Influence of Winemaking Processing Steps on the Amounts of (E)-2

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Influence of Winemaking Processing Steps on the Amounts of (E)-2-Decenal and Tridecane as Off-Odorants Caused by Brown Marmorated Stink Bug (Halyomorpha halys) Pallavi Mohekar, James Osborne, Nik G Wiman, Vaughn Walton, and Elizabeth Tomasino J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04268 • Publication Date (Web): 08 Jan 2017 Downloaded from http://pubs.acs.org on January 9, 2017

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Journal of Agricultural and Food Chemistry 1

Influence of Winemaking Processing Steps on the Amounts of (E)-2-Decenal and Tridecane as Off-Odorants Caused by Brown Marmorated Stink Bug (Halyomorpha halys) Pallavi Mohekar†, James Osborne†, Nik G. Wiman‡, Vaughn Walton‡, Elizabeth Tomasino*,†

†

Oregon State University, Department of Food Science & Technology, 100 Wiegand

Hall, Corvallis, Oregon 97331, USA ‡

Oregon State University, Department of Horticulture, 4017 Agriculture and Life

Sciences Building, Corvallis, Oregon 97331, USA *

[email protected], (T) (541)-737-4866, (F) (541)-737-1877

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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1

ABSTRACT

2

Brown marmorated stink bugs (BMSB) release stress compounds, tridecane and (E)-

3

2-decenal, that effect final wine quality. This study focuses on determining the effect

4

of wine processing on (E)-2-decenal and tridecane release in both red and white

5

wines. Wines were produced by adding live BMSB to grape clusters at densities of 0,

6

0.3, 1 and 3 bugs per cluster. Compound concentrations were measured using HS-

7

SPME-MDGC-MS. For red wines, the highest levels of stress compounds were found

8

using 3 BMSB/cluster (tridecane, 614 µg/L and (E)-2-decenal, 2.0 µg/L). Pressing

9

was found to be the critical process point for stress compound release and additional

10

pressing processes, press type and press fractions, were investigated. BMSB taint for

11

white wines was not found to be problematic to wine quality. An action control of 3

12

BMSB/cluster is recommended as this related to the known consume rejection

13

threshold for (E)-2-decenal.

14 15

Keywords: brown marmorated stink bug, press type, Pinot noir, Pinot gris, Merlot

16 17 18 19 20 21


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Introduction

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Brown marmorated stink bug (BMSB) (Halyomorpha halys Stål) (Hemiptera:

24

Pentatomidae) is an economically important insect pest of multiple agricultural crops,

25

including winegrapes. In vineyards, BMSB can effect yield and quality by direct

26

feeding on berries.1,2 This damage may further lead to secondary pest attacks or

27

infection by other pathogens1,3 and result in fruit collapse due to progressive

28

necrosis.1,4 Direct injury to grape berries at veraison and pre-harvest have been found

29

in Virginia vineyards.3 Additional damage by BMSB occurs through grape

30

contamination post-harvest and has been little explored. Effects of post-harvest

31

contamination is the major focus of this study.

32 33

Contamination of winegrapes occurs when BMSB are carried into the winery within

34

the grape clusters. BMSB are known to migrate into vineyards during the harvest time

35

(August-September in North America),5–7 greatly increasing the chance of winegrape

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contamination. The insects are harvested and go unnoticed within grape clusters due

37

to their small size, cryptic behavior and coloring. The presence of BMSB during wine

38

processing can effect juice and wine quality through the release of volatile

39

compounds produced as a stress response.8–10

40 41

BMSB stress compounds include tridecane and (E)-2-decenal, and make up more

42

than 70% of released stress compounds.8,11 Tridecane, the largest component of

43

BMSB stress compounds is an odorless compound. To the best of our knowledge

44

there is no information regarding any sensory effects of tridecane on wine quality.



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(E)-2-Decenal however is an aromatic compound with undesirable strong green,

46

coriander (or cilantro) and musty-like aromas.10 (E)-2-Decenal concentrations in

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Pinot noir at above 4.8 µg/L (consumer rejection threshold, CRT) result in a

48

significant drop in consumer preference, indicating a negative effect of this

49

compound on wine quality.10 Only wines made contaminated with BMSB contained

50

(E)-2-decenal and tridecane (data not shown).

51 52

The stress compounds produced by BMSB may be considered detrimental to wine

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quality10, as mentioned previously, and therefore should be minimized. While

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measures are being taken to control BMSB in vineyards, no treatment is completely

55

effective.1,12 Therefore post-harvest processes need to be managed to reduce any

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possible release of stress compounds into wine. This knowledge is very important to

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winemakers in devising quality control measures. Research to minimize multicolored

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asian lady beetle (MALB) taint has shown that wine processing can alter taint

59

associated compounds in the final wine, when it is not possible to remove the insects

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prior to grape processing.13 There is a good possibility that different wine processes

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will also impact release of stress compounds from BSMB in wine grapes. The

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objective of this study was to determine the wine processing steps responsible for

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BMSB stress compound release in both red and white wine, and to determine when

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levels of contamination in grape clusters reach the CRT of (E)-2-decenal (4.8 µg/L).

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There is little information available on post-harvest processing related to release of

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BMSB stress products and effect to final product quality. This work is the first to

67

investigate this aspect of BMSB contamination. The impact of BMSB was measured


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in terms of (E)-2-decenal, the main compound responsible for the BMSB odor and

69

tridecane, another BMSB stress compound. Additionally, the effect of dead BMSB

70

included during wine processing was also investigated.

71 72

Methods

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Chemicals

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HPLC/ACS-grade (E)-2-decenal, 3-octanol, and tridecane were purchased from

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Sigma Chemical Co. (St. Louis, MO, USA). n-Tetradecane-d30 was purchased from

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C/D/N Isotopes Inc. (Quebec, Canada). All compounds had a minimum purity of

77

98%. Milli-Q water was obtained from a Millipore Direct-Q® 5 UV-R water

78

purification system (EMD-Millipore, Billerica, MA, USA). Absolute ethanol (200-

79

proof) was obtained from Pharmco-AAPER (Vancouver, WA, USA) and sodium

80

chloride from VWR International LLC (PA, USA). Commercial wines used for

81

calibration curves and check standards were free of BMSB taint and include, 2010

82

Oregon Pinot noir, 2012 California Merlot and 2010 Oregon Pinot gris.

83 84

Brown Marmorated Stink Bug

85

BMSB were collected during August to September of 2013 and 2014 from various

86

locations throughout the Willamette Valley, Oregon. Insect ID was conducted using

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key characters for this species.14 Insects were stored in plastic cages at 20 oC and fed

88

organic fruits, vegetables and water. BMSB densities of 0.3, 1 and 3 per grape cluster

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were used to produce different wine treatments (Table 1). Twelve hours prior to wine

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processing, BSMB were chilled to 4.4 °C, counted and placed into plastic cages. This



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lower temperature slows metabolism, ensuring that all stress compounds released are

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due to wine processing. One hour prior to processing they were transported to the

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winery and brought to room temperature. BSMB for dead bugs were killed by

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freezing at minimum 24 hours prior to processing.

95 96

Grapes

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Vitis vinifera L. cv. Pinot noir, Pinot gris and Merlot grapes were hand-harvested

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from a small research vineyard, owned and managed by Oregon State University

99

(Monroe, OR, 44.3140°N, 123.2968°W). Pinot gris and Merlot wines were made in

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2013 and Pinot noir was made in 2013 and 2014. Grapes were inspected after harvest

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and found to be free of BMSB. Fruit was stored for 24 – 48 hours at 4 oC directly

102

after harvest.

103 104

Vinification

105

The different winemaking treatments include contamination with live BMSB, dead

106

BMSB, press type, and press fractions (Table 1). The number of BMSB per cluster

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was based on previously recorded BMSB levels in East coast vineyards 3,15. Standard

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commercial winemaking protocols (supplementary information) were used to make

109

white and red wine.

110 111

Sampling

112

Must and wine samples were collected after each processing step. Must parameters

113

are found in Table S1. A 40 ml sample was collected in 50 ml centrifuge tubes (VWR

114

International) during wine processing for stress compound analysis. Samples were


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centrifuged at 4200 rpm for 10 minutes (Allegra–22 Centrifuge, Beckman Coulter

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Inc.) and stored in 40 ml amber vials with polytetrafluoroethylene (PTFE) lined caps

117

(Sigma Aldrich) at -18 oC.

118 119

Additional wine treatments

120

Pressing

121

Differences in press types and press fractions were compared to determine their

122

impact on stress compound levels in the finished wines. We compared the stress

123

compound levels between Pinot noir using basket (2 bar for 1 min) and a bladder

124

press (1 bar for 2 min). To determine the effect of press fraction, Merlot wine was

125

processed using a bladder press (1 bar for 2 min). This wine was divided into its free

126

run and first press fractions. One third of the free run was processed separately from

127

the pressed fraction. The taint levels in the finished wine made from the press fraction

128

was calculated based on the final volume before removal of the free run.

129 130

Presence of dead BMSB

131

It is possible in vineyards that grape clusters chemically treated for pests may contain

132

dead BMSB. This study was conducted to determine if dead insects are able to release

133

stress compounds within wines during processing. For this reason, Pinot noir wine

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was made, per red wine vinification protocol, but used 0.3 dead BMSB per cluster in

135

place of live BMSB (Table 1).

136 137

Chemical Analysis



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Standard Wine Parameters

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Wine samples were analyzed for alcohol, pH, titratable acidity, reducing sugar, SO2

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(free and total) and malic acid. Alcohol percentage from 2013 and 2014 wines were

141

measured using an ebulliometer and density meter (Anton Paar DMA 4500 M-EC).

142

Titratabilew acidity was measured using titration method, pH was measured using a

143

pH meter, reducing sugar was measured using Rebelein method, and free and total

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SO2 was measured using the aspiration method.16 Malic acid was measured using

145

enzymatic kit (Vitiessential Laboratories, AU). Color and total phenolics were

146

measured by absorbance at 520 and 280 nm using an UV-3101fc spectrophotometer

147

(Shimazdu North America, Pleasanton, CA, USA).17 All samples were analyzed in

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triplicate.

149 150

BMSB taint analysis

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Sample preparation

152

Stock standard solutions and working standards of all (E)-2-decenal, 3-octanol,

153

tridecane and n-tetradecane-d30 were prepared as described in Tomasino et al.18

154 155

Sample preparation for wines and must

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All samples were prepared in a 20 mL amber vials (22.5 x 75.5 mm). 7.04 mL of

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Milli-Q water and 1.8 mL of wine sample was added to the vial to achieve a 5X

158

dilution. For juice samples, the water level was reduced to 5.24 mL and the alcohol

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level was adjusted by adding 1.8 mL of 14% (for red wine) or 11% (for white wine)

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ethanol solution, to ensure the ethanol concentration was the same in all vials. Sample


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dilution was followed by the addition of 80 µL of internal standard and 4.5 g sodium

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chloride. Vials were tightly capped with PTFE-lined caps and placed in a stack cooler

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attached to the Combi-Pal autosampler (CTC-Analytics, Zwingen, Switzerland). Vial

164

temperature was maintained at 8 oC prior to sample extraction. All samples, including

165

calibration curves, were run in triplicate.

166 167

SPME fibre, conditioning and sample extraction

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Headspace solid phase microextraction (HS-SPME) using stableflex

169

divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) combination

170

SPME fiber (50/30 µm thick, 2 cm long, 24 Ga, Sigma-Aldrich, USA) was used for

171

the measurement of BMSB stress compounds from must/wine. All new fibers were

172

incubated for 30 min at 250 oC before their first use.

173 174

Using a CTC Combi-Pal autosampler (CTC-Analytics AG, Switzerland) sample vials

175

were incubated at 40 oC and agitated simultaneously at 500 rpm (5 sec on, 2 sec off)

176

for 10 min. The SPME fiber was then inserted into the vial headspace for the

177

absorption of sample volatiles for 60 min at 40 oC with no agitation. The fiber was

178

injected into the first GC and compounds were desorbed at 250 oC for 10 min. The

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fiber was conditioned further in an NDL heater for 10 min at 250 oC.

180 181

Detection of (E)-2-Decenal and Tridecane by Heart-Cut Multidimensional Gas

182

Chromatography-Mass Spectrometry



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A gas chromatogram (GC, Shimadzu GC-2000 plus) was connected to a GCMS

184

(Shimadzu QP 2010) by a heated transfer line (Shimazdu North America, Pleasanton,

185

CA, USA). The GC in the first dimension contained a heart-cut dean’s switch

186

(Shimazdu North America, Pleasanton, CA, USA). The system was controlled using

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MDGCsolutions (Shimazdu North America, Pleasanton, CA, USA). The basic

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components of the system and operating parameters to measure (E)-2-decenal and

189

tridecane in wine are described below.

190 191

First dimension system: The column used in the first GC was an Rtx-wax column,

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30.0 m length, 0.25 mm ID and 0.5 mm film thickness (Crossbond®

193

Carbowax®polyethylene glycol, Restek Corporation, Bellefonte, PA, USA). The

194

carrier gas used was helium with the flow rate at a constant pressure of 169.5 kPa.

195

The injector was set in splitless mode. A flame ionize detector (FID) was set to

196

230 °C and used to determine that heart-cuts were consistent (no peaks in the

197

specified cut windows). The fiber was injected using the autosampler. The oven

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temperature was programmed as follows; 35 °C for 5 min, raised to 100 °C at a rate

199

of 10 °C/min and held for 10 min, increased again to 180 °c at a rate of 4 °C/min,

200

then increased to 250 °C at a rate of 10 °C/min and held at 250 °C for 10 minutes.

201

The heart cut windows used were from 12.5-15.5 min, 17.0-19.3 min, 20.0-25.0 min,

202

and 31.5-34.5min. The switching pressure used was 135 kPa.

203 204

Second dimension system: An Rxi-5MS column, 15 m x 0.25 mm ID x 0.5 m (100%

205

dimethyl polysiloxane, Restek Corporation, Bellefonte, PA, USA) was in the second


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oven and threaded through the heated transfer line and connected to the dean’s switch

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in the first system. The oven temperature program started at the same time as the first

208

dimension and is described as follows; 35 °C for 10 min, increased to 70 °C at a rate

209

of 3 °C/min held for 8 min, increased again to 115 °C at a rate of 3 °C/min and held

210

for 6 min, increased to 250 °C at a rate of 15 °C/min and held for 6 min. Flow rate in

211

this system was constant and held at 86.9 kPa.

212 213

The MS ion source and interface temperatures were 200 and 250 oC. The MS detector

214

was operated in electron impact mode (EI, 70 eV) under a scan mode (m/z 33-303

215

Da) at an absolute voltage of 0.8 kV. A detector gain of +0.6 kV was used for each of

216

the target compounds and isotopes. Compound identification was based on

217

comparison of pure standards and NIST11 (National Institute of Standards and

218

Technology) mass spectra library. Retention times, target ions and qualifier ions for

219

each compound are reported in Table 2.

220 221

Quantitation, Limit of Detection, Limit of Quantitation

222

Quantitation for (E)-2-decenal and tridecane was carried out using stable isotope

223

dilution analysis19. Limit of detection (LOD) and limit of quantitation (LOQ) are

224

described in Tomasino et al.18 Quantitation parameters for the two BMSB stress

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compounds are found in Table 2. White wine standards curves were measured in

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Pinot gris wines and the standard curve was a composite of samples run in both Pinot

227

noir and Merlot.

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Data Analysis

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Limit of Detection, Limit of Quantitation, Accuracy, Repeatability

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Calculations for LOD and LOQ were based those used by Callejón et al.20

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Recoveries of standard addition were estimated by running red and white base wines

233

with added reference standards. Percent recoveries were used to determine the

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accuracy of the measurments.18 Tridecane showed a low percent recovery in both red

235

and white wine (Table 3). Therefore a recovery factor, x4.94 for Pinot gris and x2.17

236

for Pinot noir and Merlot was applied to the final calculation as a correction. A low

237

solubility of tridecane in water and therefore in ethanol solution could be responsible

238

for its low percent recovery. Future work should focus on improving these parameters

239

for tridecane quantitation. (E)-2-Decenal measurements did not require any

240

adjustments as percent recovery was well within an acceptable range of 80-120%

241

(Table 3).21 During each run, a standard with (E)-2-decenal concentration of 3.22

242

µg/L and tridecane concentration of 6.45 µg/L was analyzed to ensure compound

243

stability.

244 245

Statistical Analysis

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All statistical analysis was run using XLSTAT-Pro 2014 (Addinsoft, New York, NY,

247

USA). Significant differences among BMSB wine treatments were measured using

248

one-way Analysis of Variance (ANOVA), student’s t-test and Tukey’s HSD.

249 250

Results and discussion

251

Basic chemical composition


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Wine chemical composition displayed a small variation among treatments but none

253

would be due to BMSB addition (Table 4). All wines reached dryness or desired

254

alcohol level. Some differences in ethanol content of the 2013 merlot were noted.

255

This is most likely due to the usage of the different press fractions for the treatments

256

versus the control. Different press fractions are known to alter juice and wine

257

composition.22 No differences were found for titratable acidity, phenolic content or

258

color (data not shown), but some differences were found for pH and residual sugar.

259

Residual sugar differences are most likely due to fermentation parameters. BMSB do

260

not absorb sugar and they cannot consume enough sugar to cause the differences

261

found. Additionally the differences in pH are unlikely to be from the additional of

262

BMSB. Research done investigating MALB in wine fermentations have shown that

263

insects did not alter any of the basic wine parameters.13

264 265

BMSB taint in white wine

266

Concentrations of (E)-2-decenal and tridecane were greatest after hard press (Figure

267

1). This was expected as BMSB would still be alive during pressing and may

268

continuously produce stress related compounds. However at finished wine only low

269

levels of tridecane, 9.69 µg/L, were measured in T2. (E)-2-decenal appeared to be

270

completely removed during alcoholic fermentation. This is most likely due to

271

compounds being driven off by carbon dioxide, (E)-2-decenal reacting with SO2 or

272

conversion to its corresponding alcohol n-decanol during fermentation.23–25 Fiola9

273

also reported loss of BMSB stress compounds post fermentation. Sensory analysis

274

comparing Pinot gris with and without 9.7 µg/L of tridecane were not found to be



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different.26 This suggest that at the tested concentration, tridecane does not appear to

276

effect white wine quality. Therefore the risk of BMSB contamination affecting white

277

wine is low, as the critical processing step, pressing which was responsible for the

278

greatest amounts of stress compounds released, occurs prior to fermentation.

279 280

(E)-2-Decenal and tridecane in red wine

281

As anticipated, the concentration of both (E)-2-decenal and tridecane increased with

282

an increase in BMSB density (Table 5, Figure 2). Destemming/crushing produced the

283

greatest concentrations of (E)-2-decenal (Table 5) and pressing produced the greatest

284

concentrations of tridecane (Figure 5). As with white wine, both compounds were

285

greatly reduced during fermentation but pressing after fermentation resulted in an

286

increase in both stress compounds. It is thought that compounds were reduced due to

287

compounds being driven off by carbon dioxide rather than the other two possible

288

mechanisms mentioned previously, as the winemakers could distinctly smell BMSB

289

stress compounds when mixing the cap with the fermenting juice (known as punch

290

downs). All final treatment wines contained tridecane and the 2014 Pinot noir T3

291

wine also contained (E)-2-decenal in the finished wine. The presence of BMSB stress

292

compounds in the red wines after pressing is of concern given the minimal decrease

293

of the taint compounds in the wine post-pressing and the low reactivity of BMSB

294

stress compounds in the wine matrix. For example, (E)-2-decenal levels decreased by

295

only a small amount after pressing in comparison to a large reduction after

296

destemming. Pressing has been found to be more problematic than destemming for

297

other taints, such as MALB.27 Pressing parameters and the use of press fractions are


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known to alter the composition of juice and wine, including sugar content,22 phenolic

299

content28,29 and varietal aromas.30,31 Due to the high significance of pressing for

300

BMSB stress compounds in red wine the study was expanded to specifically

301

investigate different pressing options.

302 303

The effect of press type

304

At a density of 3 BMSB/cluster, the use of a bladder press versus a basket press

305

produced greater concentrations of tridecane (Figure 3). A similar result was observed

306

at BMSB density of 0.3 bugs/cluster (data not shown). These preliminary findings

307

suggest that taint levels in the finished wine can be minimized by using different

308

presses. A bladder press presses the grapes from the center of the press outwards

309

while a basket press presses the grapes from the top and bottom. Both of these presses

310

have a different application of pressure which can alter final composition. Different

311

press types have been found to alter the amount of varietal aromas in juice and

312

wine.32 The application of greater or more consistent pressure appears to crush more

313

berries and BMSB which results in a greater extraction of wine and stress

314

compounds.

315 316

It should be noted that in this study the basket and bladder press were operated at

317

different pressures and times. This was due to the size of each press, with the basket

318

press being smaller than the bladder press. Therefore our results may not accurately

319

represent larger commercial sized basket or bladder presses.

320



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The effect of press fraction

322

Press fractions in red wine making are often kept separate to achieve a desired

323

composition.22,33 Certain wines, such as champagne or sparkling wines, are produced

324

from free run, without any press fraction.34 The levels of BMSB taint in free run

325

versus press fraction after alcoholic fermentation was of interest, as blending may

326

provide a solution to reduce BMSB compounds in wine. Our results show that free

327

run wines contained significantly less tridecane compared to press fraction wines (α =

328

0.05) (Figure 3). This was anticipated since pressing typically crushes the BMSB,

329

releasing more stress compounds. The use of free run and elimination of pressing step

330

is recommended for highly contaminated grape clusters and investigating blends of

331

free run and press fractions may also result in low to no BMSB stress compounds in

332

the final wine.

333 334

The impact of dead BMSB

335

Tridecane levels from wines made with dead BMSB at a density of 0.3 bugs/cluster

336

had (E)-2-decenal concentrations below the HS-SPME-MD-GCMS LOD in final

337

wine. Finished wine contained 7.74 µg/L of tridecane which was significantly lower

338

than the tridecane concentration of 26.88 µg/L, found in Pinot noir made from live

339

BMSB at the same density. Unfortunately due to a lack of bugs that year it was not

340

possible to test higher bug densities. It is anticipated that the greater the

341

BMSB/cluster the amount of stress compounds would increase. The result of this

342

treatment indicates that killing BMSB in the field may not be enough to get rid of

343

BMSB taint compounds from the wine. However, this would only be true if the


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BMSB were in a stressful situation prior to death (such as agitated due to machine

345

harvesting). If the stress compounds were not produced prior to death, then crushing

346

of dead BMSB into wine would result in little to no release of stress compounds.

347 348

BMSB threshold in grape clusters

349

We did not calculate an economic or action threshold for white wine since BMSB

350

taint does not appear to be problematic, as final wine did not contain any (E)-2-

351

decenal and in a sensory tests, the amount of tridecane was not found to alter aroma

352

perception.26 A single threshold limit is reported for both Pinot noir and Merlot since

353

(E)-2-decenal CRT in these wines is similar.26 A conservative approach has been

354

adopted for reporting BMSB threshold density of 3 BMSB/cluster due to the negative

355

effect of its taint compounds on wine quality, the variation caused by press type, and

356

the non-linear relationship between the amount of taint released and BMSB densities

357

in grape clusters. BMSB tolerance limit is estimated to be 3 bugs per cluster, resulting

358

in wine with 2 µg/L (E)-2-decenal. Additional BMSB treatments would need to be

359

researched to determine a specific number of bugs/cluster, with corresponding

360

pressing technology, resulting in 4.8 µg/L of (E)-2-decenal or greater. Higher taint

361

levels are expected during industrial pressing operation utilizing a heavier press type

362

and longer pressing time. Therefore, an upper threshold of 3 BMSB per cluster should

363

maintain (E)-2-decenal level in wine below its CRT. Consumer preference may still

364

be effected at this low level of (E)-2-decenal. A previous study has shown that 28%

365

of the consumer panel exhibits low preference towards (E)-2-decenal and therefore

366

could reject Pinot noir containing (E)-2-decenal below 0.05 µg/L.10



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367 368

The BMSB threshold levels from this study are higher than previously reported

369

threshold of 10-20 BMSB per 25 lb lugs (0.1-0.2 per cluster).9,35 This disparity is

370

most likely due to differences in wine processing. These threshold limits were based

371

on wines in which BMSB was removed prior to pressing. We have clearly shown that

372

the BMSB taint in must decreases during fermentation and that it is pressing after red

373

wine fermentation that is the critical processing point.

374 375

This study provides the first estimates of the BMSB threshold limit of 3

376

BMBS/cluster. BMSB threshold levels can be applied to guide a pesticide application

377

program in the vineyard. Additionally, it provides a valuable reference point for

378

future research on this taint. BMSB appears to be problematic to wine quality if the

379

insects are present during red wine processing, as pressing is the critical step for

380

release of stress compounds into wine at concentrations that would impact aroma

381

perception. We do not see any BMSB taint problems associated with white or rose

382

wines because pressing occurs before fermentation. However, the effect of tridecane

383

to red wine quality should be investigated. While this compound is known to be

384

odorless it may still effect wine quality through interactions with other wine

385

compounds.36

386 387

While present levels of BMSB found in vineyards do not appear great enough to alter

388

the quality of red wines, BMSB have been found in vineyards throughout the wine

389

grape growing season with levels has high as 18 bugs counted in a 3 minute time


Page 19 of 33


390

period.3 Therefore it is entirely plausible that some vineyards will experience 3

391

BMBS/cluster levels in the near future. Outcomes from this work provide important

392

information for winemakers to make informed decisions to reduce the effect of

393

BMSB stress compounds on final wine quality. It also provides an action threshold of

394

3 BMSB/cluster to vineyard managers to devise control measures.

395 396

Additionally, results from this study are not only important for wine quality but also

397

suggest that other agricultural products may experience problems due to BMSB

398

should they have the insects present with the fruit or vegetable during any post-

399

harvest processing. Food products that may be particularly affected include those that

400

have a high level of processing, such as puree and juice production, using crops that

401

are known to be hosts to BMSB.



Page 20 of 33 20

402 403

Funding Sources This work was supported by the National Institute of Food and Agriculture, U.S.

404

Department of Agriculture, [SCRI#2011-51181-30937] and the U.S. Department of

405

Agriculture, Northwest Center for Small Fruits [OWB# 4040-0001].

406 407

Supporting Information/Associated Content

408 409

This material is available free of charge via the Internet at http://pubs.acs.org.

410

Vinification details for both red and white wines.

411

Table S1. Must composition for Pinot noir, Pinot gris and Merlot before and after

412

sugar and acid adjustments made during year 2013 and 2014

413 414


Page 21 of 33


415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459

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(9) (10)

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(13)

(14)

Leskey, T. C.; Hamilton, G. C.; Nielsen, A. L.; Polk, D. F.; Rodriguez-Saona, C.; Bergh, J. C.; Wright, S. E. Pest Status of the Brown Marmorated Stink Bug, Halyomorpha Halys in the USA. Outlooks Pest Manag. 2012, 23, 218–226. Callot, H.; Brua, C. Halyomorpha halys (Stål, 1855), the marmorated stink bug, new species for the fauna of France (Heteroptera Pentatomidae). L’Entomologiste 2014, 69, 69–71. Basnet, S.; Kuhar, T. P.; Laub, C. A.; Pfeiffer, D. G. Seasonality and Distribution Pattern of Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) in Virginia Vineyards. J. Econ. Entomol. 2015, 108, 1902– 1909. Lee, D.-H. Current status of research progress on the biology and management of Halyomorpha halys (Hemiptera: Pentatomidae) as an invasive species. Appl. Entomol. Zool. 2015, 50, 277–290. Nielsen, A. L.; Hamilton, G. C. Seasonal Occurrence and Impact of Halyomorpha halys (Hemiptera: Pentatomidae) in Tree Fruit. J. Econ. Entomol. 2009, 102, 1133–1140. Nielsen, A. L.; Hamilton, G. C. Life History of the Invasive Species Halyomorpha halys (Hemiptera: Pentatomidae) in Northeastern United States. Ann. Entomol. Soc. Am. 2009, 102, 608–616. Nielsen, A. L.; Hamilton, G. C.; Shearer, P. W. Seasonal Phenology and Monitoring of the Non-Native Halyomorpha halys; (Hemiptera: Pentatomidae) in Soybean. Environ. Entomol. 2011, 40, 231–238. Baldwin VI, R. L.; Zhang, A.; Fultz, S. W.; Abubeker, S.; Harris, C.; Connor, E. E.; Van Hekken, D. L. Hot topic: Brown marmorated stink bug odor compounds do not transfer into milk by feeding bug-contaminated corn silage to lactating dairy cattle. J. Dairy Sci. 2014, 97, 1877–1884. Fiola, J. A. Brown Marmorated Stink Bug (BMSB) Part 3 - Fruit Damage and Juice/Wine Taint. Timely Viticulture. 2011. Mohekar, P.; Lapis, T. J.; Wiman, N. G.; Lim, J.; Tomasino, E. Brown Marmorated Stink Bug Taint in Pinot noir: Detection and Consumer Rejection Thresholds of trans-2-Decenal. Am. J. Enol. Vitic. 2017, 68, 120–126. Mohekar, P.; Tomasino, E.; Wiman, N. G. Defining defensive secretions of brown marmorated stink bug, Halyomorpha halys. In Annual Meeting of the Entomological Society of America; Minneapolis, MN, 2015. pp. 164 Leskey, T. C.; Short, B. D.; Lee, D.-H. Efficacy of insecticide residues on adult Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) mortality and injury in apple and peach orchards: Residual insecticide efficacy on Halyomorpha halys. Pest Manag. Sci. 2014, 70, 1097–1104. Pickering, G. J.; Ker, K.; Soleas, G. J. Determination of the critical stages of processing and tolerance limits for Harmonia axyridis for “ladybug taint” in wine. Vitis 2007, 46, 85–90. Hoebeke, E. R.; Carter. Halyomorpha halys (Stål) (Heteroptera: Pentatomidae): a polyphagous plant pest from Asia newly detected in North



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Page 24 of 33 24

Figure Captions

Figure 1. BMSB stress compound concentrations throughout Pinot gris processing, a) (E)-2-decenal (□) in T2 and (□)T1; b) Tridecane (■) in T2 and (■) T1 Figure 2. Tridecane concentrations throughout Pinot noir processing (■) in 2014 T3, (■) 2014 T1, (■) 2014 PN with dead BMSB T1, and (■) 2013 T2 Figure 3. Differences in tridecane concentration in 2014 Pinot noir T3 using (■) bladder press, 2013 Pinot noir T3 using (■) basket press, and in Merlot T2 using (□) press fraction and (□) free run.


Page 25 of 33


Table 1. Vintage, grape and treatment details for red and white wines.

Year

Wine grape

Number of BMSB per treatment (per Kg of grapes) Control

T1*

T2

T3

(0 BMSB/ cluster)

(0.25 or 0.3 BMSB/ cluster)

(1 BMSB/ cluster)

(3 BMSB/ cluster) 30

2013

Pinot noir

0

3

8

2013

Pinot gris

0

2.5

10

2013

Merlot

0

̶

̶

2013

Merlot (press fraction)

2013

Merlot (free run)

2014

Pinot noir

2014

Pinot noir with dead BMSB

̶ ̶ 0

3

6.6 ̶

Press type

3

2.5

Basket

3

6.1

Bladder

3

3.0

Basket

2

18.0

Bladder

̶

2

18.0

Bladder

30

2

30.0

Bladder

3

30.0

Bladder

̶ ̶

6.6

Batch Number of size replicates (Kg)

̶

3

*In Pinot gris, T1 corresponds to 0.25 BMSB per cluster. In rest of the wine treatments T1 corresponds to 0.3 BMSB per cluster.



Page 26 of 33

26

Table 2. Quantitation parameters for HS-SPME-MDGC-MS analysis of BMSB stress compounds.

Analyte

CAS No.

Purity (%)

Retention Target time

Ions

(min)

m/z

Confirming ions m/z

Standard

Standard

Calibration

curve

curve

range

(R2)

(R2)

(µg/L)

Red

White

Wine

Wine

Tridecane

629-50-5

99.0

37.67

57

43, 71, 85

0 ̶ 38.73

99.32

99.85

(E)-2-decenal

3913-81-3

95.0

40.28

55

70, 98

0 ̶ 19.37

99.79

98.86

3-Octanol

20296-29-1

99.0

25.05

59

55, 83

n-Tetradecane-d30

204244-81-5

98.5

42.24

66

50, 82


Page 27 of 33


27

Table 3 Limit of detection, limit or quantitation and spike recoveries for (E)-2-decenal and tridecane using HS-SPME-MDGC-MS

(E)-2-decenal

Tridecane

LOD (µg/L)

LOQ (µg/L)

Recovery (%)

LOD (µg/)

LOQ (µg/L)

Recovery (%)

White wine

0.02

0.07

90.60

0.02

0.06

20.24

Red wine

0.01

0.03

94.52

0.01

0.03

46.00



Page 28 of 33

28

Table 4. Significant differences (α=0.05) found in basic chemical parameters for finished wines. Alcohol (% v/v)

pH

Residual Sugar (g/L)

2013 PG Control

11.4 ± 0.2a

3.1 ± 0.0a

21.5 ± 1.5a

2013 PG Treatment 1 (0.25 BMSB/ cluster)

11.8 ± 0.4a

3.1 ± 0.0a

17.5 ± 3.9a

2013 PG Treatment 2 (1 BMSB/ cluster)

11.8 ± 0.1a

3.1 ± 0.0a

22.2 ± 0.3a

2013 PN Control

14.0 ± 0.2b

3.7 ± 0.0a

1.6 ± 0.1a

2013 PN Treatment 1 (0.3 BMSB/ cluster)

14.5 ± 0.5a

3.7 ± 0.0b

1.7 ± 0.2a

2013 PN Treatment 2 (1 BMSB/ cluster)

14.8 ± 0.2a

3.7 ± 0.0a

1.7 ± 0.4a


14.5 ± 0.1a

3.7 ± 0.0a

1.4 ± 0.4a

2013 Merlot Control

14.4 ± 0.5a

3.5 ± 0.0a

2.0 ± 0.1ab

2013 Merlot Press Treatment 1 (1 BMSB/ cluster)

12.9 ± 0.0b

3.5 ± 0.0a

1.9 ± 0.1b

2013 Merlot Free Run Treatment 1 (1 BMSB/ cluster)

12.9 ± 0.0b

3.5 ± 0.1b

2.1 ± 0.0ab

2014 PN Control

13.5 ± 0.2bc

3.8 ± 0.1a

1.1 ± 0.1ab

2014 PN Treatment 1 (0.3 BMSB/ cluster)

13.7 ± 0.1ab

3.6 ± 0.0b

1.4 ± 0.1a


13.1 ± 0.1c

3.7 ± 0.0ab

1.8 ± 0.6b

2014 PN Treatment (0.3 Dead BMSB per cluster)

14.1 ± 0.1a

3.8 ± 0.0a

1.3 ± 0.0a


Page 29 of 33


29

Table 5 (E)-2-Decenal (µg/L) concentrations in Pinot noir wines from 2013 and 2014 at different wine processing stages for 2 different BMSB treatment levels.

Treatment

0.3BMSB/cluster (T1)

3BSMB/cluster (T3)

Vintage

2014

2013

2014

Destemming

3.9±0.3

nd*

95.0±14.0

Cold Soak

2.0±0.1

nd

10.5±2.0

End of fermentation

nd

nd

2.0±0.1

Pressing

2.0±0.0

2.0±0.1

9.5±1.6

End of MLF

nd

2.0±0.1

2.2±0.0

Finished wine

nd

nd

2.0±0.1

*nd=none detected



Page 30 of 33

30

700 30

500 20

400 300

10

200 100

0

0 Destemming

Soft Press

Hard Press

Settling

End of fermentation

Figure 1.


Finished wine

Tridecane concentration (µg/L)

(E)-2-decenal concentration (µg/L)

600

Page 31 of 33


31


4500.0 3750.0 3000.0 2250.0 1500.0 750.0 0.0 Destemming

Cold Soak

End of fermentation

Pressing

Red wine processing steps Figure 2.


End of malolactic fermentation

Finished wine


Page 32 of 33

32


750

a

500 c 250

d

b 0 2014 Pinot noir 2013 Pinot noir (Bladder press) (Basket Press)

Figure 3.


2013 Merlot (Press fraction)

2013 Merlot (Free run)

Page 33 of 33


Graphic for Table of Contents


Influence of Winemaking Processing Steps on the Amounts of (E)-2

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