Investigation on Key Molecules of HLB Induced Orange Juice Off-flavor

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Investigation on Key Molecules of HLB Induced Orange Juice Off-flavor Johannes Kiefl, Birgit Kohlenberg, Anja Hartmann, Katja Obst, Susanne Paetz, Gerhard E. Krammer, and Stephan Trautzsch J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00892 • Publication Date (Web): 11 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Journal of Agricultural and Food Chemistry

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Investigation on Key Molecules of HLB Induced Orange Juice Off-flavor

Johannes Kiefl1*, Birgit Kohlenberg1, Anja Hartmann1, Katja Obst1, Susanne Paetz1, Gerhard Krammer1, Stephan Trautzsch1

1

Symrise AG, Flavors Division Research & Technology, P.O. Box 1253, D-37601

Holzminden, Germany

*

Author to whom correspondence should be addressed:

Phone:

+49 (0) 5531/901099

E-Mail:

[email protected]

ACS Paragon Plus Environment


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ABSTRACT

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Orange fruits from Huanglongbing (HLB) infected trees do not fully mature and show

3

a severe off-flavor described as bitter-harsh, metallic and less juicy and fruity. The

4

investigation of juice from HLB infected (HLBOJ) and healthy control oranges (COJ)

5

by gas chromatography mass spectrometry showed higher concentrations of fruity

6

esters such as ethyl butyrate and ethyl 2-methylbutyrate, and soapy-waxy alkanals

7

such as octanal and decanal in the COJ whereas the HLBOJ showed higher

8

concentrations of green aldehydes like hexanal and degradation compounds of

9

limonene and linalool like α-terpineol. Application of aroma extract dilution analysis

10

on terpeneless peel oil led to the identification of long-chained aldehydes such as

11

(E,E)-2,4-decadienal,

12

decenal and octanal with the highest flavor dilution factors among 25 odor-active

13

volatiles in the peel oil of healthy oranges.

14

Taste guided fractionation and identification of the HLBOJ secondary metabolites

15

followed by sensory validation revealed that flavanoids such as hesperidin may

16

modulate the flavor to evoke the unacceptable harsh, metallic taste impression.

17

Quantitation of the bitter components showed good correlation between the limonoid

18

and flavanoid concentration with the off-flavor and quality of the oranges obtained

19

throughout the season.

(Z)-8-tetradecenal,

trans-4,5-epoxy-(E)-2-decenal,

20 21

KEYWORDS

22

Huanglongbing (HLB), Citrus Greening, Citrus sinensis, off flavor, gas

23

chromatography olfactometry, LC Taste®, limonin, flavanoids


(Z)-4-

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INTRODUCTION

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The Florida orange industry is facing one of the most serious challenges in

26

recent decades. The orange crop is reported to be continuously decreasing from

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104.7 million boxes in 2013-2014 to 96.8 million boxes in 2014-2015 and down to

28

81.6 million boxes in the 2015-2016 season.1,

29

infection of orange trees with the bacteria Candidatus Liberibacter ssp. which is

30

transmitted by the Asian citrus psyllid, Diaphorina citri.3 Orange fruits from infected

31

trees may drop before harvest and do not get ripe thus showing a severe off-flavor

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described as bitter, harsh, metallic and less juicy and fruity.4, 5 Even after sorting out

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infected small fruits before juicing, a reasonable amount of infected regular sized

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fruits can enter the process sufficient to negatively impact the flavor.

35

2

This decline is caused by the

Approaches to prevent HLB infection by psyllid control and spraying of

36

insecticides,6,

7

nutritional foliar supplementation8,

9

37

spraying antibiotics10 are being intensely studied at the moment. These approaches

38

aim at preventing the crop decline while increasing the costs for grove management.

39

At the same time, optimization of the juice sensory quality is not targeted.

and curing of infected trees by

40

The development of resistant rootstocks is a promising field of research,11

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however, recultivation of Florida orange groves with resistant rootstocks is estimated

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to take 5-10 years. Orange trees are genetically modified to develop resistance as

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well, however, regulation issues arise with selling this juice and byproducts from juice

44

production, for example, to European markets. Therefore, no short-term solution is

45

available to stop crop decline and to remove the off-flavor of orange juice and

46

products thereof yet.

47

Sensory analysis of fruits obtained in the 2007 and 2008 season in Florida

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revealed that juice from infected trees is lower in orange, fatty and fresh flavor notes

49

and sweet taste but higher in bitter, metallic, pungent/peppery, salty/umami and ACS Paragon Plus Environment


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astringent flavor notes and taste. This was found to be more significant for juice

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produced from symptomatic fruits of early season Hamlin rather than late season

52

Valencia.12 Thereby, juice from healthy Hamlin oranges was higher in aldehyde

53

volatiles such as acetaldehyde and octanal, higher in (Z)-3-hexenol and higher in

54

esters such as ethyl acetate and ethyl butanoate.4 Juice from healthy and infected

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Valencia oranges showed no significant differences in volatiles.4 Similar results were

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obtained by Dagulo et al. who used fruits from the same seasons and concluded that

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HLB infection might be the major cause for the higher concentrations of terpenes and

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alcohols and lower levels of esters such as ethyl acetate, ethyl butanoate, methyl

59

hexanoate, ethyl-3-hydroxyhexanoate and sesquiterpenes such as valencene in

60

symptomatic juices.13

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Lower amounts of sugars and higher amounts of citric acid and limonin were

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found to have a major effect on the flavor of juice from infected fruits.4, 13 Limonin is

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known to cause delayed bitterness in sweet orange juices originating from the

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conversion of the tasteless precursor limonate-A-ring lactone in the acidic juice

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matrix in the presence of heat or enzyme limonin-D-ring-hydrolase (EC 3.1.1.36).14-16

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During maturation limonin could be converted into its tasteless precursors down to a

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residual level of limonin below 2.6 mg/L where bitter taste is not perceived.5

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Therefore, limonin is an important quality parameter and is routinely analyzed in the

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citrus industry. The role of nomilin as bitter taste enhancing component was recently

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highlighted and nomilin (8.4 mg/L bitter threshold) as well as limonin (7.0 mg/L bitter

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threshold) acted synergistically in a model solution by lowering the recognition

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threshold to 3.9 mg/L (1:1 mixture). In juice, nomilin at 2 mg/L subthreshold

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concentration reduced the limonin recognition threshold to 2.6 mg/L.5 However, in

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juice from oranges of infected trees additional off-flavors not associated with limonin

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or nomilin occur.12,

17

Little information is available on those additional off-flavor ACS Paragon Plus Environment

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compounds because current studies are focusing more on known taste components

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and sensory guided screening approaches like gas chromatography olfactometry or

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LC Taste®18 are scarcely applied.

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Commercial orange juice “not-from-concentrate” is usually made by blending

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juice from different varieties such as the early season Hamlin with a more acidic and

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green flavor profile and late season Valencia having a sweeter and overripe flavor

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profile. Blending up to 25 % juice of HLB infected fruits with less than 2.5 mg/L

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limonin with juice of healthy fruits from 2009 season did not perceivably change the

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flavor.17 However, this way of blending is limited by the availability of juice from

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healthy fruits which decreases year by year. Therefore, this approach is already self-

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limiting because limonin levels in early Hamlin juice are continuously increasing19 and

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at the same time the sugar level in late Valencia decreases which prompted us to

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study the off-flavor in more detail during the season 2014 and 2015. It was the aim to

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investigate the key flavor molecules of orange juice from healthy and infected fruits

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by sensory guided analysis of the peel and juice volatiles and non-volatiles in order to

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develop strategies for compensating juice derived off-flavor notes.

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MATERIAL AND METHODS

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Materials. The following reference compounds were obtained in the highest

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available grade of purity: hesperidin, naringenin, liquiritin, and citric acid (Sigma-

96

Aldrich,

97

Vestenbergsgreuth, Germany) and sucrose (Fluka, Neu-Ulm, Germany). Nomilin,

98

tangeretin, nobiletin, eriocitrin and limonin glucoside were gifts from Dr. Baldwin and

99

Dr. Manthey (USDA-ARS, Florida, USA).

Steinheim,

Germany);

limonin,

poncirin

and

didymin

(Phytolab,

100

LC/MS grade acetonitrile and formic acid were from Fluka (Neu-Ulm,

101

Germany) and sulfuric acid was obtained from Sigma-Aldrich (Steinheim, Germany).

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Deionized water was prepared by an arium mini UV lab water system (Sartorius,

103

Göttingen, Germany). Silica gel for column chromatography (silica gel 60, 63−

104

200 µm) was obtained from Merck KGaA (Darmstadt, Germany).

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Orange juices were spiked with hesperidin (> 98 %) obtained from Blue

106

California Ingredients (Rancho Santa Margarita, USA), a polymethoxylated flavone

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extract from orange containing 1.32 % tangeretin and 28.6 % nobiletin (Miritz,

108

Kirchgandern, Germany) and limonin and poncirin obtained from Phytolab (> 98 %

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and > 95 % respectively, Vestenbergsgreuth, Germany) for sensory evaluation. Stock

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solutions of these compounds of 1-10 % concentration in 1,2-propanediol and

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ethanol mixtures were prepared at 50 °C and finally diluted in juice.

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Hamlin and Valencia oranges (Citrus sinensis (L.) Osbeck) were collected on

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a monthly basis from commercial groves in southern Florida during the season 2014

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and 2015. The trees were selected based on visual symptoms: blotchy-mottle leaves,

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twig die back and small fruits with diameter less than 6 cm were characteristic for

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HLB infected symptomatic trees.3 Regular sized fruits from HLB infected symptomatic

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trees were classified as asymptomatic and the other small, lopsided fruits as

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symptomatic HLB fruits. Fruits were taken from the same number of healthy and ACS Paragon Plus Environment

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infected trees in a random manner. The fruits were washed, hand juiced and lightly

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pasteurized using a dish washer with 71 °C for 15 s. Before squeezing fruits were

121

checked for aborted seeds which is another criteria for classification of symptomatic

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fruits.3

123 124

Preparation and analysis of terpeneless peel oil. The contribution of

125

hydrocarbon terpenes such as limonene, myrcene and pinenes to odor perception in

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juice is limited because of their high odor thresholds.20 For this reason, column

127

chromatography was used to reduce hydrocarbons from 2 g cold pressed peel oil.

128

Chromatography was performed on a 45 cm × 40 mm i.d. glas column filled with

129

silica gel. First, the column was loaded and then eluted with 1 L of 100 % pentane

130

and finally rinsed with 1 L of 100 % diethyl ether. The pentane fraction was discarded

131

and the diethyl ether fraction with a concentration of limonene below 400 ppm was

132

used for GC-MS analysis after concentration to 1 mL by Turbovap evaporation

133

(Turbovap II, Biotage, Sweden). One microliter of the sample solution was injected

134

with a Gerstel MPS XL autosampler (Gerstel, Muelheim, Germany) in a cooled

135

injection system containing an empty glass liner and then desorbed at 180 °C in

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splitless injection mode. GC-MS analysis was performed as described in sensory

137

guided analysis of volatiles part.

138 139

Preparation of orange juice extract. Twenty liter juice from HLB

140

symptomatic fruits was centrifuged and processed by solid phase extraction. A

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column with 250 mm × 25 mm size filled with polystyrene resin was rinsed with the

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juice and medium polar components were trapped. The loaded column was washed

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with water to remove residual polar components such as sugars and organic acids

144

and was then eluted with ethanol. The eluate was concentrated by evaporation to ACS Paragon Plus Environment


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dryness and finally freeze dried to obtain 19 g extract. The pulp obtained by

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centrifugation and the polar and medium polar eluates were evaluated by five trained

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panelists on sucrose and citric acid taste solution and the medium polar fraction

148

showed highest bitter taste intensity.

149 150

Juice basic chemical parameters. The basic chemical parameters such as

151

sucrose, total soluble solids (°Brix), Scott oil and citric acid of the juice were

152

determined as follows: sucrose was quantified via an external standard method using

153

an Agilent 1100 series HPLC system (Agilent, USA) comprising a binary pump,

154

autosampler and RI detector. Chromatographic separation was carried out at 80 °C

155

on a Grom Resin ZP column (250 mm x 8 mm, 8 µm, Dr. Maisch, Germany) in

156

isocratic mode with an aqueous mobile phase. Flow rate was 0.4 mL/min and

157

injection volume was 10 µL. The Brix value was determined with an Abbe

158

refractometer at 20 °C (Atago, Tokyo, Japan). Citric acid was quantified via an

159

external standard method using an Agilent 1290 Infinity HPLC system (Agilent, USA)

160

comprising a binary pump, autosampler and DAD detector. Chromatographic

161

separation was carried out at 40 °C on an Aminex HPX-87H column (300 mm x

162

7.8 mm, 9 µm, Bio Rad, USA) in isocratic mode with 0.004 M sulfuric acid water.

163

Flow rate was 0.8 mL/min and injection volume was 10 µL. The eluent was monitored

164

at 210 nm. Peel oil content was quantified by the Scott bromate titration method.21

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Briefly, 25 g of the juice sample and 25 g of ethanol were distilled with a few boiling

166

stones until the solvent ceased to reflux. Then, 10 mL of 4 N HCl with a drop of 0.1 %

167

methyl red indicator were added and peel oil content was determined by titrating the

168

distilled fraction with 0.025 N bromide-bromate solution until the color disappeared.

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Sensory guided analysis of volatiles. Extraction of juice components was

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performed using Gerstel® PDMS Twister with 1 mm layer thickness and 10 mm

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length (Gerstel, Muelheim, Germany). One gram of juice was weighed in a 10 mL

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headspace vial and 5 mg/kg of 2-nonanol as internal standard was added and the

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vial was finally filled with 10 grams of deionized water. The Twister was added to this

175

solution and stirred at room temperature maintained at 20 °C for one hour. The

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Twister was then taken out of the solution and rinsed with few milliliters of deionized

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water and carefully dried with clean paper towels.

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Sample volatiles were transferred to the GC by desorbing the Twister at

179

180 °C for 8 min in the Gerstel® thermal desorption unit (TDU), then the analytes

180

were cryofocussed at -50 °C in the cooled injection system inlet containing a Tenax

181

filled glass liner and then desorbed at 200 °C for 10 min onto the GC column, where

182

the analytes were separated on a ZB-Wax plus column (60 m x 0.32 mm, 0.25 µm,

183

Phenomenex, Torrance, California, USA) placed in a Agilent 6890A gas

184

chromatograph (Waldbronn, Germany) using helium as the carrier gas.

185

The effluent was transferred by a Graphpack®-3D/2 sulfonated crosspiece

186

(Gerstel, Muelheim, Germany) into three deactivated fused silica capillaries directed

187

to a sniffing port set at 250 °C (ODP 3, Gerstel, Germany), FID set at 300 °C, and

188

MS (Agilent 5973 MSD) for simultaneous detection. A constant flow of 2 mL/min was

189

applied. The GC temperature was programmed starting at 40 °C, held for 2 min, then

190

raised at 4 °C/min to 240 °C, and held for 38 min. Retention indices were calculated

191

by cochromatography of a homologous series of n-alkanes from C6-C30. The effluent

192

was transferred to the MS via a 2.2 m deactivated capillary column held at 280 °C

193

and the MS was set in scan mode (EI ionization 70 eV, m/z 25 – 400, 3.4 scans/sec)

194

The volatile analytes were identified by matching the retention index (constraint of ±5

195

set for positive match) and mass spectrum (constraint of >800/1000 match factor set ACS Paragon Plus Environment


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for positive match) with an in-house database which was built by analysis of

197

authentic references on standard instruments with defined protocols. The peak area

198

of 2-nonanol obtained by the FID signal was used for peak response normalization.

199

Data were acquired by Agilent MSD ChemStation version E.02.02.

200

Aroma extraction dilution analysis22 of the cold pressed and terpeneless peel

201

oils was performed as follows: the oil was stepwise diluted 1:1 with diethyl ether, and

202

aroma-active compounds were located by sniffing each dilution (HRGC-O) and by

203

calculating retention indices on a ZB-Wax plus column (60 m x 0.32 mm, 0.25 µm)

204

(Phenomenex, Torrance, California, USA). Evaluation of the dilutions was performed

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until no odorant could be sniffed in the diluted extract and the flavor dilution (FD)

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factor was, thus, denoted for each compound. The extract with FD 1 was sniffed by

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three trained assessors independently to define a list of odor active components.

208 209

LC Taste® fractionation18 and taste dilution analysis. Orange juice bitter

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components were studied by chromatographic fractionation of an extract of HLB

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infected fruits followed by taste analysis of three sequential dilutions. LC Taste®

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fractionation was performed using high temperature liquid chromatography (HTLC)

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on a polymer-based PRP-1 column in a semipreparative scale (250 mm × 10 mm;

214

10 µm particle size; Hamilton, Bonaduz, Switzerland) at elevated temperature (80 °C

215

isotherm) and detection on a diode array detector (SunChrom SpectraFlow;

216

wavelength range 200-550 nm, SunChrom, Friedrichsdorf, Germany). The mobile

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phase consisted of water (A) and ethanol (B) and a gradient elution of 100 % A at

218

0 min to 25 % B in 25 min to 100 % B at 25-40 min was used. Flow rate was

219

10 mL/min, injection volume 1 mL and the eluent was monitored at 230 nm.

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The fractions were dissolved in 1,2-propanediol and diluted in sucrose 3 %

221

and citric acid 0.1 % taste solution and sequentially diluted with taste solution 1:10, ACS Paragon Plus Environment

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1:50 and 1:500. The genuine concentration of the bitter components in orange juice

223

was mimicked by the dilution of 1:500. The serial dilutions of each of these fractions

224

were then presented to the sensory panel in order of ascending concentration. The

225

intensity of the perceived bitterness of each dilution and fraction was evaluated on a

226

scale of 0-10.

227 228

Identification of taste active components. The composition of the fractions

229

obtained by LC Taste® analysis was analyzed first by liquid chromatography high

230

resolution mass spectrometry (LC-HRMS). Mass spectra were recorded using a

231

mass spectrometer Bruker microTOFQII (Bruker, Bremen, Germany) with ESI+ and

232

ESI- ionization and scan range of 50-1600 Da in combination with a Waters Acquity

233

UPLC system (Waters, Eschborn, Germany). Chromatographic separation was

234

carried out on a C-18 column (Kinetex, 1.7 µm, 100 mm × 2.1 mm; Phenomenex,

235

Torrance, California, USA) at a temperature of 50 °C and a flow rate of 0.55 mL/min

236

using an acetonitrile/water gradient. Two microliter was injected and the gradient

237

started with 100 % water containing 0.1 % formic acid, increasing to 95 % acetonitrile

238

within 22 min. This concentration was kept for 5 min. Peak identity was confirmed

239

either by cochromatography of the reference compounds or by NMR analysis. 1H,

240

13C, COSY, HMQC, and HMBC measurements were performed on a Bruker

241

Avance-III 600 spectrometer (Bruker, Rheinstetten, Germany). Data processing was

242

performed with MNova (version 9, Mestrelab Research, Santiago de Compostela) or

243

Topspin (version 3.2; Bruker, Rheinstetten). Spectra were recorded in DMSO-d6 and

244

referenced towards tetramethylsilane.

245 246

Quantitative HPLC analysis of hesperidin. Hesperidin was quantified via an

247

external standard method using an Agilent 1290 Infinity HPLC system (Agilent, USA), ACS Paragon Plus Environment


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consisting of binary pump, autosampler and DAD detector. Chromatographic

249

separation was carried out at 40 °C on a poroshell 120 SB-C18 column (100 mm x

250

2.1 mm, 2.7 µm, Agilent, USA). The mobile phase consisted of 0.1 % formic acid

251

water (A) and acetonitrile (B) and a gradient elution of 5-5 % B at 0-0.1 min; 5-50 %

252

B at 0.1-10 min; 50-100 % B at 10-12 min and 100-100 % at 12-15 min was used.

253

Flow rate was 0.4 mL/min and injection volume was 2 µL. The eluent was monitored

254

at 280 nm.

255 256

Quantitative LC-MSMS analysis of bitter components. The LC-MSMS

257

system consisted of an Agilent HP 1200 system and a 3200 QTRAP MSMS (Applied

258

Biosystems, Darmstadt, Germany) with an electrospray ionization (ESI) source. The

259

data were processed using the Analyst 1.5 software. Chromatographic separation

260

was carried out at 50 °C on a Luna C18(2) column (150 mm x 2.0 mm, 3 µm,

261

Phenomenex, USA). The mobile phase consisted of 0.1 % formic acid water (A) and

262

acetonitrile (B) using a gradient elution for method 1 (M1) of 5-30 % B at 0-5 min; 30-

263

95 % B at 5-30 min; 95-95 % B at 30-35 min and for method 2 (M2) of 5-70 % B at

264

0-15 min; 70-95 % at 15-19 min; 95-95 % at 19-20 min. Flow rate for both methods

265

was 0.2 mL/min and injection volume was 5 µL. Mass spectrometer was operated in

266

negative mode for M1 including liquiritin (internal standard, IS), eriocitrin, limonin

267

glucoside, didymin, poncirin, naringenin, limonin, nomilin and for M2 in two MSMS

268

scan segments: first segment 0-13 min in negative mode for liquiritin (IS) and second

269

segment 13-20 min in positive mode for nobiletin and tangeritin. In the multiple

270

reaction monitoring (MRM) mode, the compounds and IS were analyzed using the

271

mass transition and MSMS operating conditions described in Table 1. The optimum

272

instrumental parameters were determined by analysis of analytes and IS (10 µg/mL)

273

solutions into ESI source via a syringe pump. The following MS parameters were ACS Paragon Plus Environment

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used for the measurements: source temperature: 500 °C, ion spray voltage: -4.5 kV

275

in negative mode and 5.5 kV in positive mode, nitrogen served as curtain gas

276

(20 psi).

277

For quantitative analysis, aliquots (0.5 g/2 g, depending on analyte concentration) of

278

the OJ spiked with 150 µL of IS solution (60 µg/mL), were extracted with methanol

279

(up to 4 g) in an ultrasonic bath for 15 min. The extract was separated from solids by

280

centrifugation; the supernatant was passed through a 0.2 µm membrane filter and

281

analyzed by means of LC-MSMS. For calibration purpose, reference standards

282

(100 µg/mL for eriocitrin, limonin glucoside, didymin, poncirin, naringenin, limonin,

283

nomilin, nobiletin and tangeretin) and IS (60 µg/mL for liquiritin) were dissolved in

284

methanol. A series of working calibration solutions spiked with IS solution (60 µg/mL)

285

were prepared by appropriate dilution of standard stock solution with methanol to

286

yield final concentrations of 0.2, 0.5, 1, 3, 7 µg/mL for eriocitrin; 0.8, 2, 5, 12,

287

38 µg/mL for limonin glucoside; 0.4, 1, 3, 6, 15 µg/mL for didymin; 0.05, 0.2, 0.5, 1,

288

3 µg/mL for poncirin and tangeretin; 0.06, 0.1, 0.2, 0.5, 1, 3 µg/mL for naringenin and

289

nobiletin; 3, 7, 17, 50 µg/mL for nomilin and 1, 3, 6, 16 µg/mL for limonin. The limits

290

of quantitation (LOQ) ranged from 0.01 to 5 µg/mL (Table 1). Recovery experiments

291

were performed in a commercial OJ by spiking the OJ with standard stock solution

292

and IS solution (60 µg/mL). Mean recovery rates ranging from 90 % (nobiletin) to

293

115 % (eriocitrin) were found.

294 295

Comparative sensory analysis. Sensory tests were carried out with a panel of 10

296

trained panelists without known taste disorders. Panelists were informed about the

297

procedure and intention of the project, and written consents were obtained. Four

298

samples including a reference juice were presented and the panelists were asked to

299

rank and score the attributes sweet, acidic and HLB bitter on a scale of 0-10. The ACS Paragon Plus Environment


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samples were tested in sensory panel rooms under single-blinded and standardized

301

conditions and a fully randomized presentation design among product, panelist and

302

replications was chosen. Only four samples including the reference juice were tested

303

in one session to allow proper rinsing of the mouth and to avoid carry-over of taste

304

impressions.

305

The comparative sensory test was independently run three times with two

306

different juice bases spiked with hesperidin, limonin, poncirin or polymethoxylated

307

flavons. Significance levels were not calculated because less than 10 panelists

308

attended. First, panelist were trained by descriptive profiling of orange juices with low

309

250

54

1,6

255

140

22

16 250

49

13

0,3

2,3

3,5

230

240

15

10

0,1

0,5

3,3

270

>250

16

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Investigation on Key Molecules of HLB Induced Orange Juice Off-flavor

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