Evaluation of Nonvolatile Chemistry Affecting Sensory Bitterness

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Chemistry and Biology of Aroma and Taste

A Comprehensive Evaluation of the Nonvolatile Chemistry Affecting the Sensory Bitterness Intensity of Highly Hopped Beers Christina Hahn, Scott Lafontaine, Clifford Pereira, and Tom H. Shellhammer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05784 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 11, 2018

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Journal of Agricultural and Food Chemistry

1 Evaluation of the Nonvolatile Chemistry Affecting the Sensory Bitterness Intensity of Highly Hopped Beers Christina D. Hahn¹, Scott R. Lafontaine¹, Cliff B. Pereira¹, Thomas H. Shellhammer¹* ¹Oregon State University Department of Food Science & Technology 234 Wiegand Hall Corvallis, OR 97330 *Corresponding author. Phone: 1-541-737-9308. E-mail: [email protected]

ACS Paragon Plus Environment


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1

Abstract

2

The range of different nonvolatile constituents extracted from hops in highly hopped

3

beers suggests that isohumulones may not be the sole contributor to beers’ bitterness. Among

4

brewers producing hop-forward beer styles there is concern that the Bitterness Unit (BU) is no

5

longer an accurate predictor of beer bitterness. This study examined factors within the beer

6

matrix that influence sensory bitterness perception in highly hopped beers. Over 120 commercial

7

beers were evaluated using sensory and instrumental techniques. Chemical analysis consisted of

8

the BU via spectrophotometry, hop acids via HPLC, total polyphenols via spectrophotometry,

9

and alcohol content plus real extract via an Alcolyzer. Sensory analysis was conducted over two

10

studies, and the beers’ overall bitterness intensity were rated using a 0-20 scale. This study

11

identified that the BU measurement predicts sensory bitterness with a nonlinear response, and it

12

proposed an alternative approach to predicting bitterness based on isohumulones, humulinones,

13

and ethanol concentrations. The study also revealed the importance of oxidized hop acids,

14

humulinones, as a significant contributor to beer bitterness intensity.

15

Keywords: Hops, bitterness, brewing, beer, isohumulones, humulinones.


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Introduction

17

With the rise in popularity of hop-forward beer styles there has been a growing concern

18

in the beer industry surrounding the accuracy of instrumental bitterness analyses. Anecdotally,

19

some in the brewery quality assurance field feel that current methods for predicting beer

20

bitterness, such as the Bitterness Unit (BU) method, a liquid/liquid extraction of bitter

21

compounds, and the direct measurement of isohumulones via high performance liquid

22

chromatography (HPLC), often fail to accurately predict the sensory bitterness of beer of highly

23

hopped beer styles The projects presented herein were designed to test existing methodology and

24

to search for potential new approaches to instrumentally predicting sensory bitterness intensity in

25

beer.

26

Hops (Humulus lupulus) are the primary source of bitter compounds in beer, and these

27

bitter compounds can be categorized into three groups: isohumulones, oxidized hop acids, and

28

polyphenols.1 There is little question that isohumulones are the main contributor to beer

29

bitterness, however all three groups have the ability to impact both sensory bitterness intensity

30

and the BU value of a beer.2-5 How a brewer uses the hops will determine their concentration in

31

finished beer. The timing and mass of hop additions in the brewing process will influence the

32

overall aroma, bitterness, and astringency of the finished beer.6 In the production of hop-forward

33

beer styles, such as American craft Pale Ales and India Pale Ales (IPAs), brewers add

34

significantly more hop material to beer relative to traditional lager beer styles. This shifts both

35

the concentration and ratio of bitter compounds present in the finished beer from solely

36

isohumulones (as in American light lager beer) to a broad array of hop-derived compounds such



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as humulones, oxidized humulones (humulinones), oxidized lupulones (hulupones), and

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polyphenols, in addition to isohumulones.

39

Generally, early additions of hops during wort boiling impart more bitterness and less

40

aroma while the inverse is true for hops added late in wort and/or beer production.3 A traditional

41

hopping scheme for lager beer would be a single early addition in the range of 1.5 g/L at the

42

beginning of a 60-90 minute wort boil. By contrast, a hopping scheme for an American craft IPA

43

will involve the addition of hops at multiple stages in the brewing process and to the finished

44

beer itself, a technique referred to as “dry-hopping”. The Brewers Association reported that in

45

2014 the top selling American Imperial IPAs contained between 9 and 11.5 g/L of hop material,7

46

while American light lager beer contains approximately 1 g/L. Brewers looking to increase hop

47

aroma by adding large quantities of hops late in process also potentially increase the

48

concentration of oxidized hop acids and total polyphenols in the finished beer.

49

Oxidized hop acids are present in freshly baled, aged or improperly stored hops at

50

varying levels.8-10 Recent work has shown that oxidized hop acids, hulupones and humulinones,

51

are 84% and 66% as bitter as isohumulones, respectively.2 Separately, polyphenols can

52

contribute both bitterness and astringency to a wide variety of food and beverages depending on

53

their degree of polymerization and molecular weight, and have been shown to have an additive

54

effect on beer bitterness in the presence of isohumulones.5,

55

polyphenols contribute to sensory bitterness alone is not understood. A 2008 study showed that

56

at concentrations less than 300 mg/L, total polyphenols influence BU values in lager beer


11-12

The magnitude to which

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systems. For every 15-20 mg/L increase in total polyphenols, a one-unit increase in BU was

58

observed.5

59

At relatively high concentrations in beer (ranging 14 mg/L to 28 mg/L) humulones, the

60

precursors to isohumulones, have been shown not contribute to beer bitterness.13 However, at

61

these elevated concentrations they may influence the BU value. Although the BU method

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captures these compounds in its measurement, there is no accounting for their relative abundance

63

or relative bitterness. Furthermore, beer matrix effects, such as alcohol, residual extract and pH,

64

may also influence bitter taste sensation.14-17 Residual extract and ethanol concentrations are well

65

known factors brewers manipulate to balance a beer’s bitterness. Typically, beers with a high

66

concentration of bittering compounds will have higher levels of residual extract.6 Ethanol

67

concentration may also play a role in bitterness perception, as numerous studies in wine show

68

that ethanol enhances bitter taste sensation.11, 17-19

69

To predict sensory bitterness intensity, the International Bitterness Unit, or BU method, is

70

most frequently employed. The BU method is a manual liquid-liquid extraction technique that

71

utilizes polarity to separate the bittering components of beer using a non-polar solvent (2,2, 4-

72

trimethylpentane).20 The absorbance of the extracted components in the non-polar phase is

73

measured via a spectrophotometer at 275 nm and multiplied by a factor of 50 to obtain the BU

74

value. The BU method was developed in 1955 by Rigby and Bethune with the intent of rapidly

75

measuring the concentration of isohumulones in beer prior to the advent of HPLC.21 By 1968,

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the BU was internationally recognized as a standard method by the American Society of Brewing

77

Chemists (ASBC) and the European Brewery Convention.22 In addition to measuring



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isohumulones, other ultraviolet-adsorbing compounds may influence the BU value.4,

20, 23

79

inclusion of non-isohumulone substances in the BU assay presents a challenge to brewers,

80

particularly when using this technique to evaluate beer styles that have been heavily hopped.4, 10

The

81

Advances in instrumentation now allow for the rapid and easy measurement of

82

isohumulones in beer using HPLC. The measurement of isohumulones by solid phase extraction

83

and HPLC was first published by Donley in 1992 and became an official method of the ASBC in

84

1993 (Beer-23, C).24 By 2011 the ASBC approved a direct injection method for the measurement

85

of isohumulones by HPLC that eliminated the need for solid-phase extraction (Beer-23, E).20, 25

86

Although HPLC allows for the measurement of a wide range of different hop acids, historically

87

the focus has been solely on isohumulones.

88

Regardless of the advances made in beer analyses tools, the American craft beers

89

produced today are unlike the beers for which the current methods (BU and isohumulones by

90

HPLC) were designed to measure. American craft beers are complex in their non-volatile

91

chemistry relative to light lager beer, and there is concern surrounding the BU’s ability to

92

adequately predict sensory bitterness in these new, highly hopped beer styles. This study aimed

93

to address this concern by (1) evaluating a broad array of chemical factors known to affect bitter

94

taste sensation along with sensory evaluation of bitterness using commercially-available,

95

American beers with a focus on hop-forward styles, (2) performing multiple linear regression

96

modeling to determine the significant drivers of beer bitterness, and (3) proposing a new,

97

analyte-specific, predictive model of beer sensorial bitterness intensity.


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Materials and methods

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To gain insight into the drivers of sensory bitterness in beer, 121 unique commercial

100

beers from 42 breweries across the United States were tested for 7 factors known to impact the

101

sensory perception of bitterness in beers. Sensory analysis was conducted in two separate studies

102

with specific objectives for each. A multiple replication study focused on evaluating a smaller

103

number of beers (30) with a small (10 member) trained sensory panel. The beers’ overall

104

bitterness intensity were rated using a 0 to 20 scale. This study revealed the chemical drivers of

105

sensory bitterness, confirmed the inadequacy of total isohumulone content as a complete

106

measure of beer bitterness, and proposed an alternative, liquid chromatography approach to

107

predicting bitterness based on the combination of isohumulones, humulinones, and ethanol

108

concentrations. A separate, single replication study was used to validate the results from the

109

multiple replication study and was focused on evaluating a larger number of commercial beers

110

(104 in total, 13 of which had been evaluated in the multiple replication study) with a larger (19

111

member) trained sensory panel.

112

Reagents and standards

113

Ferric

ammonium

citrate

(green)

was

purchased

from

Fisher

Chemicals.

114

Ethylenediaminetetraacetic acid disodium salt, dihydrate (EDTA),

115

carboxymethylcellulose, and octyl alcohol were obtained from Sigma-Aldrich Chemical Co (St.

116

Louis, MO). HPLC grade methanol was obtained from VWR International, BDH analytical

117

(West Chester, PA, USA). Hydrochloric acid, 2,2,4-trimethylpentane, phosphoric acid and

118

ammonium hydroxide obtained from Avantor performance materials (Center Valley, PA).


medium viscosity


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DCHA-Iso ICS-I3 and international calibration extract ICE-3 standards were obtained from

120

ASBC. DCHA humulinone and hulupone standards were produced2 and pure standards were

121

obtained through Robert Smith at S.S. Steiner, Inc.

122

Beer analysis

123

The concentrations of hop acids in beer samples were assayed using a modified version

124

of ASBC method Beer-23E.20 The modified conditions were as follows: analysis was performed

125

on a 2.6 um EVO C-18 100 A LC column 100 x 4.6 mm (Phenomenex, Torrance, CA) at 40°C

126

measuring absorbance at 275 nm for the isohumulones and humulinones, 330 nm for the

127

hulupones, and 314 nm for the humulones. These wavelengths were chosen considering the

128

absorbance spectrum of each hop acid.2 Prior to analysis beer was degassed and 7 µL of each

129

beer sample was injected into a mobile phase consisting of 10% A (reagent water) and 90% C

130

(75% MeOH, 24% H2O, 1% H3PO4) flowing at 1.6 ml/min. Ten minutes following the injection

131

the mobile phase was switched to 100% B (100% MeOH), and at 14 minutes it was switched

132

back to 10% mobile phase and A 90% mobile phase C.

133

Total polyphenols were measured spectrophotometrically according to the EBC

134

Analytica methods (9.11).26 Bitterness units were measured according to ASBC methods of

135

analysis Beer 23A.20 Spectrophotometric analysis for total polyphenols and bitterness units were

136

carried out using a Shimadzu PharmaSpec UV-1700 spectrophotometer, Shimadzu Corporation

137

(Columbia, MD). Ethanol, residual extract, and pH were analyzed using an Anton-Paar

138

Alcolyzer with supporting pH module (Anton Paar USA, Ashland, VA).


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Sensory Methodology: Multiple replication study

140

For the multiple replication study, the sensory panel consisted of 10 members - 6 males

141

and 4 females, ranging from ages 21 to 54, with a median age of 28. Thirty (30) unique beers

142

were evaluated, 13 of which were also evaluated in the single replication study. The panel

143

included 4 individuals with previous training on bitterness intensity and 6 self-identified frequent

144

craft beer drinkers with minimal training on bitterness intensity. All panelists were over the age

145

of 21 and self-identified beer consumers.

146

Of the 30 beers evaluated, the majority were commercially categorized as pale ales, India

147

pale ales (IPA), session IPAs, and imperial or double IPAs. Beer selection began by evaluating

148

the chemistry of 60 unique beers from 17 American craft breweries. The beers were sorted based

149

on the highest and lowest values of the seven chemical factors of interest. The 30 beers selected

150

consisted of the extreme values of ethanol, residual extract, pH, isohumulones, humulinones, and

151

humulones in the larger beer set and represented the wide range of variation found in heavily

152

hopped beer styles.

153

The panel met as a group for five one-hour training sessions. Training sessions focused

154

on differentiating and identifying peak bitterness intensity in different beers. To minimize

155

sensory fatigue only 10 beers were evaluated per one-hour session with three separate sessions

156

making a full replication. The 30 beers were evaluated over the course of 12 sessions for a total

157

of 4 replications. To minimize variation in bitterness intensity among the sessions, a randomized

158

incomplete block design was used to ensure that the beers evaluated per session had

159

approximately the same variance in BU values (See Supporting Information). Each 10-beer



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group was evaluated in its own session, and Qualtrics software (Qualtrics, Provo, UT) was used

161

to generate a random presentation order for each panelist within each session. Beers samples (45

162

ml) were presented in open 120 ml clear glasses, blind coded with random three-digit numbers.

163

Beers were stored at 4°C for the duration of the study and removed from cold storage and poured

164

30 minutes prior evaluation. Training and testing took place in the same location; a conference

165

room held at 20°C and lit with Fluorescent lighting.

166

At the beginning of each session, panelists were assigned a Chromebook tablet (Google,

167

Mountain View, CA), and directed to an online Qualtrics survey. Each testing session began with

168

the panelists warming up and calibrating bitterness intensity scaling by tasting four reference

169

samples. External reference samples for anchoring the overall bitterness scale were chosen by

170

using four commercially available beers of varying sensory bitterness intensities. The sensory

171

scores for these references were determined by consensus during training. The zero reference

172

point (0) was anchored by Michelob Ultra-Light (Anheuser-Busch, St. Louis, MO) (BU = 4), the

173

low reference point (4) was anchored by Session Lager (Full Sail Brewing Co., Hood River, OR)

174

(BU = 18)., the medium-low reference point (8) was anchored by Mirror Pond Pale Ale

175

(Deschutes Brewery, Bend, OR) (BU = 40), and the medium-high reference point (15) was

176

anchored by Torpedo IPA (Sierra Nevada Brewing Co., Chico, CA) (BU = 66). No reference

177

was selected to anchor the high end of the scale (20) thereby reducing potential sensory fatigue.

178

After the reference samples were evaluated, panelists were instructed to taste the samples

179

in the order in which they appeared on their ballot. Panelists scaled the overall bitterness

180

intensity of a given sample using a sliding bar scale (0 = none, 20 = extreme), and the scaling


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data were collected using Qualtrics. After assessing a sample, the panelists were instructed to

182

rinse their mouths with a 0.1% pectin solution to aid in the reduction of bitterness carry-over,35

183

and then to rinse their mouths again with deionized water. Panelists were instructed to wait until

184

all perceivable bitterness had dissipated before tasting the next sample. Panelists repeated this

185

procedure for each sample. After all samples had been assessed, panelists were allowed to re-

186

taste the samples (if necessary) in any order deemed necessary to most accurately assess a given

187

sample’s overall sensory bitterness intensity.

188

Sensory Methodology: Single replication study

189

For the single replication study, the panel consisted of 19 members - 14 males and 5

190

females, ranging from ages 22 to 65, with a median age of 28. The panel contained 4 individuals

191

with previous training on bitterness intensity and 15 self-identified frequent craft beer drinkers

192

with minimal training on bitterness intensity. Of the 19 panelists, 4 also participated in the

193

multiple replication study. The study started with 116 unique beers from 42 American breweries

194

but was reduced to 104 unique beers from 38 American breweries, due to quality defects

195

(preliminary data not shown). Thirteen (13) of the 104 beers had also been evaluated in the

196

multiple replication study. This study included a greater number of beer styles in an effort to

197

capture the variability within styles produced by craft and macro breweries alike. The beers were

198

not screened based on their chemical composition prior to sensory evaluation.

199

The panel met as a group for three one-hour training sessions. Training was followed by

200

twelve testing sessions conducted over the course of ten weeks. Panelists evaluated 10 beers per

201

session and scaled bitter intensity using a paper ballot. Paper ballots were utilized due to the



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limited number of available Chromebooks and the increased number of panelists. XLSTAT

203

software was used to randomly assign the beers to each session, and to randomize the sample

204

presentation order for each panelist. Two beers were selected to be replicated throughout the

205

study thereby serving as internal controls: Fat Tire (New Belgium Brewing, Fort Collins, CO)

206

served as a low bitterness intensity control (overall bitterness intensity = 3) (BU = 20)and

207

Torpedo IPA (Sierra Nevada Brewing Co., Chico, CA) (BU = 66) served as the medium-high

208

bitterness intensity control (overall bitterness intensity = 15). These beers were randomly

209

replicated three times during the study. The remainder of the samples were not replicated.

210

Panelists scaled the overall bitterness intensity of a given sample using a sliding bar scale (0 -

211

20) on a paper ballot. Sensory training, commercial reference beers, environmental conditions,

212

and general sensory protocol was carried out as described previously for the multiple replication

213

study.

214

Statistical analysis

215

In the multiple replication study, each of the 30 beers was evaluated 40 times,

216

independently (10 panelists over 4 replications). Panel performance ANOVA revealed

217

significant main effects for panelist, beer, and panelist by beer interaction (Table 1). A

218

significant panelist effect is not unexpected in sensory analysis as individual panelists interpret

219

stimuli differently. No effects were observed for replication, replication by panelist, or

220

replication by beer. The 40 bitterness intensity scores generated for each beer were averaged into

221

a single value and assigned to that beer as an overall bitterness intensity score. Results were

222

evaluated using a mixed model, two-way, analysis of variance (ANOVA) model. Fixed effects


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included beer. Random effects included panelist, replication, panelist by beer, replication by

224

beer, and panelist by replication interactions. Data analysis was carried out using XLStat 2016

225

(Addinsoft, New York, NY, U.S.A.).

226

In the single replication study, most panelists missed at least one session during the study,

227

thus each of the 104 unique beers was evaluated singly by 9 to 19 panelists. This unbalancedness

228

with respect to number of panelists per beer required the generation of adjusted means for each

229

beer (adjusted for panelist differences) to be used as the Overall Bitterness Intensity score for

230

each beer.

231

Multiple linear regression using stepwise (forward) model selection in the SAS

232

GLMSELECT procedure was carried out for the seven predictive chemical factors of interest

233

(linear and quadratic in each factor as well as linear-by-linear interactions) to develop a model to

234

predict the overall bitterness intensity scores that were derived from each of the two sensory

235

panel studies. The model selection criterion used was AICC (Corrected Akaike’s Information

236

Criterion) and it was found that the same conclusions would be reached with other criteria

237

(Akaike’s Information Criterion (AIC), Schwartz’s Bayesian Criterion (SBC) or prediction sum

238

of squares (PRESS)). A model hierarchy requirement was used so that quadratic in a predictor

239

could enter the model only when linear in that predictor was already present in the model and a

240

linear-by-linear interaction could enter the model only when the two linear terms were already

241

present in the model. Resampling and model selection for each resampled set of data was used

242

to assist in determining the relative importance of predictors (effect selection percentage).



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Second order polynomial models and other linear models were fit with the SAS

244

GLMSELECT procedure. Nested models were compared using F tests (numerator = the change

245

in model sum of squares divided by the change in number of parameters; denominator = residual

246

mean square from the full model).

247

Results and Discussion

248

The beers evaluated in this study presented a broad range of chemistry and sensory

249

bitterness (Figure 1). Furthermore, the beer subsets as a group for each study were not

250

statistically different as assessed by a means comparison (2-sided t-test, p>0.05).

251

Across both studies, residual extract concentrations ranged from 2.5 to 8.1% w/w with an

252

average concentration of 4.9% w/w (Figure 1.A). Residual extract concentration is largely

253

determined by beer style. Lager beers often have lower residual extract relative to ales such as

254

porters, stouts, pale ales and IPAs. Residual extract is mostly comprised of the un-fermentable

255

carbohydrates that remain following the alcoholic fermentation, and these dextrins are primarily

256

lower molecular weight polymers of D-glucose units linked by α-(1→4) or α-(1→6) glycosidic

257

bonds. The breakdown of these dextrins in the oral cavity can lead to the sweet taste of some

258

beers.14-15,

259

extract to influence the flavor, mouthfeel, and bitterness of beer. Beers with increased bitterness

260

tend to be brewed with a higher residual extract, a technique used by brewers to create

261

“balanced” beers.6 Across all food systems, sweet taste suppresses bitter taste, and bitter taste

262

suppresses sweet taste.14,

263

taste qualities exhibit this inhibitory pattern.29-30

27

The residual extract also contains protein and minerals. Brewers adjust residual

28

This phenomenon is known as mixture suppression, and all basic


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Like residual extract, the ethanol concentration of a beer may be determined by beer

265

style, however significant variation in alcohol concentration may also be found within styles. It is

266

not uncommon to find “sessionable” (low ethanol) or “imperial” (high ethanol) versions of a

267

brewery’s flagship beer seasonally. The beers in this study displayed a wide range of ethanol

268

concentrations from 3.8% to 10.1% v/v. The average concentration of ethanol across both studies

269

was 6.4% v/v (Figure 1.B). The influence of ethanol on beer flavor is multifaceted, and its effect

270

on bitterness intensity is one example. Ethanol is considered to have a sweet taste at low

271

concentrations,18 however numerous studies have shown that the presence of ethanol increases

272

bitter taste sensation in alcoholic beverages.11,

273

evaluating bitterness intensity of commercial beer, as many beers tend increase in both residual

274

extract and ethanol concentration as hopping rates increase.

16-19, 28

This can be a confounding factor when

275

The range of pH values was somewhat wider than expected at pH 4.0 to 5.4 (Figure 1.C).

276

The elevated pH values are likely the result of beers with high levels of dry-hopping.8 It is worth

277

noting, that as pH increases, so does the solubility of humulones (α-acids), lupulones (β-acids),

278

and humulinic acid. A 1955 study by Spetsig showed that the solubility of these hop acids

279

increased exponentially with pH.31 Thus, the effect of elevated pH via dry hopping may increase

280

the concentration of humulones in finished beer.

281

The solubility of humulones in wort and beer is low, and thus it was not unexpected to

282

find very low levels of these compounds in the beers in this study. While the solubility of

283

humulones is pH dependent, it has been reported to be approximately 3 mg/L in water.32 In a

284

traditional brewing process, where the hop material is boiled in wort for an extended period of



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time (60-90 minutes), very little humulone will persist into the finished beer.1 In dry-hopped beer

286

styles, the solvating power of ethanol increases the solubility of humulones. The majority of the

287

beers tested in the study presented herein had little to no humulones, however four of the beers

288

had humulone concentrations greater than their solubility in beer (14 mg/L)13 with a maximum of

289

24.2 mg/L (Figure 1.F). Although humulones did not contribute bitterness to beer at these levels,

290

they do adsorb at 275 nm and may contribute to a beer’s BU value if present. Because

291

humulones may contribute to the BU value, but are not bitter, they may affect the BU’s ability to

292

accurately predict bitterness intensity.13, 23

293

As previously stated, isohumulones are the primary source of bitter taste in beer. They are

294

isomers of hop-derived humulones which are transformed with heat and time via a first order

295

reaction that is temperature-dependent.3 Isohumulone concentrations (as determined by HPLC)

296

averaged 36 mg/L across both studies and ranged from 7.0 to 74.2 mg/L (Figure 1.D). The most

297

prominent forms of isohumulone are isohumulone, iso-co-humulone and iso-ad-humulone,33-35

298

and it is standard practice to sum their concentrations to obtain the reported value of

299

isohumulone.20

300

The role of oxidized hop acids has been the focus of several studies related to hop and

301

beer quality.2, 36-38 Although there is little evidence to support the theory, there is a widespread

302

belief within the brewing community that as hops age and decrease in total α-acids the increase

303

in oxidized lupulones (hulupones) prevents the loss of bittering potential in the hop itself.32 Of

304

the 121 unique beers tested in this study, most had no detectible levels of lupulones or their


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oxidation products. In a small number of cases, very low levels of hulupones were detected, but

306

these levels were below the limit of quantification.

307

In contrast, both the prevalence and concentration of the oxidized humulones

308

(humulinones) (Figure 1.E) were higher than previously reported in literature. The average

309

concentration of humulinones was 17 mg/L and 117 out of the 121 unique beers contained

310

detectible levels of humulinones (≥1 mg/L). The concentration of humulinones in finished beer

311

is dependent on the concentration of humulinones present in the hop varieties used, total hop

312

mass, and hop contact time.4, 8 A recent study by Algazzali and Shellhammer confirmed the bitter

313

quality of humulinones as previously published.35,

314

detect humulinones as low as 8 mg/L, and it was proposed that humulinones have a relative

315

bitterness intensity of 66% when compared to isohumulones.2 A recent study by Oladokun et

316

al.40 examined the impact of hop bitter acids and polyphenols on beer bitterness in 34 global

317

commercial lager beers. In this study humulinone concentrations were considerably lower (≤ 3

318

mg/L) in the lager beers tested in comparison to the American hoppy ales and lagers tested in

319

this study.40

39

The study’s trained sensory panel could

320

The concentration of total polyphenols in finished beer is dependent on hop mass, hop

321

type (extract, pellet or whole cone), the hop varieties used, the temperature of dry-hopping and

322

contact time.9-10 Polyphenols comprise a vast group of different compounds that contribute to

323

bitterness and/or astringency of beer.41 Typically, heavily late-hopped and/or dry-hopped beer

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styles contain elevated levels of total polyphenols.40 A study by McLaughlin and Shellhammer5

325

showed polyphenols to have an additive effect on bitter taste perception when combined with 10



Page 18 of 36

16 326

mg/L isohumulones in lager beer. The magnitude in which polyphenols alone influence the

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bitterness of beer is currently not understood. However, Oladokun et al.40 showed polyphenols to

328

influence the bitter quality of lager beer. In the study, beers with increased concentrations of

329

polyphenols were perceived as having ‘harsh’ and ‘progressive’ bitterness. It is worth noting that

330

these studies were conducted in lager beers with maximum total polyphenol concentrations

331

below 300 mg/L. In this study, total polyphenol concentrations ranged from 135 to 697 mg/L

332

with an average of 329 mg/L (Figure 1.G). Over 60% of the beers tested in the study presented

333

herein contained total polyphenol concentrations in excess of 300 mg/L. Additional research is

334

needed to understand the impact of polyphenols in beer at these concentrations.

335

The BU method was developed in 1955 as a way to rapidly measure isohumulones prior

336

to the advent of the HPLC.21 When analyzing beers in which isohumulones are the primary

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source of bitter compounds, the BU is an accurate predictor of sensory bitterness. In hop-forward

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beer styles, such as American craft IPAs and pale ales, brewers add hops to the wort late in

339

production or to finished beer. This technique preserves volatile hop aroma compounds that

340

would otherwise be lost during wort boiling. In addition to adding hop aroma, late additions of

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hops also add non-volatile compounds such as humulones, oxidized hop acids, and

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polyphenols.42 These compounds may contribute to a beer’s BU value, and at elevated

343

concentrations may potentially impact a beer’s bitterness intensity. In this study, bitterness units

344

ranged from 10 to 118 with an average BU of 60 (Figure 1.H). A mistaken conventional wisdom

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is that 1 mg/L of isohumulones is equal to 1 BU. Although this may hold true for low bitterness

346

lager beers, the relationship did not hold true for the beers evaluated in this study (Figure 2).


Page 19 of 36


17 347

The beers tested had a wide range of sensory bitterness intensities (Figure 1.I). A variety

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of breweries, brands, and beer styles where tested, but an emphasis was placed on hop-forward

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beer styles, as one of objectives the study was to understand if the BU could accurately predict

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sensory bitterness intensity in these beers. At low levels of BU, the relationship between sensory

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bitterness intensity and the BU appeared to be linear. However, for BU values of 50 and above,

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the relationship became curvilinear, and increases in sensory bitterness intensity became smaller

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as the BU values increased (Figure 3, D & 4 D). This result may be from a saturation

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phenomenon, that is, as stimuli increases, it becomes increasingly difficult to differentiate

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changes in the stimuli as the senses become overwhelmed.30 Alternately, it may be that the

356

compounds affecting increases in the BU at these elevated levels (> 50 BU) have a reduced

357

impact on sensory bitterness intensity, or some combination of the two.

358

For each of the two studies, model selection for predicting sensory bitterness intensity

359

was conducted using all seven factors of interest (linear, quadratic and linear-by-linear

360

interactions). For both studies, the most important predictors were linear in isohumulones, linear

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in humulinones, linear in alcohol by volume (ABV) and second-order terms involving

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isohumulones and/or humulinones). Because of the importance of linear and second-order effects

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involving isohumulones and humulinones, the second order response surface was investigated by

364

fitting a full second-order model to the data (Eqn. 1).

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BI = β₀ + α₁(ISO) + γ(HU) + δ(ISO * HU) + α₂(ISO)² + γ₂(HU)²

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where BI = sensory bitterness intensity (0-20),

367

ISO = Isohumulones and HU = humulinones (mg/L)


(Eqn. 1)


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18 368

Within each study, the fitted model had the following characteristics:

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1) linear coefficients were similar to each other (α1 ≅ γ1);

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2) quadratic coefficients were similar to each other (i.e. α2 ≅ γ2);

371

3) the interaction coefficient (δ) had the same sign as the quadratic terms (α2 & γ2) and

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(given the large standard error) was not very different from the sum of the quadratic

373

coefficients.

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These three characteristics (and contour plots of the fitted surface) suggested that a much simpler

375

model with a single predictor (sum(ISO + HU) = the sum of the isohumulones and humulinones)

376

could predict sensory bitterness intensity well (Eqn. 2). Indeed, in both studies the simpler model

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fit well (R2= 0.913 in the multiple rep study and R2 = 0.772 in the single-rep study) and the more

378

complex model (Eqn. 1) did not significantly improve the fit of the simpler model (F < 1, p >

379

0.49 for both studies).

380

= + + + +

381

The effectiveness of sum(ISO + HU) as a predictor is shown graphically in Figures 3 and

382

4. Individually, isohumulone and humulinone concentrations are relatively poor predictors of

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sensory bitterness (Figures 3 & 4, A & B), while the sum of isohumulones and humulinones

384

(Figure 3 & 4, C) is almost as good a predictor of sensory bitterness as BU (Figure 3 & 4, D).

385

These same figures also highlight a need for the model to capture curvilinearity as well as the

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greater variation about the relationship in the single-rep study (Figure 4) compared to the

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multiple rep study (Figure 3).


(Eqn. 2)

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19 388

With the effectiveness of a single predictor established, model selection was repeated

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with sum(ISO + HU) as an 8th potential predictor. In both studies, the most important predictors

390

were linear and quadratic in sum(ISO + HU) and linear in ABV (Eqn. 3). The addition of ABV

391

increased the R-squared slightly, but significantly (p = 0.0002 for single-rep study and p=0.041

392

in multiple rep study). The positive influence of ethanol on bitterness perception is not

393

unexpected as it has been reported elsewhere that ethanol increases bitter perception in beer and

394

wine.16-19, 27-28

395

= + + + + + ₃

396

It is important to note that although the simple sum of isohumulones and humulinones

397

was effective as a single predictor, this does not mean that future studies should necessarily give

398

equal-weight to isohumulones and humulinones. That is, a weighted sum of isohumulones and

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humulinones could potentially be even more effective as a predictor in future studies despite not

400

being the case in this study.

(Eqn. 3)

401

As it was devised, the BU describes the bitterness of beer in a linear fashion, and for low

402

BU beers this relationship holds true. However, when using a linear BU response to predict

403

bitterness intensity over the entire data set within each study, there was obvious systematic lack

404

of fit with the tendency to under-predict in the mid-range of bitterness and over-predict at the

405

extremes. Adding a quadratic term significantly improved the fit (p F Beer 29 11826 407.8 32.98

Evaluation of Nonvolatile Chemistry Affecting Sensory Bitterness

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