Copper(II)-Mediated Hydrogen Sulfide and Thiol Oxidation to

Subscriber access provided by Warwick University Library

Article

Copper(II) mediated hydrogen sulfide and thiol oxidation to disulfides and organic polysulfanes, and their reductive cleavage in wine: Mechanistic elucidation and potential applications Gal Y Kreitman, John C Danilewicz, David William Jeffery, and Ryan J. Elias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05418 • Publication Date (Web): 05 Mar 2017 Downloaded from http://pubs.acs.org on March 5, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Agricultural and Food Chemistry

Copper(II) Mediated Hydrogen Sulfide and Thiol Oxidation to Disulfides and Organic Polysulfanes, and their Reductive Cleavage in Wine: Mechanistic Elucidation and Potential Applications Gal Y. Kreitman†, , John C. Danilewicz‡, David. W. Jeffery§, Ryan J. Elias*,† ⊥

†

Department of Food Science, College of Agricultural Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡

44 Sandwich Road, Ash, Canterbury, Kent CT3 2AF, United Kingdom

§

School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia

Present address: Mondelēz International, Inc., 100 Deforest Avenue, East Hanover, NJ 07936, United States ⊥

* To whom correspondence should be addressed. Tel: +1 (814) 865-5371 Fax: +1 (814) 863-6132 E-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

2

Fermentation-derived volatile sulfur compounds (VSCs) are undesirable in wine and are

3

often remediated in a process known as copper fining. In the present study, the addition of Cu(II)

4

to model and real wine systems containing hydrogen sulfide (H2S) and thiols provided evidence

5

for the generation of disulfides (disulfanes) and organic polysulfanes. Cu(II) fining of a white wine

6

spiked with glutathione, H2S, and methanethiol (MeSH) resulted in the generation of MeSH-

7

glutathione disulfide and trisulfane. In the present study, the mechanisms underlying the

8

interaction of H2S and thiols with Cu(II) is discussed, and a prospective diagnostic test for releasing

9

volatile sulfur compounds from their non-volatile forms in wine is investigated. This test utilized

10

a combination of reducing agents, metal chelators, and low oxygen conditions to promote the

11

release of H2S and MeSH, at levels above their reported sensory thresholds, from red and white

12

wines that were otherwise free of sulfidic off-odors at the time of addition.

13 14

KEYWORDS: Hydrogen sulfide, thiols, copper, polysulfanes, wine aroma

15

2 ACS Paragon Plus Environment

Page 2 of 32

Page 3 of 32

16


INTRODUCTION

17

Sulfidic off-odors in wine present a serious quality issue due to their adverse effects to

18

overall wine aroma.1,2 When detected in the course of winemaking, such off-odors are routinely

19

treated through the use of copper fining prior to bottling.3–5 During the post-bottling period, even

20

in wine deemed to be free of faults at the time of bottling, VSCs that are responsible for the

21

undesirable odors may accumulate during storage, especially when oxygen ingress through the

22

closure is limited.6–9 The most common VSCs responsible for so-called “reductive” sulfidic off-

23

odors that can arise post-bottling are H2S, MeSH, and dimethyl sulfide (DMS).1,10 The generation

24

of DMS, whose aroma is often described as “canned corn”, is linked to non-enzymatic hydrolysis

25

of S-methylmethionine over a wine’s aging period;11,12 however, the pathways for the generation

26

of H2S and MeSH, described as “rotten egg” and “rotten cabbage”, respectively, have yet to be

27

established. Several hypotheses have been proposed to explain the non-enzymatic mechanism(s)

28

responsible for the generation of H2S and MeSH in finished wine (i.e., post-fermentation). These

29

mechanisms have been suggested to include bisulfite reduction,13,14 thioacetate and thioether

30

hydrolysis,3,15 and Strecker degradation of sulfur-containing amino acids.16,17 The reduction of

31

symmetrical disulfides has also been proposed leading to MeSH and EtSH, with these thiols having

32

markedly lower (ca. 10-50 times) sensory detection thresholds than their respective disulfides.1,18

33

While the oxidation of thiols plausibly explains disulfide formation, the underlying mechanisms

34

by which H2S and thiols are subsequently released remain to be elucidated.

35

Recent work suggests that the interaction of transition metals, particularly copper, with

36

sulfur-containing compounds under anaerobic (reductive) conditions is correlated with the release

37

of H2S and MeSH.6,9,19,20 In order to understand, in mechanistic terms, the role of copper in the

38

generation of potential latent sulfur forms, our group has recently focused on elucidating the initial



39

reactions involving the metal-catalyzed oxidation of sulfhydryls that are responsible for the

40

“removal” of undesirable VSCs (Figure 1).21 In that work, we presented evidence that Cu(II)

41

initially coordinates with two sulfhydryl moieties to produce complex (1), followed by an electron

42

transfer reaction from sulfur to give Cu(I). The generation of the disulfide and polysulfanes appears

43

to occur in a concerted manner in close proximity to the metal and without the release of free thiyl

44

radicals, such as in dimeric complex (2).21 The resulting copper-thiolate (Cu(I)-SR) species (3)

45

subsequently aggregates to further stabilize Cu(I). Initially, in the absence of oxygen, both thiol

46

and H2S oxidation products are produced as well as Cu(I) complexes. In a similar manner

47

formation of a mixed complex involving H2S (4) would result in formation of a hydrodisulfide (5).

48

Incorporation of this product into a Cu-complex (6) would in turn result in trisulfane (7). The

49

resultant very fine (Cu(I)-SR)n aggregates remain reactive, behaving as soluble species wherein

50

Cu(I) is highly reductive and is readily oxidized by Fe(III) and O2 upon exposure to air. It is

51

apparent, therefore, that the VSCs responsible for sulfidic off-odors cannot be readily removed by

52

filtration from wine as an insoluble complex after copper fining, but rather generate redox-active

53

compounds that remain in the wine effectively as soluble components.

54

The dissociation of metal-sulfide complexes could be partially responsible for the release

55

of H2S and MeSH under reductive wine conditions; however, in some wines it has been shown

56

that up to 42% and 76% of H2S and MeSH, respectively, are generated through other pathways.20

57

However, it is also possible that the cleavage of disulfides via sulfitolysis could serve as a pathway

58

for VSC release during storage.14,22 With respect to disulfide formation, recent work seemed to

59

show that Cu(II) had a remarkable effect (more so than oxygen) on the generation of disulfides

60

that arose from oxidation of varietal thiols 3-sulfanylhexan-1-ol and 3-sulfanylhexyl acetate in

61

wines, leading to the hypothesis that disulfides play a key role as aroma reservoirs in wine.23 In


Page 4 of 32

Page 5 of 32


62

another study, Cu(II)-catalyzed oxidation of model compound 6-sulfanylhexan-1-ol (6SH) in the

63

presence H2S not only resulted in the formation of 6SH-disulfide, but also polysulfanes consisting

64

of up to five linking sulfur atoms between the 6SH constituents.24

65

Due to the relative abundance of thiol-containing compounds such as glutathione (GSH)

66

and cysteine (Cys) in wine, and in light of the preceding results, it is likely that the use of Cu(II)

67

to treat off-odors due to MeSH and H2S would generate a variety of asymmetrical (i.e., mixed)

68

disulfides and organic polysulfanes (Figure 1). The generation of polysulfanes in hydroalcoholic

69

media has been hypothesized to occur in the presence of H2S and thiols via a perthiol

70

intermediate.25 Recent work demonstrated the presence of transient species of hydropolysulfides

71

which, some have argued, contribute to the “minerality” of wine and can further react to generate

72

perthiols.26 Furthermore, polysulfide species are thiophilic species that may further react to

73

generate additional mixed polysulfanes.27,28 The presence of polysulfanes and polysulfides in wine

74

is therefore indicative of there being forms of latent VSCs beyond simple disulfides that have yet

75

to be fully considered.

76

The observations outlined above lead to the following aims for the present study: (1) to

77

confirm metal-catalyzed generation of disulfides and organic polysulfanes in model and real

78

wines; and (2) to adapt a method to force the the dissociation of metal sulfides and reduction of

79

disulfides and organic polysulfanes to release H2S and thiols. The ultimate goal being to provide

80

a tool for winemakers to determine whether their product is susceptible to the appearance of

81

sulfidic off-aromas in the post-bottling period.

82 83



84

MATERIALS AND METHODS

85

Materials. L-Cysteine (Cys), L-cystine, ethanethiol (EtSH), diethyl disulfide (DEDS), sodium

86

thiomethoxide (as a source of MeSH), ferrous sulfate hexahydrate, tris(2-carboxyethyl)phosphine

87

(TCEP) and bathocuproinedisulfonic acid (BCDA) disodium salt) were obtained from Sigma-

88

Aldrich (St. Louis, MO). L-Tartaric acid and L-glutathione (GSH) were obtained from Alfa Aesar

89

(Ward Hill, MA). Cupric sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown,

90

NJ), TRIS hydrochloride was from J.T. Baker (Center Valley, PA), sodium hydrosulfide hydrate

91

(as a source of H2S) was from Acros Organics (Geel, Belgium) and ferric chloride hexahydrate

92

was from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore Q-

93

Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical

94

or HPLC grade, and solutions were prepared volumetrically, with the balance made up with Milli-

95

Q water unless specified otherwise. Six commercial Pennsylvania wines (three white and three red

96

wines) from the Lake Erie American Viticultural Area were purchased locally.

97 98

Preparation of model wine and real wine samples

99

Disulfide and polysulfane generation in model wine. Model wine was prepared by

100

dissolving tartaric acid (5 g/L) in water, followed by the addition of ethanol to yield a final

101

concentration of 12% v/v. The solution was adjusted to pH 3.6 with sodium hydroxide (10 M) and

102

brought to volume with water.

103

Either glutathione (GSH, 500 µM) or cysteine (Cys, 500 µM) were added to model wine

104

and mixed thoroughly. H2S (250 µM) and/or MeSH (250 µM) were subsequently added to the

105

solutions to give a total of four treatments: (1) Cys+H2S, (2) Cys+H2S+MeSH, (3) GSH+H2S, and

106

(4) GSH+H2S+MeSH. Once the sulfhydryl compounds were added to their respective solutions,


Page 6 of 32

Page 7 of 32


107

Fe(III) (100 µM) and Cu(II) (50 µM) were added and the solutions were thoroughly mixed. The

108

solutions (25 mL) were stored in the dark at room temperature in capped 50 mL capacity

109

polypropylene tubes under air. The samples were analyzed after 24 hours by ultra fast liquid

110

chromatography (UFLC) tandem QTOF-MS, as described below.

111

Disulfide and polysulfane generation in white wine. GSH was added to commercial

112

white wine blends to achieve a final concentration of 50 µM. H2S and MeSH were subsequently

113

added to achieve the following three treatment concentrations of each sulfhydryl: 100 µg/L, 500

114

µg/L, and 5000 µg/L. Following the addition of the sulfhydryl-containing compounds, Fe(III) (5

115

mg/L) and Cu(II) (1 mg/L) were added and the resulting solutions were mixed thoroughly. The

116

samples (100 mL) were stored in the dark in stoppered 100 mL volumetric flasks and analyzed

117

after 24 hours by UFLC-QTOF-MS.

118

Release and reduction of bound VSCs. Initial experiments were conducted in either air

119

saturated model wine (dissolved [O2]: 7–8 mg/L) or in an anaerobic chamber (dissolved [O2]:

Copper(II)-Mediated Hydrogen Sulfide and Thiol Oxidation to

Recommend Documents