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Determination of key volatiles in Australian Black Truffles aroma by combined GCMS and organoleptic techniques

Professor Garry Lee Centre For Forensic Science

Garry.Lee@uwa.edu.au

0402 342090

Progress Report: 18th August, 2012 • Flamen Nominee

Support

Associate Professor Hannah Williams

Acknowledgements

Dr Gavin Flematti Kenny Choo Sophiana Benali

Three Components

1. Investigation of the quantitative changes in

truffle volatiles at different stages of fruiting

and development by chemical instrumentation

techniques.

2. Sensory Evaluation. This includes the

characterisation of the essential aroma

qualities of truffles of various quality by truffle

experts.

3. Correlate the Sensory and Chemistry using

GC-O-MS . Develop a grading scale for

Australian black truffles.

Project Summary

Western Australia – 3 Regions, Manjimup – 4 truffieres

Truffle Samples

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New South Wales – 3 Regions

Truffle Samples

Tasmania – 2 Regions, Canberra – 1 Region

Truffle Samples

Truffles from Canberra

• Received fresh or vacuum packed

• Stored at 4 °C wrapped in paper towel in glass container.

• Used within 2 days of possession

Truffle Samples

Solid Phase Microextraction

Analytical Methodology

• Varying temperatures.

• Four different SPME fibres were investigated.

• Combination of three different stationary phases

(divinyl benzene (DVB), carboxen (CAR) and

polydimethylsiloxane (PDMS).

• GC-MS column - polyethylene glycol (AT-wax)

stationary phase

Analytical Methodology

• Custom Built Headspace apparatus

• Whole truffle analysis

• Freshly dug truffle with soil analysis

What have we done so far…

Fresh and Healthy Truffles have

Higher Concentrations of:

Literature

2-methyl Butanal 2-Methyl Butanal

Ethyl acetate 3-Methyl Butanal

Dimethyl sulphide Dimethyl disulfide

9,12-octadecanoic acid and its

esters

2-Methyl I propanol

Acetaldehyde 1-octen-3-ol

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Results Results

Results Results

• 75 volatile compounds

• The main volatile compounds identified from fresh truffles:

– Mixture of esters, alcohols and aldehydes as well as dimethyl sulphide.

– 2-methylbutanal, 3-methylbutanal, dimethyl sulfide, 2-methyl-1-propanol and 1-

octen-3-ol.

• Trends.

– High levels of formic acid esters. These have only been identified as minor

components of truffle odour in other parts of the world. They were present as minor components in Tasmanian truffles as well.

• 1-Octen-3-ol

– observed in higher concentrations in medium and lower grade Australian truffles than high

graded Australian truffles.

– Manjimup truffles, had 20 to 47% in medium and low grade truffles but only as 2.5 – 2.7% in

high grade truffles.

– Canberra truffles (aged at least 1 week before analysis) – high level of 1-octen-3-ol

– Tasmanian truffles contained the least amount (0.2 – 1.8% of the top 20 volatiles).

Results – Chemical Analysis Results – Chemical Analysis

3-methyl, 6-propyl phenol. • High levels in Tasmanian Truffles

• Minor component from other states

4-ethyl-2-methoxy-phenol. (4-ethyl guaiacol) • High levels in Tasmanian Truffles

• Absent in WA Truffles

Spicy

2,3-Dimethoxy toluene. • High levels in Tasmanian Truffles

• Also found in some NSW Truffles

• Absent in WA Truffles

Cooked Vegetable

1-methoxy-3-methyl benzene. • Main component in Tasmanian Truffles (37 – 51%

of all volatile components).

• Major component of NSW truffles (42.4% of the

volatiles from truffles grown in Braidwood)

Floral

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Results – Chemical Analysis

Methoxy benzene (Anisole) • May be a major compound – see in most truffles.

Phenolic

2,6-dimethyl anisole • High levels in some truffles and absent in others

Butanoic Acid 2 methyl-, 2-methylbutyl ester • Only in WA Truffles

Green

2-Methyl-2-hexanol • Only in NSW truffles

• Account for 33% of volatiles in truffles from

Bredbo.

Flavour compound

found in

ripening of

banana

• 1,3-Pentadiene,

– Odour similar to kerosene or acrylic paint,

– Present as a minor component in all the high quality graded truffles.

– This compound has been reported as part of the aroma profile of T. simonea.

– It has not been observed in the aroma profile of T. melanosporum.

– When diced, 1,3-pentadiene is released as a major component of the aroma

profile (present 27.3% of total volatiles)

Results – Chemical Analysis

• dimethyl sulphide, 2-methyl butanal, 3-

methyl butanal, dimethyl ether, formic acid

1-methyl propyl ester, 2-methyl butanol and

1-octen-3-ol.

Characteristic Australian Truffle Odor

• 1,2-dimethoxy benzene and 1,3-

dimethoxy benzene were also found in

approximately 70% of all truffles

analysed.

Hazelnut, Grade: Extra

Western Australian Truffles

A20-120 DAYS

R time Compounds 1 2 3 6 7

1.790 Dimethyl sulfide 4.08 5.57 7.58 5.76 5.44

1.990 Propanal 1.28 0.46 0.67 0.77 0.65

2.120 4-aminoheptane 3.73 1.92 2.21 4.27 2.82

3.140 Butanal, 2-methyl 28.15 13.81 17.31 30.91 22.11

3.750 Formic acid, 1-methylpropyl ester 11.70 9.94 9.67 11.77 12.06

6,845-6,920 UNKNOWN 2.43 3.19 2.72 1.57 2.69

11.660 1-butanol, 2-methyl 32.19 43.35 43.05 30.57 38.72

14.545 Butanoic acid, 2-methyl, 2-methylbutyl ester - 1.67 1.30 - 0.53

16.910 Benzene methoxy (Anisole) 3.77 5.95 5.00 3.62 3.23

17.660 1-hexanol 0.75 1.43 - - -

20.420 UNKNOWN 1.09 2.22 1.70 0.52 1.15

21.165 1-octen, 3-ol 0.58 1.03 0.92 0.67 0.67

23.050 Benzaldehyde 0.85 2.11 1.12 - 0.59

28.470 Benzene 1,2-dimethoxy 1.59 3.01 2.34 1.62 1.39

32.370 Phenylethyl alcohol 0.41 1.10 0.79 0.42 0.47

Hazelnut, Grade: Extra

Western Australian Truffles

Oak, Little Aroma Detected

Western Australian Truffles

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Green – mould observed.

Tasmanian Truffles

Tasmania A DAYS (age +4) Retention time Compounds 1 2 3 6 8

1.515 1,3 pentanediene 3.12 2.77 1.64 6.56 5.04

1.790 Dimethyl sulfide (DMS) 0.54 0.83 0.79 2.67 2.27

2.150 2-propanamine, 2-methyl - - 3.56 - -

2.435 Propane, 2-ethoxy 0.59 - 0.56 2.95 2.10

3.140 Butanal, 2-methyl - 2.23 1.01 1.14 0.77

3.750 Formic acid, 1-methylpropyl ester 6.03 7.45 7.00 19.20 21.60

4.080 Formic acid butyl ester 1.02 0.57 - 2.35 2.16

11.660 1-butanol, 2-methyl 5.23 7.45 6.74 7.44 15.47

16.910 Benzene methoxy (Anisole) 3.58 2.20 2.28 - -

20.680 Benzene 1-methoxy, 3-methyl (Anisole m-methyl) 46.29 42.83 43.70 25.20 21.77

23.250 2,3-dimethylanisole 15.50 15.41 17.92 8.24 7.45

28.470 Benzene 1,2-dimethoxy 1.68 1.77 1.85 1.91 2.90

28.930 Benzene 1,3-dimethoxy 4.10 3.79 3.91 4.17 3.66

30.060 Phenol, 4-ethyl, 2-methoxy 1.38 1.36 1.33 1.04 1.03

30.330 2,3-dimethoxytoluene or 3,4 3.22 3.19 3.05 2.68 3.66

Tasmanian Truffles

Canberra DAYS Retention time Compounds 1 2 5 6 7 8 9 12

1.790 Dimethyl sulfide 23.03 16.33 15.31 15.79 14.37 9.91 8.11 5.65

2.120 4-aminoheptane - - - - 2.71 3.78 3.47 3.63

2.435 Propane 2-ethoxy 7.59 7.93 4.08 3.36 1.52 - - -

3.140 Butanal, 2-methyl - - 4.62 5.71 15.35 19.53 21.80 20.48

3.470 Dimethyl ether 0.60 - - - - - - -

3.750 Formic acid, 1-methylpropyl ester 32.81 42.14 43.88 43.90 33.16 22.81 23.13 25.12

4.080 Formic acid butylester 6.46 7.58 4.68 - - - - -

6.555 Disulfide dimethyl 2.15 1.97 1.72 2.24 1.69 0.86 0.59 0.43

8.190 1-butanol, 3-methyl formate(89%) 1.61 1.78 - - - - - -

11.660 1-butanol, 2-methyl 3.13 0.64 6.63 8.51 13.51 25.28 24.63 27.00

Formic acid hepthyl ester (96%) ou heptanol (95%) 1.54 2.17 - - - - - -

16.910 Benzene methoxy (Anisole) 12.18 10.15 8.73 7.78 8.91 7.05 7.85 4.88

17.660 1-hexanol 1.20 0.86 0.76 0.41 0.40 0.65 0.60 0.86

18.120 Dimethyl trisulfide 1.85 2.07 2.12 3.18 2.47 1.03 0.98 0.85

21.165 1-octen, 3-ol - 0.53 0.84 0.86 0.52 0.85 - -

20.630 Anisole m-methyl - - - - - - - 0.86

23.050 Benzaldehyde - - - - - 0.44 0.56 2.30

28.470 Benzene 1,2-dimethoxy 1.18 1.26 1.31 1.26 1.34 1.45 2.03 -

32.370 Phenylethyl alcohol 0.22 - 0.32 0.44 0.68

Canberra Truffles Canberra Truffles

NSW A DAYS Retention time Compounds 1 2 3 6 8

1.515 1,3 pentadiene 1.91 1.16 2.21 6.08 6.82

1.790 Dimethyl sulfide 4.02 4.46 4.29 11.00 15.57

2.120 4-aminoheptane 5.07 - - - -

2.150 2-propanamine, 2-methyl - - 1.81 1.77 1.49

2.435 Propane 2-ethoxy 1.55 1.45 1.78 1.85 1.06

3.140 Butanal, 2-methyl 6.84 1.65 - - -

3.750 Formic acid, 1-methylpropyl ester 17.59 21.28 22.77 26.55 12.91

4.080 Formic acid butylester 1.80 2.51 4.50 7.72 4.11

6.555 Disulfide dimethyl - - - 1.97 2.29

11.660 1-butanol, 2-methyl 14.81 26.29 12.76 - -

16.910 Benzene methoxy (Anisole) - - - - 11.62

20.680 Benzene 1-methoxy, 3-methyl (Anisole m-methyl) 20.71 19.34 24.81 21.65 20.18

23.250 2,3-dimethylanisole 5.35 5.26 8.23 7.37 6.62

28.470 Benzene 1,2-dimethoxy 0.74 0.84 0.81 0.78 1.38

28.930 Benzene 1,3-dimethoxy 2.50 2.94 3.72 5.06 5.23

30.060 Phenol, 4-ethyl, 2-methoxy 0.72 0.90 1.07 1.40 1.74

30.330 3,4-dimethoxytoluene or 2,3 0.89 1.05 1.03 1.11 1.33

NSW Region A Truffles NSW Region A Truffles

0

5

10

15

20

25

30

4 5 6 7 8 9 10 11 12 13

Rel

ati

ve

are

a %

Days following harvest

Truffle from NSW – Region A

1,3 pentadiene

Dimethyl sulfide

Butanal, 2-methyl

Formic acid, 1-methylpropyl ester

1-butanol, 2-methyl

Benzene 1-methoxy, 3-methyl (Anisole m-

methyl)

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Hazelnut vs Oak

Compounds Oak Hazel Oak Hazel Oak Hazel

1 5 9

1,3-pentadiene - 0.63 - 0.34 0.56 0.58

Dimethyl sulfide 6.59 5.09 4.83 9.06 3.93 9.62

Propane 2-ethoxy 1.00 1.20 0.51 3.89 - 6.97

Butanal, 2-methyl 4.77 8.27 12.17

Dimethyl ether 1.05 3.00 2.27 2.50 2.64 1.51

Formic acid, 1-methylpropyl ester 32.15 21.27 21.75 34.62 11.08 36.05

UNKNOWN 5.24 3.80 -

1-butanol, 3-methyl formate(89%) 0.49 - -

1-butanol, 2-methyl 32.24 38.23 42.41 19.21 36.49 6.08

Benzene methoxy (Anisole) 7.08 4.98 2.61 5.86 1.86 11.10

1-hexanol 1.33 2.70 1.52 3.59 1.28 1.80

Benzene, 1-methoxy, 3-methyl - 8.19 5.14 7.65 20.39 11.08

1-octen, 3-ol 0.41 0.96 1.02 0.44 0.89 0.52

2,6-dimethylanisole 2.00 - -

Benzene 1,2-dimethoxy 2.02 1.37 1.48 1.49 1.57 1.23

Benzene 1,3-dimethoxy 0.44 - -

Phenylethylalcohol 0.62 3.45 0.94 5.03 1.09 3.61

Fruity/aromatic

Overripe

Over ripe banana

Earthy

Dry Dry Yeast

Fermented Woody

Winery Musty

Mouldy

Cooked Fresh baked fruit cake Fungal

Treacle

Wet

Field Mushroom

Confectionary Caramel Forest Floor

Vanilla Soil

Fresh Green fruity cacao Compost

Citric acid Rotting leaves

Floral Fresh Green/sweet Damp wood

Spicy Sweet

Vanilla

Vegetal & Herbs

Cooked Woody

Clove-like Cooked Vegetable

Aniseed Fresh

Sweet corn

Nutmeg Pine

Savoury Roast

Cold Roast Meat Green grass

Roasted

Dried

Wet hay

Baked Baking bread Straw

Maillard

Sweet

Malt Tobacco

Chocolate (Dark)

Sulfurous

Seaweed

Caramel Garlic

Raison Sulfury Onion

Aldehydic Hydrogen sulfide

Savoury

Hot Coffee

Chemical

Medicinal Medicinal

Ground Coffee Metallic Metallic

Smokey

Pungent

Phenolic

Nutty Nuts

Cashews Gasoline

Pistachio Kerosene

Walnuts Acrylic paint

Trees Oak Ammonia

Acidic Sour Sour

Acetic Acetic

Animalic Wet Dog Wet Dog