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

Professor Garry LeeCentre For Forensic ScienceGarry.Lee@uwa.edu.au0402 342090

• Blue Frog Truffles

• Flamen Nominee

• Macenmist

Support

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

1. Grading Scale for Australian Black Truffle

2. An accurate aroma profile for Australian Black Truffles– Used to develop oils– Train Dogs

3. Electronic Nose.

Project & Future Outcomes

Why are we doing this?

The West Australian, February 15th, 2011

What is it that we desire from a

Truffle?

• Based on physical and morphological Aspects.

• Grade A –Truffles in this class should be of the highest quality. They should be of regular shape and sufficiently mature to have the characteristic aroma, taste and colour of the species, with only very slight damage.

• Grade B –Truffles in this class may include those that have been damaged or broken but are sufficiently mature to have the characteristic aroma, taste and colour of the species. They may have some shape imperfections and predator damage. It includes truffle pieces greater than 100 g.

• Grade C –Truffles in this class comprise smaller truffle pieces, which have been broken or cut from larger truffles but are sufficiently mature to have the characteristic aroma, taste and colour of the species, but will contain defects and damage marks.

• Grade D –Truffles in this class are immature truffles with little or no aroma and primarily aimed at the manufacturing industries.

Australian – New Zealand Standard for Truffles

• Other Provisions.

• Size – Bands: Special (750g +), 250-750g, 100-250g, 20-100g, 0-20g. .

• Quality – Maturity: The aroma must be sufficiently developed and must display satisfactory ripeness for the grade allocated.

• Minimum Requirements – Australian origin, firm, clean, intact, free from pest, damage, foreign visible matter etc.

• Tolerances

Australian – New Zealand Standard for Truffles

• Based on physical and morphological Aspects.

• “Extra” Class –Truffles in this class must be of superior quality. They must be characteristic of the species.They must have a rounded shape, more or less regular and lobed.They must be free from defects, with the exception of very slight superficial defects as well as very slight

defects in appearance, shape and colour, provided these do not affect the general appearance of the produce, the quality, the keeping quality and presentation in the package.

• Class I – Truffles in this class must be of good quality. They must be characteristic of the species.The following slight defects, however, may be allowed, provided these do not affect the general

appearance of the produce, the quality, the keeping quality and presentation in the package:• a slight defect in shape• a slight defect in development• slight defects in colouring• slight superficial bruising.

• Class II– This class includes truffles that do not qualify for inclusion in the higher classes but satisfy the minimum requirements specified above.The following defects may be allowed, provided the truffles retain their essential characteristics as

regards the quality, the keeping quality and presentation:• defects in shape• defects in development• defects in colouring• superficial bruising• slight superficial damages caused by pests provided they are not developing.

UNECE STANDARD FFV-53

Which one is the Market Grade?

• Damage to Industry Reputation

• Industry not getting it’s worth in smaller truffles

• Customers not getting what they paid for

Cost to Industry and Customers

Good Quality or Not?

Size?

• Truffles:– Western Australia: 3 regions– New South Wales: 2 regions– Canberra

• Immature, Mature & Rottten

• Grade A, B and C (Graded by individual Sources)

• Received fresh or vacuum packed

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

• Used within 2 days of possession

Chemical Profile of Truffles

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

Purge and Trap

Analytical Methodology

• Solid SampleTruffle subsample was chopped into fine pieces, placed

in a 40 mL purge and trap vial, heated to 80 oC and sparged with helium into a purge and trap concentrator equipped with a Supelco Vocarb 3000 absorbent trap and attached to a GC-MS.

• Water Extraction15 mL of water was added to one gram of chopped truffle

sample in a 40 mL vial and sonicated prior to sparging with helium.

• Methanol ExtractionOne gram of chopped truffle sample was extracted with

10 mL of methanol. 1 mL of this solution was added to 41 mL of water. 5 mL of this solution was then sparged with helium.

Results

*March et al., International Journal of Mass Spectrometry, 249-250 (2006), p60-67

Compound T. melanosporum

(Relative % from top 30 peaks)

Region 1 Region 1 (1 week old)

Region 2 *Literature

dimethylsulphide 8.7 1.4 2.82 17.5Formic acid, 1‐methylethyl ester 10.17 0.88 0 0

2‐butanone 1.92 1.23 1.08 15.6Butanal, 2‐methyl 1.66 20.22 2.66 0ethanol 1.66 3.29 12.42 13.5Formic acid, 1‐methylpropyl ester 29.48 10.45 1.34 0

2‐butanol 0.29 0 0 5.251‐propanol, 2‐methyl 6.99 7.29 10.24 0

1‐Butanol, 2‐methyl 7.88 27.37 45.89 6.18

Benzene, methoxy

(anisole) 4.18 1.61 0.94 0

Benzene, 1‐methoxy‐3‐methyl 3.91 7.78 0.63 0

Ethyl Acetate

4.76 4.78 4.80 4.82 4.84 4.86 4.88 4.90 4.92 4.94 4.96 4.98 5.00 5.02 5.04 5.060

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TIC: T35_610_Acetone_2306.D\data.msTIC: T042_Acetone_2306.D\data.ms

TIC: T28_510_Acetone_2306.D\data.ms• Black, Fresh truffle• Red, Old truffle (3 weeks)• Blue, rotten truffle

7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 90

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TIC: T35_610W_S.D\data.msTIC: T042W_S.D\data.ms

TIC: T28_510W_S.D\data.ms

Hexanal

• Black, Fresh truffle• Red, Old truffle (3 weeks)• Blue, rotten truffle

• Fresh truffles had relatively fewer peaks = relatively few compounds in its volatile profile.

• Ageing and decomposing truffles had many compounds and hence many more peaks in their chromatograms.

• Rotten truffles appeared to be more odorous than the fresher truffles.

• Fresh truffles had higher concentrations of heavier molecules such as 9,12-octadecanoic acid and its esters.

• The less fresh and rotten samples had lower concentrations of these heavier fractions, but higher concentrations of smaller, and apparently the decomposition compounds of these heavier molecules.

Evolving Volatiles

Butanoic Acid

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TIC: T35_610_Acetone_2306.D\data.msTIC: T042_Acetone_2306.D\data.ms

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• Black, Fresh truffle• Red, Old truffle (3 weeks)• Blue, rotten truffle

Preliminary Indicator Compounds

Fresh and Healthy Truffles have Higher Concentrations of:

Decomposing and Rotten Truffles have Higher Concentrations of:

9,12-octadecanoic acid and its esters

2-methyl propionic acid

Ethyl acetate 2,3-butanediolDimethyl sulphide Linoleic acid ethyl ester2-methyl butanal 3-pentene-2-oneAcetaldehyde 3-hexene-2-one

Butanoic acidPropanoic acidButanoic acid-2-methylHexanoic acid-2-methylEthanol

Chef Sensory Workshop

Experts Sensory Workshop

• Each participant will be given a sample of green and red bell peppers.

• Participants are not allowed to discuss their answer with other participants.

• Open the container given and sniff the bell peppers.

• Write down the words best describe bell peppers in the description forms provided.

• Please allow 5 to 10 seconds rest before the next sniff.

• Each participants will be asked to provide one description that is different from other participants.

• Discussion and scoring of descriptors

• Run the test again using truffle sample instead of bell peppers.

Methodology

Results – Grade “A” Truffles

Truffle Vote A Vote B Vote  C Marks Final Grade

Chef Experts Chef Experts Chef Experts Chef Experts Chef Experts

272 0 1 5 1 2 3 12 8 B C

274 1 2 6 2 0 1 15 11 B B

492 5 1 1 0 1 4 18 7 A C

494 1 0 5 4 1 1 16 9 B B

292 1 1 3 1 3 3 12 8 B C

194 2 0 4 4 1 1 15 9 B B

374 3 1 1 4 3 0 14 11 B B

614 4 4 2 0 1 1 17 13 A A

634 2 0 2 4 3 1 13 9 B B

524 6 3 1 1 0 1 20 12 A B

TruffleVote A Vote B Vote  C Marks Final Grade

Chef Experts Chef Experts Chef Experts Chef Experts Chef Experts

271 1 0 4 5 2 0 13 10 B B

273 0 0 4 2 3 3 11 7 C C

471 2 2 2 1 3 2 13 10 B B

473 4 1 2 3 1 1 17 10 A B

291 5 2 1 2 1 1 18 11 A B

193 0 0 2 2 5 3 9 9 C B

171 1 2 5 1 1 2 14 10 B B

373 3 2 2 2 2 1 15 11 B B

393 5 0 1 1 1 4 18 6 A C

513 0 0 0 3 7 2 7 8 C C

511 3 2 4 3 0 0 17 12 A B

Results – Grade “B” Truffles

TruffleVote A Vote B Vote  C Marks Final Grade

Chefs Experts Chefs Experts Chefs Experts Chefs Experts Chefs Experts

496 2 1 5 0 0 4 16 7 B C

495 4 1 3 1 0 3 18 8 A C

296 0 0 7 2 0 3 14 7 B C

295 5 0 2 2 0 3 19 7 A C

476 1 1 5 1 1 3 14 8 B C

175 3 2 3 1 1 2 16 10 B B

376 1 2 5 1 1 2 14 10 B B

396 5 1 2 1 0 3 19 8 A C

Results – Grade “C” Truffles

Results – Descriptors, Grade “A” TrufflesDescriptor Additional descriptor Strength Chemical

Dark Chocolate caramelizedmalt Strong to medium

2‐methyl butanal3‐methyl butanalOctanoic

acidSeaweed Medium dimethylsulfideEarthy Fungus/soil Strong 1‐octan‐3‐olMusty MediumYeast Microbiological MediumMushroom Strong 1‐octen‐3‐one

Fruity Fruit cake WeakFormic acid, 1‐methyleythl esterEthyl ButyrateEthyl‐3‐methyl‐butyrate

Ground Coffee Woody Weak 5‐Methyl‐2‐phenyl‐2‐hexenalShearing shed/dampWet hay/straw VegetativeWet rotting leavesNutty Strong to medium Decanoic

acid ethyl esterFresh tobacco dried vegetative weakCompost WeakTreacle weak

Green/sweet hexanalhexanol

Results – Descriptors, Grade “B & C” Truffles

Ammonia/Pungent MediumDamp/Moist WeakClay WeakCaramel WeakDry StrongMushroom StrongFungus Strong to MediumCold Roast Meat WeakSour/Acidic WeakDark Chocolate WeakNutty WeakCompost  StrongEarthy StrongMusky Strong

Wet Fur (dog) Weak

Rotting Leaves/  Compost

Weak

Mould Medium

Dry Weak

Ammonia Strong

Musky Weak

Yeast Medium

Sweet Corn Weak

Mushroom Weak

Damp Weak

Earthy Weak

Grade “B” Grade “C”

Sensory Panel Training

What next?

• Link between sensory data and chemical data

• Data reduction technique – main compounds responsible for sensory stimulus can be separated from other ‘extraneous’ information

• Chemical data can be ‘intelligently’ related to sensory attributes to obtain ‘meaningful’ data to guide industry and solve complex questions

Voice Recorder

Sniff Port

GC/MS output

GC/MS

Time IntensityPerceptual

Measurement

Tenax Trap

GC-O Technique

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Abundance

TIC: LC2E72A.DTIC: CO-72C.D (*)

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OEthyl Butyrate GC/MS profile

Principal aromagram profile

N

NO

“earthy”

O

“violets”

HOO

“caramel”

“fruity”

Volatile and aromagram profiles

Nano-sensor

1. Traditionally, the active sensing material of gas sensors is based on semiconducting metal oxides

2. Limitations of these sensors include:• High operating temperatures • Increased energy consumption• Relatively large physical dimensions required for sufficient gas adsorption

3. In addition, other materials have been utilised including silicon devices, polymer-carbon black composites and organic materials.

Traditional Gas and Vapour Sensors

• High surface area and large aspect ratios• Ability to reduce sensor platform dimensions• Room temperature sensing potential• Low power consumption — i.e. cost effective• Chemical modification capabilities for increased sensitivity and specificity

to target analytes

Carbon Nanotube

• Dynamic vapour deliver system:– Vapour generation from multi-phase analyte (gases, vapours of liquids & solids)– Large dynamic vapour concentration range – ppt to ppm (analyte dependent)– Magnitude of gas analytes & environments– Automated time-based command prompts– Chemical interference studies – humidity, environmental spikes, etc.

Sensor Testing Facility

• Kenny Choo, Honours Student, Curtin University

• Associate Professor Hanna Williams, Curtin University

• Al Blakers – Manjimup Truffles

• Harry Eslick – Wine and Truffle Company

• Adam Wilson – Great Southern Truffles

• Damon Boorman

• Wayne Haslam – Blue Frog Truffles

• Jenna Valentin, University of Western Australia

Acknowledgements