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					     Technical notes
     Identification of a new cork taint compound

     Cork taint continues to cause significant loss of quality in bottled wine. Consequently,
     most Australian wineries invest considerable time and money on quality assessments
     of corks, and only accept corks that meet the requirements of their quality control
     procedures. Duncan (1995) has given an account of the quality control procedures
     developed at Southcorp Wines for closures together with a listing of cork taints and
     their possible origin. TCA was considered to be the main cause of cork taint, a view
     consistent with that of most other authors.

     One of the taints described by Duncan (1995) was referred to as ‘fungal must’.
     This character was considered to be distinct from the taint caused by TCA. The ability
     of the winery panelists to distinguish ‘fungal must’ from other forms of cork taint was
     confirmed by gas chromatography/odour (GC/O) assessments of extracts from corks
10   that had been assessed as affected by ‘fungal must’. These extracts invariably gave a
     discrete, intensely musty, odorous zone in the GC/aromagram that did not correspond
     to TCA or any other previously described compound associated with cork taint.
     However, no peak could be detected in this region of the chromatogram and, for more
     than ten years, the causative agent remained unknown.

     In order to identify the compound responsible for ‘fungal must’ cork taint, it was
     necessary to prepare highly concentrated extracts of contaminated corks for analysis.
     Accordingly, steam distillation of 140 contaminated corks was chosen as the first step
     in the isolation and concentration process. This process reduced the quantity of silicone
     and paraffin components originating from the cork coatings in the final extracts.
     Pentane extracts of the steam distillate were then concentrated and analysed, and
     although several peaks could now be seen in the region of the chromatogram where
     the ‘fungal must’ aroma eluted, none corresponded to compounds that could be
     responsible for this aroma. The causative agent remained ‘hidden’ under these peaks
     in the chromatograms.

     During this phase of our investigations we also identified, for the first time in cork,
     two spiroethers (Simpson et al. 2004). However, these compounds only displayed weak
     aromas at the concentration found in the extracts. Spiroethers have been identified in
     the bark of certain angiosperm trees and they are important in the insect communication

     Technical Review No.156                                                        June 2005
of several coniferophagous bark beetles. The bark beetles are able to distinguish between
host and non-host trees by the presence of these compounds and the males of certain
species of beetle synthesise these compounds, which are used to repel rival males.

Next, we trapped the ‘fungal must’ material, which eluted from the GC column, in
water and determined its chemical properties, based on the effect on aroma. Mustiness
was retained in the presence of potassium hydroxide, sodium borohydride and copper
sulphate, but was lost on addition to phosphoric acid. These tests indicated that the
compound was basic, and was not an aldehyde, ketone or thiol.

Knowing that the ‘fungal must’ compound was likely to be basic, the acid soluble
components were isolated from a second steam distillation of 140 contaminated corks.
This approach provided the means for removal of all the acidic and most of the neutral
components, and gave considerable concentration of the ‘fungal must’ compound.
This time, a new peak with a retention time corresponding to the ‘fungal must’ taint
was obtained on the GC column. The peak contained only one component, with a mass
spectrum that indicated that it was a dimethylmethoxypyrazine.

Mottram et al. (1984) had reported 2-methoxy-3,5-dimethylpyrazine (MDMP) as an
intensely musty-smelling bacterial metabolite some 20 years earlier. We, therefore,
synthesised this compound and confirmed its identity as the cause of ‘fungal must’
taint (Simpson et al. 2004).

Sensory properties of 2-methoxy-3,5-dimethylpyrazine (MDMP)
Using a panel of 33 judges from the AWRI, a group aroma threshold of MDMP in a
neutral white wine was determined to be 2.1 ng/L. The most common descriptors used
by the judges were ‘dirty’, ‘dusty’, ‘musty’ and ‘mouldy’. A few assessors also described
the higher concentrations as ‘chocolate’ or ‘coffee’. The group aroma threshold of
MDMP compares closely with that of TCA, which has an aroma threshold in wine of
1–4 ng/L and is recognised as one of the most potent taint compounds affecting a wide
range of foods and beverages. Among the panel, some judges appeared to be particularly
sensitive to this compound and were able to detect it in the white wine at a concentration
below 1 ng/L. Others were relatively insensitive to MDMP and could only detect it
above 10 ng/L.

June 2005                                                        Technical Review No.156
     Analysis of MDMP in cork soaks and commercial wines
     Several corks from a batch assessed as having a high incidence of ‘fungal must’ were
     each separately soaked in 100 mL of white wine over 48 hours. Those wines that
     exhibited a musty taint were then analysed for MDMP using a deuterium-labelled
     analogue synthesised in our laboratory as an internal standard. MDMP was detected
     in all wine soaks which exhibited a ‘fungal must’ taint. The concentration ranged from
     20–60 ng/L, well above the sensory detection threshold. As there was no MDMP in
     the wine prior to soaking, this showed that it had come from the corks. Subsequently,
     two bottles of commercial wine thought to be affected by cork taint were analysed, this
     time for both TCA and MDMP. One wine contained similar concentrations of MDMP
     and TCA (10 and 8 ng/L, respectively) while the other contained a higher concentration
     of MDMP (42 ng/L). This was likely to have masked any TCA, which was present at
     its threshold concentration of 2 ng/L.

     Co-occurrence of MDMP with TCA in wine corks
     Among the types of cork taint described by Duncan (1995), the frequency of the sensory
12   detection of TCA alone by soaking trials of over 150 000 wine corks was 1.06%, while
     for FM (i.e. MDMP) alone the incidence was 0.82% (Simpson et al. 2005). The incidence
     of taint where both TCA and FM were recognisable in the same cork soak was 0.19%,
     corresponding to about 9% of the total TCA and FM taint (Simpson et al. 2005). Almost
     certainly, there would have been corks containing both TCA and MDMP but with one
     of the taints not recognised because of its lower concentration. Consequently, the
     proportion of corks containing instrumentally detectable quantities of both of these
     two compounds together was likely to have been higher. As indicated above, TCA and
     MDMP have been shown to co-occur in some bottled wines.

     The co-occurrence of several other musty compounds in cork samples has also been
     described by Amon et al. (1989). Three or more of the taint compounds were detected
     together by GC/O assessment of the extracts of wines (57%) and associated corks
     (73%), although the contribution of the individual compounds to taint was uncertain.
     Active growth of mixed microflora occurring at particular sites on the corkwood,
     concentration of metabolites by movement of water or moisture, or storage of cork
     slabs on the ground and aerial contamination of the more exposed slabs might explain
     the tendency of taint compounds to occur together in the corks.

     Technical Review No.156                                                      June 2005
Origin of MDMP
The first report of MDMP, by Mottram et al (1984), described this compound as being
responsible for an obnoxious ‘musty, foul drains, or sour dishcloths’ odour present in
certain machine cutting emulsions used in engineering workshops. They isolated an
aerobic, Gram-negative bacterium which, when grown, gave MDMP as the only major
component of the volatile fraction isolated from the culture broth. Mottram et al. (1984)
were not, however, able to characterise the organism with known bacterial species.
Certain bacteria (Pseudomonas spp.) are known to produce pyrazine off-odours and
a mould (Aspergillus flavus) produces compounds in the same chemical class.

MDMP, together with 2-methoxy-3-isopropylpyrazine, has also been identified in coffee
(Czerny and Grosch 2000), where it was described as having an ‘earthy’ aroma. It was
shown to be important to the aroma of both raw and roasted coffee and to possess an
intense odour; its odour threshold in water was determined as 0.4 ng/L. These authors
also suggested that the two methoxypyrazines they detected in raw coffee had
a bacterial origin.
However, the origin of MDMP might not necessarily be bacterial. It has been recognised
for many years that certain aroma-intense microbial metabolites occurring in foods
and beverages can be produced by different types of microflora. For example, geosmin
can be produced by Actinomycetes (bacteria) (Gerber and Lechevalier 1965),
cyanobacteria (Safferman et al. 1967) and a number of Penicillium spp. (moulds) (Larsen
and Frisvad 1995). Davis et al. (1981) isolated the microflora from wine corks imported
into Australia and showed that numerous moulds, bacteria and yeast were present on
the corks but the metabolites produced by these microorganisms were
not investigated.

Mottram et al. (1984) concluded that MDMP was likely to be a relatively common
cause of off-odour in the environment. However, there has been no further report of
MDMP as a cause of off-odour in the published literature since then, and only the one
report of its occurrence in a food product (Czerny and Grosch 2000). Perhaps this is
because of the difficulty in analysing for this compound and the exceedingly low
concentration at which it can cause an off-odour. Now that we have developed improved
techniques to synthesise and analyse MDMP, this might lead to more reports of this
compound being a cause of off-odour in the environment.

June 2005                                                       Technical Review No.156
     Importance of MDMP as a cause of cork taint
     There is little doubt that TCA remains the most important cause of cork taint in wines.
     The importance of MDMP is less clear, although the data of Southcorp Wines indicates
     that it might make a significant contribution to cork taint. Solubility considerations
     suggest that the impact of MDMP to the aroma of cork soaks, compared to that of
     TCA, is likely to be exaggerated by the conditions of the soaking trials. However, as
     indicated above, the detection of MDMP in bottled wine by sensory means might not
     be obvious if it co-occurs with TCA. Only by instrumental analysis of a large number
     of commercial wines will the importance of MDMP to cork taint become clearer.


     Amon, J.M.; Vandepeer, J.M.; Simpson, R.F. (1989) Compounds responsible for cork taint in wine. Aust. N.Z.
     Wine Ind. J. 4(1): 62-69 (AWRI publication #337)

     Czerny. M.; Grosch, W. (2000) Potent odorants of raw Arabica coffee. Their changes during roasting. J. Agric.
     Food Chem. 48: 868-872.

     Davis, C. R.; Fleet, G. H.; Lee, T. H. (1981) The microflora of wine corks. Aust. Grapegrower Winemaker
     208; 42, 44.

     Duncan, B. Closure quality control at Southcorp Wines. (1995) Proceedings ASVO Oenology Seminar Corks
     and Closures, Leske, P.A.; Eglington, J.M., eds. Adelaide, SA: Australian Society of Viticulture and Oenology;

     Gerber, N. N.; Lechevalier, H. A. (1965) Geosmin, an earthy-smelling substance isolated from Actinomycetes.
     Appl. Microbiol. 13; 935-938.

     Larsen, T. O.; Frisvad, J. C. (1995) Characterization of volatile metabolites from 47 Penicillium taxa. Mycol.
     Res. 99; 1153-1166.

     Mottram, D.S.; Patterson, R.L.S.; Warrilow, E. (1984) 2,6-Dimethyl-3-methoxypyrazine:
     a microbiologically-produced compound with an obnoxious musty odour. Chem. Ind. 448-449.

     Safferman, R. S.; Rosen, A. A.; Mashni, C. I.; Morris, M. E. (1967) Earthy-smelling substance from a
     blue-green alga. Environ. Sci. Technol. 1; 429-430.

     Simpson, R.F.; Capone, D.L.; Sefton, M.A. (2004) Isolation and identification of
     2-methoxy-3,5-dimethylpyrazine, a potent musty compound from wine corks. J. Agric. Food Chem.
     52: 5425-5430 (AWRI publication #789).

     Simpson, R.F.; Capone, D.L.; Duncan, B.C.; Sefton, M.A. Incidence and nature of ‘fungal must’ taint in wine
     corks. Aust. N.Z. Wine Ind J. 20(1): 26–31; 2005 (AWRI publication #826).

     Bob Simpson                   Dimi Capone                    Mark Sefton
                                   Chemist                        Principal Research Chemist

     Technical Review No.156                                                                           June 2005