Determination of key volatiles in Australian Black Truffles aroma by combined GCMS and organoleptic techniques
Professor Garry LeeCentre For Forensic [email protected] 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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Abundance
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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Abundance
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
7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.500
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Abundance
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
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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1e+07
1.05e+07
1.1e+07
1.15e+07
1.2e+07
1.25e+07
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Abundance
TIC: LC2E72A.DTIC: CO-72C.D (*)
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0 5 10 15 20 25 30 35 40Time (mins)
Odo
ur In
tens
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LCBCO
O
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