyama No. 3

filter paper (Kiriyama Glass Co., Tokyo, Japan), and the filtrate was used for the taste sensor analysis

(SA402B, Intelligent Sensor Technology Co., Ltd.).

In this study, the detection sensor, called C00, was part of the taste sensor consisted of electrode

composed of lipid/polymer membranes and attached to a mechanically controlled robot arm. The

analysis procedure using the electrode of taste sensor is shown in Fig. S1. The device performed the

following operations: (1) measurement of the reference solution (corresponding to saliva) to obtain

reference electric potential (Vr); (2) measurement of the sample solution to obtain electric potential of

sample (Vs); (3) measurement of the reference solution after light rinsing in the reference solution to

obtain electric potential (Vr′); and (4) rinsing of the detection sensor with the alcohol solution for

cleaning. The values (Vs − Vr) and (Vr′ − Vr) were calculated, giving the relative membrane potential

and the change in membrane potential caused by adsorption (CPA), respectively. Each sample

solution was measured four times, the first measurement result being excluded from the analysis. The

bitterness response values of the four different coffee brews and fraction_1 to fraction_4 were

calculated using the CPA value of C00 sensor and the following formula:

Bitterness response value = –0.210 × CPA (C00) value

2.6. LC-MS/MS analysis of six VCOs

The LC-MS/MS analysis of six VCOs (Fig. S2) was performed as previously reported (Frank et al.,

8
2007) with some modifications. A solid phase STRATA C18-E extraction column was used to remove

impurities in the sample solutions before the VCOs were analyzed via LC-MS/MS. Then, 500 mg of

the STRATA C18-E column was preconditioned with 4 mL of ethanol and 4 mL of water containing

0.1% formic acid and 20% ethanol before the experiment. A mixture of 0.5 mL of the sample solution

and 4.5 mL of 20% ethanol was applied to the STRATA C18-E column. The solution passed through

the column via the addition of 3 mL of a wash solution containing 0.1% formic acid and 20% ethanol,

which was discarded. Subsequently, approximately 5 mL of 0.1% formic acid in acetonitrile was added

to the column and the eluate collected and solidified using a centrifugal concentrator (CVE-3100,

Tokyo Rikakikai Co., Ltd, Tokyo, Japan), and then dissolved in 500 µL of 50% ethanol for LC-MS/MS

analysis of the VCOs.

The Nexcera X2 apparatus (Shimadzu Corporation, Kyoto, Japan), which consisted of a pump,

column oven, auto sampler, and degasser, was connected to an LCMS-8060 (Shimadzu Corporation)

with electrospray ionization (ESI). Separations were achieved on a CAPCELL PAK C18 MGⅢ (100

mm × 2.0 mm i.d., 3 μm, Shiseido Co., Ltd., Tokyo, Japan) with a flow rate of 0.2 mL/min at a column

oven temperature of 40 °C. The mobile phase was composed of eluent A (water containing 0.1%

formic acid) and eluent B (acetonitrile containing 0.1% formic acid), and the gradient program was set

as follows: 0–5.0 min, 25%–28% (v/v) B; 5.0–20.0 min, 28%–30% (v/v) B; 20.0–30.0 min, 90% (v/v)

B; 30.0–40.0 min, 25% (v/v) B. The injection volume was 1 µL. Tentative identification of VCO peaks

in the samples was carried out by comparison with multiple reaction monitoring (MRM) transition data

previously reported (Frank et al., 2007). Supplementary table S3 represents the MRM transition for

analyzing the VCOs. In this analysis, not all of the isomers could be identified, so they numbered in

order of elution from the LC column: VCO_1, VCO_2, VCO_3, VCO_4, VCO_5, and VCO_6. The

negative ion spray voltage was −3 kV. The nebulizer gas was nitrogen flowing at 3 L/min. The heater

gas flow was 10 L/min, and the temperature was 400 °C; the collision gas was argon at a pressure of

9
270 kPa. Mass calibration was performed using a standard sample containing PEG, and the accuracy of

MS was 100 ppm or less. The relative amount of VCOs contained in each coffee brew and fraction

were normalized with the peak area of the VCOs contained in the coffee brew with an L value of 15.

2.7. LC-MS/MS analysis of nine kinds of CGAs and seven kinds of CGLs

The LC-MS/MS analysis of nine CGAs (three caffeoylquinic acids (CQAs), three feruloylquinic

acids (FQAs), and three dicaffeoylquinic acids (diCQAs)), and seven CGLs (three CQA lactones

(CQLs), three FQA lactones (FQLs), and one diCQA lactone (diCQL)) was performed as previously

reported (Kucera et al., 2016) with some modifications.

The sample solutions were diluted 100 times with 20% ethanol before being analyzed via LC-

MS/MS. The Nexcera X2 apparatus, consisting of a pump, column oven, auto sampler, and degasser,

was connected to a LCMS-8060 with ESI. Separations were achieved on an Ascentis Express C18 (100

mm × 2.1 mm i.d., 2.7 μm, Sigma-Aldrich Japan, Tokyo, Japan) with a flow rate of 0.35 mL/min and a

column oven temperature of 25 °C. The mobile phase was composed of eluent A (water containing

0.1% formic acid) and eluent B (methanol containing 0.1% formic acid), and the gradient program was

as follows: 0–5.0 min, 0% (v/v) B; 5.0–25.0 min, 0%–40% (v/v) B; 25.0–26.0 min, 40%–90% (v/v) B;

26.0–31.0 min, 90% (v/v) B; 31.0–32.0 min, 90%–0% (v/v) B; 32.0–40.0 min, 0% (v/v) B. The

injection volume was 1 µL. Identification of the peaks for the CGAs (3-CQA, 4-CQA, 5-CQA, 3-FQA,

4-FQA, 5-FQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) in the samples was achieved by comparisons

with MRM transition data previously reported (Kucera et al., 2016) and the retention times of nine

standard materials. The 3-CQA, 4-CQA, 5-CQA, 3-FQA, 4-FQA, and 5-FQA were from Nagara

Science Co., Ltd. (Gifu, Japan); the 3,4-diCQA and 3,5-diCQA were from Cayman Chemical Company

(Michigan, U.S.A.), and the 4,5-diCQA was from ChemScene LLC (New Jersey, U.S.A.). Tentative

identification of CGL peaks in the samples was carried out via comparison of MRM transition data

10
previously reported (Kucera et al., 2016). Supplementary table S4 represents the MRM transition for

analyzing the CGLs. In this analysis, not all of the isomers could be identified, so they were numbered

as follows in order of elution from the LC column: CQL_1, CQL_2, CQL_3, FQL_1, FQL_2, FQL_3,

and diCQL. The negative ion spray voltage was −3 kV. The nebulizer gas was nitrogen flowing at 3

L/min. The heater gas flow was set at 10 L/min, and the temperature was 400 °C. The collision gas was

argon at a pressure of 270 kPa. The mass calibration of MS was performed using a standard sample

containing PEG, and the accuracy of MS was 100 ppm or less. The relative amounts of CGAs and

CGLs contained in each of the coffee brews and fractions were normalized with the peak area of the

CGAs and CGLs contained in the coffee brew with an L value of 15.

2.8. LC-PDA analysis of the three alkaloids

The analysis of the three alkaloids (nicotinic acid, nicotinamide, and trigonelline) contained in each

coffee brew and fraction was carried out via LC-PDA. First, 1 mL ultrapure water was added to 1 mL

of the above-mentioned sample solutions, and the mixtures were lyophilized using a freeze dryer. Then,

10 mL ultrapure water was added to the lyophilized samples for analysis by LC-PDA. The LC-PDA

apparatus (GL Science Inc., Tokyo, Japan) consisted of a pump, auto sampler, column oven, and PDA

detector. Separations were performed on an Inertsil ODS-3 (150 mm × 4.6 mm i.d., 5 μm, GL Science

Inc.) with a flow rate of 1.0 mL/min and a column oven temperature of 35 °C. The mobile phase was

composed of eluent A (0.05 mol/L acetic acid) and eluent B (acetonitrile containing 0.05 mol/L acetic

acid), and the gradient program was as follows: 0–30.0 min, 5%–20% (v/v) B; 30.0–45.0 min, 20%–

35% (v/v) B; 45.0–50.0 min, 35%–80% (v/v) B; 50.0–50.1 min, 80%–5% (v/v) B; 50.1–60 min, 5%

(v/v) B. Nicotinic acid and nicotinamide were detected at 265 nm, and trigonelline was detected at 270

nm. The injection volume was 10 μL. Identification of the compounds was confirmed by analyzing the

standard solutions. The relative amounts of the alkaloids contained in each of the coffee brews and

11
fractions were normalized with the peak area of the alkaloids contained in the coffee brew with an L

value of 15.

2.9. LC-UV analysis of five organic acids

The analysis of five organic acids was performed according to methods previously reported (Narita

et al., 2013). The citric acid, L-malic acid, quinic acid, glycolic acid, and L-lactic acid contained in

each of the coffee brews and fractions were analyzed by LC-UV. First, 1 mL ultrapure water was added

to 1 mL of the above-mentioned sample solutions, and the mixtures were lyophilized. Then, 10 mL

ultrapure water was added to the lyophilized samples for analysis via LC-UV. The LC-UV apparatus

(GL Science Inc.) consisted of a pump, auto sampler, column oven, and UV detector. Separation was

achieved by connecting four ion exchange columns (RSpak KC-811, 300 mm × 8.0 mm i.d., Showa

Denko Co., Tokyo, Japan) with a flow rate of 1.0 mL/min. The eluent was water containing 3 mmol/L

perchloric acid. The effluent stream from the column was mixed with a stain solution (water containing

0.2 mmol/L BTB, 15 mmol/L disodium hydrogen phosphate, and 2 mmol/L sodium hydroxide). The

detection wavelength was 445 nm, and the injection volume was 20 μL. Identification of the

compounds was confirmed by analyzing the standard solutions. The relative amounts of the organic

acids contained in each of the coffee brews and fractions were normalized with the peak area of the

organic acids contained in the coffee brew with an L value 15.

2.10. Multivariate analysis

The results obtained by the taste sensor, LC-MS/MS, LC-PDA, and LC-UV were analyzed using

PLS-R, a multivariate analysis, to investigate the bitter substances to which the taste sensor responded.

Statistics in Microsoft Excel software (RIKEN, Saitama, Japan) was used to perform the PLS-R using

data obtained from the taste sensor as response variables and data obtained from the LC-MS/MS, LC-

12
PDA, and LC-UV as explanatory variables. An auto scale was used as the scaling type. The number of

factors used for PLS analysis was 7.

2.11. Addition of L-lactic acid, nicotinic acid, or nicotinamide to the coffee brews

Coffee with or without added L-lactic acid, nicotinic acid, or nicotinamide was analyzed with a taste

sensor to measure the bitterness response values. The tests used roasted coffee with an L value of 20

extracted as described in Section 2.3. L-lactic acid, nicotinic acid, or nicotinamide was added to the

coffee brews so that the final concentrations were 1160 mg/L, 190 mg/L, and 46 mg/L, respectively,

corresponding to about 10 times the concentrations contained in the non-supplemented coffee brews.

Four coffee brews (control, with L-lactic acid, nicotinic acid, or nicotinamide) were analyzed using the

taste sensor, and their bitterness response values calculated, as described in Section 2.5.

3. Results and Discussion

3.1. Isolation of the coffee brews

Each coffee brew was fractionated into four fractions via liquid–liquid extraction with ethyl acetate,

chloroform, and 1-butanol, then each coffee brew and fraction was lyophilized to measure its dry

weight (Table 1). The total dry weight of the fractions was approximately 10% higher than the dry

weight of the coffee brew using coffee with an L value of 15. This tendency was also observed for the

other coffee brews with L values of 20, 25, and 30 and their fractions. The ratio of the total dry weight

of all fractions to the dry weight of the four types of coffee brew was 112% ± 3%. Fraction_4 had the

largest dry weight among the fractions for coffee brews at all four L values. The dry weights of the

fractions from each coffee brew with L values of 15, 20, 25, and 30 were in the order of fraction_4 >

fraction_3 > fraction_1 > fraction_2. Fraction_4 was the most similar in color to the dark brown color

of the coffee brews themselves with L values of 15, 20, 25, and 30. The main component constituting

13
the dark brown color of brewed coffee is coffee melanoidins, which are dark brown polymers produced

during the roasting process (Nunes et al., 2001). Bekedam et al. (2006) suggested that phenolic

compounds, e.g., CGAs, are present as part of the coffee melanoidins. These coffee melanoidins

include metabolites such as sugars, amino acids, and CGAs, and are highly hydrophilic, potentially

explaining their appearance in fraction_4 of each coffee brew (L values of 15, 20, 25, and 30). It has

been reported that the weight of melanoidins accounts for 25% of the dry matter of brewed coffee

(Borrelli et al., 2002). However, in this study, the dry weight of fraction_4, which was considered rich

in coffee melanoidins, was over 60% of the total dry weight. Thus, fraction_4 might have contained

hydrophilic compounds other than coffee melanoidins.

(Table 1)

3.2. Relative quantitation of the compounds contained in each coffee brew and fraction

Table 2 shows the relative amounts of the compounds in each coffee brew and fraction after

normalization with the peak area of the compounds contained in the coffee brews with an L value of

15. The trigonelline and nicotinic acid in the coffee brews were transferred to in fraction_3 and

fraction_4; in both these fractions the relative amount of trigonelline decreased, and that of nicotinic

increased, as the L value decreased. Casal et al. (2000) reported that the loss of trigonelline is strongly

dependent upon the degree of roasting and is associated with the formation of nicotinic acid, findings

that are consistent with the results of this study. Most of the citric acid, L-malic acid, quinic acid,

glycolic acid, and L-lactic acid in the coffee brews were also detected in fraction_3 and fraction_4. The

relative amounts of citric acid and L-malic acid in the coffee brews, fraction_3, and fraction_4

decreased as the L value decreased. Meanwhile, the amount of L-lactic acid in them increased as the L

value decreased.

14
 As the L value decreased, the relative amount of all CQAs, FQAs, and diCQAs in the coffee brews

decreased. Most of the CQAs and FQAs were also transferred to fraction_3, while most of the diCQAs

were transferred to fraction_2 and fraction_3. The reason for this was assumed to be because diCQAs

are compounds that are more hydrophobic than CQAs and FQAs.

Most of the CQLs and diCQLs in each coffee brew were transferred to fraction_2, while the FQLs in

each coffee brew were divided between fraction_1 and fraction_2. In addition, the CQLs and FQLs

hardly transferred to fraction_4. The VCOs in each coffee brew were transferred to fraction_1 and

fraction_2 but not to fraction_3 and fraction_4.

(Table 2)

3.3 Bitterness response value of each coffee brew and fraction

Table 3 shows the bitterness response values of each coffee brew and fraction measured using the

taste sensor. The lower the L value, the higher the bitterness response value of the coffee brews and

fractions. The bitterness response value of fraction_3 was the highest (except for L value of 25)

followed by fraction_1. Fraction _3 obtained from the coffee brew with an L value of 15 had the

highest bitterness response value among all 12 fractions. It contained large amounts of nicotinic acid,

nicotinamide, L-lactic acid, and glycolic acid compared to the other 11 fractions (Table 2). The reason

that the bitterness response value became negative in some fractions is because the reference solution

of the taste sensor contained KCl. KCl has been reported to have a bitter taste (Bartoshuk, et al., 1988).

When the bitterness response value of the sample is lower than the bitterness response value of the

reference solution of the taste sensor, the bitterness response value of the sample is considered to be

negative.

15
 (Table 3)

3.4. Investigation of bitter compounds in the coffee brews by PLS-R analysis

PLS-R analysis was performed using data obtained from the taste sensor, LC-MS/MS, LC-PDA, and

LC-UV, and the candidate bitterness compounds responding to the taste sensor were investigated with

reference to the variable importance in projection (VIP) value and coefficient of the PLS-R analysis.

Conventionally, variables that are highly relevant for explaining the bitterness of coffee brews can be

extracted from VIP values. The average of the 95% confidence interval of VIP is equal to 1.0.

Therefore, compounds with VIP values larger than 1 are often considered relevant for explaining a

PLS-R analysis. The coefficient describes how a model fits a set of predicted data related to class

separation. In this study, compounds with positive coefficients meant correlation to the bitterness

response value. Compounds with a VIP value of 1 or more and a positive coefficient were judged to

contribute positively to the bitterness response value of the taste sensor. Table 4 and Fig. S5 show the

components with a VIP value exceeding 1 and a positive coefficient, and score plot and loading plot,

respectively.

FQL_1, VCO_4, and VCO_6 were known bitter compounds of the six kinds of candidate ingredients

extracted by the PLS-R analysis. Their coefficients were 0.23, 0.14, and 0.33, respectively. The

coefficient of nicotinamide (0.52) was largest, followed by that of nicotinic acid (0.44) and L-lactic

acid (0.34). The generic term for nicotinamide and nicotinic acid is niacin, also called vitamin B3

(Arnum, 2000). Niacin deficiency in the human body causes a disease called pellagra, which can result

in death from multiple organ failure if not treated (Hegyi et al., 2004). Niacin is useful as a treatment

for pellagra (Raghuramulu et al., 1965). Nicotinic acid has a sour taste, and nicotinamide is generally

known to be bitter. However, there has been little research examining how niacin and nicotinamide

contribute to taste in food. Recently, it was reported that nicotinic acid in water and in the presence of

16
sodium gluconate initially tastes sweet; however, it probably tastes sweet-bitter or bitter at a higher

concentration of sodium gluconate (Mishra et al., 2017). Settle et al. (1986) reported that bitterness was

found to be the largest non-sour sensation produced in the sensory evaluation of lactic acid dissolved in

deionized water by 13 subjects who could recognize the normal taste recognition threshold. In addition,

this tendency for bitterness to be the taste felt most strongly besides sourness has also been reported for

other acids such as citric acid, hydrochloric acid, sulfuric acid, malic acid, phosphoric acid, and tartaric

acid. Lactic acid contributes weakly to the strength and persistence of bitterness in white wine

(Sokolowsky et al. 2012).

(Table 4)

3.5. Bitterness response value of coffee brews with or without L-lactic acid, nicotinic acid, or

nicotinamide

L-lactic acid, nicotinic acid, and nicotinamide and were added to coffee brews with an L value of 20

and analyzed using a taste sensor to confirm that these compounds were recognized as bitterness

compounds by the sensor. A coffee brew with an L value of 20 was used as the control; we added 10

times the amount of nicotinic acid, nicotinamide, and L-lactic acid contained in the control (1,900

mg/L, 460 mg/L and 1,160 mg/L, respectively) to the other coffee brews and analyzed them using the

taste sensor (Table 5). The bitterness response value of the coffee brew used in this verification was

2.61 (Table 5). This value was higher than the value (2.13) of the prepared sample obtained by re-

dissolving the freeze-dried powder of the coffee brew in water for the above fractionation test.

Although the reasons this difference occurred are not clear, it is conceivable that a plurality of factors

such as the following were involved. Some of the aroma components with volatility contained in

brewed coffee, such as furfuryl alcohol, 5-hydroxymethyl-2-furanaldehyde, and 2,5-diketopiperazines,

17
contribute to its bitterness (Shibamoto et al., 1981; Belitz, 1977; Ginz et al., 2000). Therefore, it was

considered that one of the factors that caused the low bitterness response value of the latter was that the

volatile components contributing to the bitterness were reduced by the freeze-drying process. Another

factors may have been the differences in storage period of the roasted coffee beans until the start of the

test. The coffee beans were all prepared under vacuum and stored in aluminum packaging at 4 °C, but

those used in the fractionation test were stored for about 1 month while the those used in the addition

test were stored for about 6 months. Packaged arabica roasted coffee beans vacuum stored for 18

months at ambient temperature have been reported to have an increased bitterness of about 70%

(Kreuml et al., 2013).

The bitterness response value of the coffee brew with L-lactic acid (2.98) was greater than that of the

control coffee brew (2.61). The bitterness response values of coffee brews with nicotinic acid (2.69)

and nicotinamide (2.62) were also slightly higher than that of the control coffee brew. These results

imply that L-lactic acid, nicotinic acid, and nicotinamide contribute to the bitterness of brewed coffee.

In this study, the levels of added L-lactic acid, nicotinic acid, and nicotinamide were 10 times greater

than those in the coffee brew with an L value of 20. Since the amounts of each compound added to the

coffee brews was different, it is considered that the coefficients of L-lactic acid, nicotinic acid, and

nicotinamide obtained by PLS regression analysis were different in behavior from the bitterness

response values of the taste sensor.

(Table 5)

4. Conclusion

To investigate the bitterness compounds in coffee brews to which the taste sensor responded, we

performed relative quantification of the compounds in each coffee brew and fraction and performed a

multivariate analysis. We succeeded in investigating the candidate compounds that positively

18
contributed to the bitterness response value of the taste sensor. Results of the addition experiment

clarified that a compound that positively contributed to bitterness could be analyzed with the taste

sensor and actually contributed positively. The findings and techniques of this study can be applied not

only to coffee brews but also in the development of other foods. When evaluating the bitterness of

coffee in a sensory test, it is actually an evaluation performed in a state wherein a plurality of tastes

such as acidity, sweetness, saltiness, and umami are experienced simultaneously. Therefore, when

evaluating the bitterness of coffee brews with lactic acid or nicotinic acid in a sensory test, it is almost

impossible to accurately evaluate the bitterness because the acidity of the lactic or nicotinic acid is too

strong. However, the taste sensor has a sensor corresponding to each taste, and the bitterness sensor is

used to independently evaluate only bitterness. Thus, it was possible for us to independently evaluate

the bitterness of coffee brews even in the presence of lactic acid, nicotinic acid, and nicotinamide. The

taste and aroma of coffee involve complex mixtures of multiple compounds. While the sensory test is

considered the most important when evaluating the taste of coffee, evaluation using a taste sensor may

be superior when studying certain tastes.

Previous reports (Jumhawan et al, 2013; Putri et al, 2019) have shown that multivariate analysis of

compounds in coffee brews is a useful tool for determining the origin of green coffee beans and for

identifying Asian palm civet (Kopi Luwak) coffee. It has also been reported (Wei et al., 2014) that

combining quantitative values of compounds in coffee brews with bitterness response values from

sensory tests reveals candidate bitter and sweet compounds in coffee. In this study, we have shown that

a combination of quantitative values of compounds in coffee brews and bitterness response values from

a taste sensor analyzed by multivariate analysis is useful for identifying bitter compounds in coffee

brews. This technique may also be useful in determining other tastes in coffee, such as sourness and

sweetness.

However, the PLS-R analysis performed in this study did not list CGLs and VCOs as candidate bitter

19
compounds despite them generally being known as such. The prediction accuracy of the PLS-R

analysis is likely to be improved by taking into account the coffee plant species (Coffea arabica or

Coffea canephora), geographic origin of the coffee beans, and the degree of roasting, and by analyzing

a larger number of coffee samples.

Acknowledgments

We express our deepest appreciation to Noriko Ishibashi for supporting our experiments. The authors

would like to thank Enago (www.enago.jp) for the English language review.

Conflict of interest

The authors have no conflict of interest to declare.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or

not-for-profit sectors.

20
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Figure Caption

Figure 1. Schematic showing procedure for fractionation of coffee brew by liquid–liquid

extraction

26
Tables

Table 1. Dry weights (g/500 mL) of coffee brew and each fraction

L value
15 20 25 30
Coffee brew 5.7 ± 0.7 5.3 ± 0.7 5.0 ± 0.4 6.0 ± 0.9
Fraction_1 0.4 ± 0.1 0.4 ± 0.2 0.3 ± 0.0 0.4 ± 0.2
Fraction_2 0.3 ± 0.0 0.3 ± 0.1 0.2 ± 0.1 0.3 ± 0.1
Fraction_3 1.8 ± 0.1 1.8 ± 0.3 2.0 ± 0.6 2.3 ± 0.5
Fraction_4 3.8 ± 0.6 3.3 ± 0.5 3.3 ± 0.2 3.7 ± 0.8

Values are mean ± SD of triplicate analyses. The L value indicates the degree of roasting degree; the

lower the value, the higher the degree of roasting/the darker the color.

27
Table 2. Relative amounts of the compounds contained in the coffee brew and each fraction
Coffee brew Fraction_1 Fraction_2 Fraction_3 Fraction_4
L 15 L 20 L 25 L 30 L 15 L 20 L 25 L 30 L 15 L 20 L 25 L 30 L 15 L 20 L 25 L 30 L 15 L 20 L 25 L 30
Trigonelline 100.0 ± 9.6 347.4 ± 42.8 435.6 ± 48.4 557.9 ± 48.0 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 10.0 ± 0.9 39.3 ± 3.5 47.1 ± 4.2 56.3 ± 2.3 127.4 ± 11.3 432.8 ± 37.5 544.4 ± 52.9 643.8 ± 57.3
Nicotinic acid 100.0 ± 4.5 66.7 ± 6.9 46.1 ± 1.8 45.6 ± 3.8 N.D. N.D. N.D. N.D. 4.1 ± 0.5 2.2 ± 0.2 1.4 ± 0.1 1.4 ± 0.1 51.2 ± 2.6 24.6 ± 1.8 13.5 ± 0.8 10.4 ± 0.9 16.7 ± 1.0 11.9 ± 0.7 10.8 ± 1.1 11.2 ± 0.9
Nicotinamide 100.0 ± 8.8 200.9 ± 27.5 224.3 ± 17.7 273.0 ± 32.4 N.D. N.D. N.D. N.D. 9.1 ± 1.1 5.2 ± 0.3 N.D. N.D. 126.5 ± 10.6 132.6 ± 15.3 110.0 ± 9.3 100.4 ± 12.5 96.5 ± 4.6 110.9 ± 10.7 88.3 ± 3.5 73.0 ± 6.0
Citric acid 100.0 ± 12.8 159.2 ± 22.2 181.3 ± 13.0 237.9 ± 19.9 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 31.8 ± 2.6 53.8 ± 2.9 62.3 ± 7.1 76.6 ± 4.0 65.3 ± 6.7 105.0 ± 12.1 122.1 ± 9.4 160.4 ± 14.0
L-Malic acid 100.0 ± 14.5 233.0 ± 32.7 296.7 ± 27.6 368.3 ± 36.0 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 45.2 ± 4.1 95.3 ± 7.3 147.1 ± 10.4 151.1 ± 9.3 51.4 ± 5.6 120.9 ± 10.0 152.0 ± 13.8 184.4 ± 15.3
Quinic acid 100.0 ± 10.5 69.0 ± 7.3 62.2 ± 6.0 67.0 ± 5.7 N.D. N.D. N.D. N.D. 2.3 ± 0.3 0.8 ± 0.1 0.5 ± 0.0 0.6 ± 0.0 30.3 ± 3.2 16.9 ± 1.5 16.8 ± 1.7 14.4 ± 1.5 73.0 ± 8.1 52.8 ± 5.4 45.5 ± 3.9 46.9 ± 3.5
Glycolic acid 100.0 ± 9.7 78.2 ± 8.3 59.5 ± 5.2 36.8 ± 4.0 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 32.2 ± 3.6 24.0 ± 1.9 29.2 ± 2.5 23.3 ± 2.2 30.4 ± 2.2 28.7 ± 2.4 23.2 ± 1.9 19.9 ± 1.3
L-Lactic acid 100.0 ± 8.3 45.3 ± 3.5 26.2 ± 2.3 15.6 ± 1.6 N.D. N.D. N.D. N.D. 0.6 ± 0.1 0.6 ± 0.1 2.0 ± 0.1 3.2 ± 0.2 66.9 ± 4.9 19.7 ± 0.9 17.6 ± 1.0 13.1 ± 1.0 29.3 ± 0.9 6.7 ± 0.4 5.2 ± 0.4 4.5 ± 0.4
3-CQA 100.0 ± 10.0 434.5 ± 52.4 704.3 ± 80.2 1046.6 ± 114.2 0.4 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 7.3 ± 0.7 15.9 ± 1.3 8.9 ± 0.7 12.5 ± 0.9 101.1 ± 9.3 392.9 ± 42.3 640.2 ± 56.1 965.6 ± 83.3 6.5 ± 0.7 30.4 ± 2.8 47.0 ± 4.4 76.5 ± 5.1
5-CQA 100.0 ± 11.2 556.5 ± 43.7 987.9 ± 88.6 1552.5 ± 184.6 0.6 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 1.1 ± 0.2 6.6 ± 0.5 20.5 ± 1.1 35.8 ± 2.4 118.8 ± 22.5 536.1 ± 50.0 934.1 ± 82.3 1463.8 ± 103.7 1.2 ± 0.1 4.2 ± 0.4 6.4 ± 0.5 10.5 ± 1.4
4-CQA 100.0 ± 8.3 481.1 ± 39.7 772.3 ± 68.4 1163.3 ± 93.7 0.5 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 0.1 ± 0.0 5.9 ± 0.4 14.4 ± 1.2 12.8 ± 0.8 19.6 ± 2.2 112.6 ± 11.1 446.9 ± 38.8 730.5 ± 79.3 1075.1 ± 100.5 2.2 ± 0.2 9.2 ± 0.9 12.9 ± 1.1 21.5 ± 2.4
3-FQA 100.0 ± 4.2 254.1 ± 26.5 340.0 ± 34.0 434.2 ± 32.8 0.5 ± 0.0 0.9 ±0.1 0.7 ± 0.1 0.7 ± 0.0 3.9 ± 0.4 6.3 ± 0.4 3.7 ± 0.3 5.4 ± 0.4 82.1 ± 8.9 225.9 ± 20.4 323.2 ± 21.6 439.7 ± 48.6 2.7 ± 0.2 7.8 ±0.7 9.8 ±0.9 14.4 ± 1.2
5-FQA 100.0 ± 9.0 296.1 ± 24.0 430.0 ± 46.7 592.5 ± 53.1 0.5 ± 0.0 0.3 ± 0.0 1.2 ±0.2 1.3 ±0.2 2.0 ± 0.1 9.5 ±0.8 25.0 ± 2.7 43.0 ± 3.5 105.5 ± 5.9 310.2 ± 23.1 453.9 ± 29.4 608.5 ± 59.3 0.9 ± 0.1 1.7 ± 0.1 2.1 ± 0.1 3.2 ±0.2
4-FQA 100.0 ± 6.8 264.6 ± 19.5 359.6 ± 40.2 481.4 ± 52.4 0.5 ± 0.0 0.7 ± 0.0 0.6 ±0.0 0.6 ± 0.0 4.6 ± 0.4 9.5 ± 0.7 10.3 ± 1.5 16.2 ± 0.9 82.8 ± 7.9 232.9 ± 20.3 333.6 ± 31.0 456.5 ± 40.2 1.0 ± 0.1 2.3 ± 0.2 2.9 ± 0.1 4.3 ± 0.4
3,5-diCQA 100.0 ± 3.2 973.2 ± 58.7 2476.0 ± 308.1 4735.7 ± 548.3 3.2 ± 0.5 1.2 ± 0.2 8.9 ± 1.9 6.7 ± 1.6 30.4 ± 3.0 467.1 ± 38.1 1330.4 ± 174.2 3273.6 ± 225.6 43.9 ± 3.1 285.2 ± 19.8 371.5 ± 28.9 743.7 ± 64.8 1.2 ±0.1 1.0 ± 0.1 1.2 ± 0.1 1.9 ± 0.2
3,4-diCQA 100.0 ± 4.8 896.3 ± 104.6 1983.6 ± 218.7 3452.2 ± 311.2 2.4 ± 0.5 0.8 ±0.2 2.0 ± 0.3 1.5 ± 0.1 13.5 ± 1.1 165.2 ± 14.8 489.8 ± 49.4 1154.3 ± 122.7 63.0 ± 3.9 499.4 ± 49.1 1039.9 ± 99.3 2055.0 ± 194.0 2.3 ± 0.2 1.4 ± 0.1 1.5 ± 0.1 1.8 ± 0.1
4,5-diCQA 100.0 ± 4.2 1087.8 ± 126.8 2621.1 ± 282.5 4575.0 ± 448.5 5.3 ± 1.4 1.6 ± 0.3 7.6 ± 1.3 5.5 ± 1.0 19.3 ± 1.3 335.2 ± 32.5 1057.3 ± 118.4 2662.6 ± 203.8 47.8 ± 3.9 315.2 ± 25.6 485.8 ± 38.5 1006.9 ± 89.2 2.4 ± 0.2 1.3 ±0.1 1.5 ± 0.2 1.9 ±0.1
CQL_1 100.0 ± 7.5 171.0 ± 13.2 112.5 ± 10.4 68.1 ± 7.0 0.2 ± 0.0 0.3 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 57.8 ± 4.2 119.1 ± 11.0 72.9 ± 6.1 48.0 ± 4.5 11.0 ± 0.9 25.8 ± 2.1 12.3 ± 1.1 8.4 ± 0.7 0.1 ± 0.0 0.3 ± 0.0 0.3 ± 0.0 0.9 ± 0.1
CQL_2 100.0 ± 12.1 420.6 ± 39.9 475.5 ± 42.6 475.9 ± 51.1 1.0 ± 0.1 2.4 ± 0.1 2.6 ± 0.3 2.5 ± 0.4 121.8 ± 9.4 484.9 ± 50.4 468.6 ± 47.7 531.4 ± 4.8 6.4 ± 0.3 23.9 ± 1.8 19.1 ± 1.5 28.2 ± 2.9 0.1 ± 0.0 0.6 ± 0.0 0.7 ± 0.1 0.8 ± 0.1
CQL_3 100.0 ± 8.1 207.1 ± 23.7 236.6 ± 21.8 208.6 ± 24.6 0.5 ± 0.2 0.8 ± 0.2 1.1 ± 0.2 1.0 ± 0.2 78.1 ± 3.2 203.4 ± 18.4 216.8 ± 11.2 228.1 ± 20.0 8.2 ± 0.7 12.4 ± 0.8 10.2 ± 0.7 10.1 ± 0.8 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.0
FQL_1 100.0 ± 11.7 231.4 ± 18.6 230.5 ± 25.6 211.6 ± 18.5 99.1 ± 10.2 198.9 ± 16.8 169.1 ± 15.8 159.9 ± 14.8 46.9 ± 2.6 110.3 ± 10.3 94.5 ± 6.2 93.8 ± 8.3 4.9 ± 0.2 15.9 ± 1.1 11.2 ± 1.0 17.5 ± 1.4 0.1 ± 0.0 0.4 ± 0.0 0.4 ± 0.0 0.4 ± 0.0
FQL_2 100.0 ± 10.4 203.4 ± 16.8 226.3 ± 19.0 185.6 ± 18.3 56.3 ± 5.2 97.1 ± 8.8 89.6 ± 8.1 84.8 ± 8.1 77.5 ± 5.1 146.6 ± 16.2 139.7 ± 10.5 146.7 ± 18.1 4.4 ± 0.3 7.2 ± 0.8 4.3 ± 0.2 5.3 ± 0.4 0.1 ± 0.0 0.2 ± 0.0 0.2 ± 0.0 0.1 ± 0.0
FQL_3 100.0 ± 10.6 226.7 ± 19.3 230.3 ± 18.6 206.1 ± 20.0 63.9 ± 5.9 138.4 ± 15.2 128.0 ± 13.2 120.8 ± 12.5 55.3 ± 4.6 139.6 ± 8.2 139.3 ± 12.6 138.3 ± 12.3 4.7 ± 0.3 15.2 ± 1.2 13.3 ± 0.9 20.1 ± 1.6 0.1 ± 0.0 0.4 ±0.0 0.4 ± 0.1 0.4 ± 0.1
diCQL 100.0 ± 17.4 341.5 ± 25.0 233.8 ± 28.3 237.0 ± 24.8 2.7 ± 0.3 8.9 ± 1.8 8.2 ± 1.9 6.0 ± 1.6 29.0 ± 2.2 351.5 ± 32.7 250.4 ± 18.3 746.4 66.9 2.7 ± 0.2 14.7 ± 1.5 26.8 ± 1.7 66.5 ± 5.7 0.5 ± 0.1 1.8 ± 0.2 3.7 ± 0.2 5.5 ± 0.3
VCO_1 100.0 ± 7.9 38.3 ± 2.4 30.2 ± 2.4 5.5 ± 0.9 60.1 ± 6.4 9.4 ± 0.8 0.5 ± 0.1 3.4 ± 0.3 117.9 ± 12.0 37.9 ± 2.4 16.4 ± 0.8 14.6 ± 1.3 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
VCO_2 100.0 ± 8.1 30.5 ± 2.6 14.7 ± 1.3 8.8 ± 1.5 42.1 ± 3.7 15.3 ± 1.3 1.5 ± 0.2 3.8 ± 0.3 112.4 ± 9.3 36.2 ± 3.6 15.6 ± 0.5 12.5 ± 1.3 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
VCO_3 100.0 ± 13.3 181.3 ± 20.0 148.8 ± 15.0 122.7 ± 18.4 69.8 ± 7.3 103.4 ± 11.0 73.6 ± 7.0 74.6 ± 6.5 113.0 ± 8.7 191.3 ± 15.0 131.2 ± 10.2 113.0 ± 15.0 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
VCO_4 100.0 ± 9.4 42.1 ± 4.4 22.4 ± 1.8 13.6 ± 3.3 93.5 ± 6.3 39.9 ± 3.8 15.2 ± 1.1 12.7 ± 1.2 78.1 ± 4.3 32.9 ± 2.8 12.3 ± 1.0 8.1 ± 0.6 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
VCO_5 100.0 ± 9.8 57.2 ± 3.9 19.1 ± 0.8 N.D. 97.7 ± 9.8 30.1 ± 2.0 14.9 ± 0.9 N.D. 133.5 ± 10.4 54.2 ± 4.4 16.4 ± 0.5 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.
VCO_6 100.0 ± 20.1 30.4 ± 2.0 17.9 ± 1.3 11.0 ± 2.8 118.4 ± 20.4 34.0 ± 4.1 15.5 ± 1.0 15.5 ± 1.0 26.2 ± 1.9 7.5 ± 0.4 3.2 ± 0.1 1.7 ± 0.1 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

N.D.: Not Detected. Values are mean ± SD of triplicate analyses.

The relative amounts of compounds contained in each coffee brew and the fractions were normalized with the peak area of the compounds

contained in the coffee brew with L value of 15.

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Table 3. Taste sensor bitterness response values for the coffee brew and each fraction

L value
15 20 25 30
Coffee brew 2.67 ± 0.06 2.13 ± 0.01 1.96 ± 0.04 1.40 ± 0.10
Fraction_1 1.72 ± 0.04 1.44 ± 0.04 1.27 ± 0.06 0.73 ± 0.03
Fraction_2 0.19 ± 0.08 0.13 ± 0.04 -0.07 ± 0.04 -0.13 ± 0.06
Fraction_3 2.10 ± 0.03 1.78 ± 0.05 1.18 ± 0.06 0.93 ± 0.06
Fraction_4 0.18 ± 0.05 0.12 ± 0.08 0.09 ± 0.02 -0.13 ± 0.04

Values are mean ± SD of triplicate analyses.

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Table 4. Candidate compounds that contribute to the bitterness response value of the taste sensor

Compounds VIP Coefficient

Nicotinic acid 1.49 0.44
L-Lactic acid 1.48 0.34
VCO_6 1.36 0.33
FQL_1 1.14 0.23
Nicotinamide 1.07 0.52
VCO_4 1.05 0.14

These compounds were calculated to be the most important variables in the construction model as a

whole based on their VIP (variable importance in projection) values.

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Table 5. Bitterness response values of coffee brew with added nicotinic acid, L-lactic acid, or

nicotinamide

Control Control with nicotinic acid Control with L-lactic acid Control with nicotinamide
Bitterness response value 2.61 ± 0.08 2.69 ± 0.09 2.93 ± 0.11 2.71 ± 0.06

Values are mean ± SD of triplicate analyses.

31
Hirofumi Fujimoto: Conceptualization, Formal analysis, Investigation, Writing - Original Draft,
Visualization. Yusaku Narita: Methodology, Investigation, Writing - Original Draft, Writing - Review
& Editing. Kazuya Iwai: Resources. Taku Hanzawa: Formal analysis. Tsukasa Kobayashi: Validation.
Misako Kakiuchi: Validation. Shingo Ariki: Validation. Xiao Wu: Visualization. Kazunari Miyake:
Validation. Yusuke Tahara: Supervision. Hidekazu Ikezaki: Resources. Taiji Fukunaga: Resources,
Project administration. Kiyoshi Toko: Supervision.

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Highlights:

 Aim of this study is investigating for bitter compounds in coffee brew.

 The results of instrument analysis were subjected to multivariate analysis.

 It was suggested that some alkaloids and L-lactic acid contributed to bitterness.

 Additional experiment confirmed that these compounds increased the coffee bitterness.

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