Feb 2014 Standardising the Aroma Bouquet of Australian Black Truffles (T. Melanosporum) Garry S.H. Lee Centre for Forensic Science, The University of Western Australia. Introduction A quality grading system developed using sensory technology and based on chemical signatures (aroma profiles) is proposed to underpin the current truffle grading standards employed by the Australian Truffle Industry. Funded by the Rural Industries Development Corporation and the Australian Truffle Growers Association, in collaboration with the University of Western Australia, the objective of this research was to define the aroma profile (measureable volatile organic compounds) of Extra class Australian black truffles (Tuber Melanosporum) across the range and stages of its development, from immaturity to maturity, and including rot, using a combination of mass spectrometric and sensory techniques. A quality grading scale based on aroma will minimise guesswork (human element) from truffle grading and ensure consistency of product and the maintenance of the highest quality, an essential in such a high value product. In addition to defining the optimum aroma profile of an Extra class truffle, chemical markers specific to growing regions were also identified allowing regional variations of Australian truffles to be defined. This has implications for market preference and can be used to defend the local truffle industries from interstate or international threats The Problem with Grading Standards Australia is in a unique position in that its truffle season occurs during the European summer when fresh truffles are not available. Thus there is a real opportunity for Australia to market its truffles at the European market as a high value product. For Australia to take advantage of its position, it must ensure that its truffles are of the highest quality for export. The issue of quality is not straightforward, as truffle quality is highly subjective and dependent on a number of factors. Currently the Australian grading standard requires truffles to be graded based on physical appearance size, shape and integrity. In practice most truffieres employ the time honoured tradition of experienced truffle sniffers to assess truffles for sufficient characteristic aroma as part of the grading process. This system is wrought with danger as some truffieres have been known to be prejudiced towards their own produce and have knowingly sold lower grade truffles as Extra Class. Furthermore the adopted grading system is susceptible to errors, as demonstrated by the Truffle Catcher exercise and the sensory panel training sessions conducted within this
research. Truffle Catcher showed that 13 out of 18 truffle customers had been disappointed with their truffle purchase at one time. Most of these were associated with grading inconsistencies, specifically poor aroma qualities of an Extra Class truffle. The focus group sessions used in the sensory training panels revealed that the Chef group and Truffle Expert group agreed on the grading of only 5 of 29 truffles when aroma was the only indicator used. Additionally, industry insiders have detailed recent cases where exports to overseas or interstate customers have been the subject of complaints or even rejected due to the poor aroma quality of the truffle. Incidences such as these harm the Australian and individual company brand and have the potential to cause irreversible damage to the Australian truffle industry as French, Italian, Spanish and Asian trade partners look elsewhere. Truffle Catcher Exercise The Truffle Catcher exercise was a simple 6 question survey of 30 well known restaurateurs in the Perth and surrounding regions. It was conducted to get an indication of the truffle needs; specifically aroma, of the main group of customers the Chefs. Due to the small size of the survey group, the results should be used with caution. The results of the survey showed that only 60% of the participants (18 out of 30) used or had access to truffles in 2012. Two of the non-users replied that even though they tried, they could not get a supplier. When asked Where do you source your truffles from?, five of the 18 truffle users, in addition to local, also replied overseas. Of those five, three said that the overseas truffles were better quality than Australian truffles in terms of aroma. Although all 18 truffle users replied that they were happy with Australian truffles, 13 out of the 18 replied that at some stage in the last 5 years (to 2012) they have did not believe that the truffle that they were purchasing was an Extra class or first grade truffle. This was based on their opinion of the aroma. Focus Group and Sensory Panel A list of descriptors (Table 1 and 2) for Extra, first and second class truffles (truffles were graded by the vendor) were developed during the 2011 truffle season with the help of 10 Chefs and a number of seasoned truffle experts. The truffle experts included truffle growers, dog trainers, scientists and people experienced with the handling and sales of truffles. The descriptors were then used to train a panel of volunteers to detect the aroma qualities desired in truffles. The focus group data showed interesting trends in terms of truffle grading and aroma quality. Altogether 29 truffles were used in the focus group from a number of suppliers across Australia. Of those, ten were graded as Extra Class, eleven graded as First Class and eight graded as Second Class by their respective truffieres. Judging on aroma alone, the Chefs ranked ten of the truffles Extra Class, 16 as First Class and three as Second Class. Of the ten ranked by the Chefs as Extra Class, only three of these were graded as Extra Class by the truffiere of origin. In total the Chefs agreed with only seven of the gradings offered by the truffieres (24.1%). In contrast, the Experts group agreed with 51.7% of the gradings offered by the truffiere of origin. This indicates the level of subjectivity that exists in the industry alone.
When asked to grade the truffles on aroma alone, the Chefs and Experts agreed on a grade for only 14 of the 29 truffles. Of the 15 contentious truffles, the Chefs awarded a higher grade for 14 of them. Only one truffle was given a higher grade by the Experts group. These results are in stark contrast to the Truffle Catcher exercise and anecdotal evidence offered by Chefs where the quality of the truffle is believed to be less than its grading. The focus groups were held in a room devoid of external influences, the nasal passages were neutralised after each truffle and the truffle was judged solely on aroma. Thus it is likely that the size and general shape of the truffle has no bearing on the quality of the truffle. Table 1: Descriptors and representatives food for fresh truffle. Main Descriptor Sub Descriptor Representative Samples Sweet Dark Chocolate Lindt Excellence 70% Cocoa Chocolate Hot Toffee Raisin Hot Toffee Sunbeam Raisin Seeded Earthy Flat field Mushroom Flat Field Mushroom Fresh Baked Fruit Cake Coles Smart Fruit Cake Microbiological Dry Yeast Tandaco Yeast Herbs & Leaves Seaweed Obento Sushi Sheets Seaweed Dried Tobacco Champion Ruby Tobacco Table 2: Descriptors and representatives food for second grade truffles. Main Descriptor Sub Descriptor Representative Samples Fruity Over-ripe Fruit Over-ripe Banana Earthy Flat field Mushroom Flat Field Mushroom Woody Damp Wood Damp Wood Microbiological Mould Moulded Mandarin Rotting Leaves Rotting Leaves Fresh Compost Debco Compost For Organic Garden Sour Citric Acid McKenzie s Citric Acid Chemical Pungent/Ammonia Ammonia Volatile Profile of an Australian Black Truffle To determine the characteristic aroma profile that makes up a highly desired truffle, all truffles were re-graded by a trained sensory panelist after 2011. This was necessary as the grading system used by the truffieres proved too inconsistent and highly subjective. For example, the volatile profile of two Extra class truffles from the same truffiere lifted in the same year could differ considerably. Similarly, an extra class truffle and a second class truffle from different truffieres could produce aroma profiles with a match of up to 90%.
Re-grading the truffles by a trained panellist (trained to assess the aroma of truffles like a Chef) removed the subjectivity from the grades. It also ensured that the truffles were judged on aroma alone against a measureable and scientifically robust scale. To avoid confusion, the terminology high, medium and low refer to the grades given by the trained panellist as opposed to extra, first and second class currently used by the industry. All together, over 140 volatile organic compounds were identified from Australian black truffles sourced from 12 different locations across Australia. The key volatiles that define a high class Australian black truffle were found to be dimethylsulphide, 2-methylbutanal, 3- methylbutanal, dimethyl-ether, formic acid 1-methylpropylester, 2-methylbutanol and 1- octen-3-ol. Additionally, 1,2-dimethoxybenzene and 1,3-dimethoxybenzene were also found in approximately 70% of all truffles analysed. These aroma compounds were found to be present consistently over a three year period (2011 2013) from over 98% of all high and medium class truffles. The concentrations of these compounds are dependent on a number of natural and processing variables. Natural variables include growing region, maturity and time after lifting. Processing variables include packaging, storage, modified atmosphere (vacuum) and washing. Notwithstanding processing variables, a suggested characteristic aroma profile of a typical high class Australian black truffle (Tuber Melanosporum) is given in the Table 3. Table 3: Characteristic Australian Truffle Odour Compound Sensory Notes Percentage* dimethyl sulphide Organic, wet Earth, sulfur 8.1 2-methyl butanal Musty, chocolate, nutty 32.5 3-methyl butanal Cacao, almond 0.8 dimethyl ether Etheral trace formic acid 1-methyl propyl ester Cheese 11.7 2-methyl butanol Roasted 2.0 1-octen-3-ol Mushroom, Fungal 0.6 *Typical percentage only. This will differ depending on its natural variables. It is interesting to note that 1-Octen-3-ol, which is one of the five important truffle aroma compounds identified in previous studies, was observed in higher concentrations in medium and lower class Australian truffles than high class graded Australian truffles. For example, for truffles grown in Manjimup, 1-octen-3-ol was found to be present in approximately 20 to 47% of the main components of lower class grade truffles but only as 2.5 2.7% in high class graded truffles. Coincidentally truffles from Canberra (which would have aged at least 1 week before analysis, also showed a high level of 1-octen-3-ol while truffles from Tasmania contained the least amount (0.2 1.8% of the top 20 volatiles). 1-octen-3-ol characteristically gives a mushroom odour and is described as being earthy, green, oily, vegetative or fungal.
Regional Variations in the Volatile Profile of Australian Black Truffles Like all agricultural food products, the environment plays a key role in its chemistry and biochemistry. For truffles, this is especially true for black truffles or Tuber Melanosporum which displays a low degree of genetic diversity. That is, black truffles grown far away from one another are not more genetically diverse than black truffles grown in the same place. Thus any differences in the aroma of black truffles grown in different locations are most likely due to the environment and not genetics. Soil, climate and associated tree species are thought to play an important role. A number of key impact aroma compounds have been identified which may be used to identify the region of origin of Australian Black truffles. Tasmanian truffles contain a high percentage of 3-methyl, 6-propyl phenol. This compound was found as a minor component in a small percentage of truffles grown in other states. They also contained appreciable levels of 4-ethyl-2-methoxy-phenol (or 4-ethyl guaiacol) and 2,3-dimethoxy toluene. The latter was also found in some truffles from NSW. Both compounds were absent in the aroma profile of Western Australian truffles. 4-ethyl-2- methoxy-phenol gives a spicy note while 2,3-Dimethoxy toluene has a hint of cooked vegetables. The main distinguishing aroma component in the volatile profile of Tasmanian truffles, however, was 1-methoxy-3-methyl benzene which gives a floral odor. It comprised of 37 51% of all volatile components. It was also a major component of NSW truffles making up 42.4% of the volatiles from truffles grown in Braidwood. Anisole was found in truffles from Western Australian and NSW but not Tasmania. Anisole imparts a phenolic, gasoline, ethereal or anise type smell. 2,6-dimethyl anisole was found in a high percentage in a number of truffles but was absent in others. Butanoic acid 2 methyl-, 2-methylbutyl ester which gives rise to a fresh greeny type note was found only in Western Australian truffles. 2-Methyl-2-hexanol was only found in the truffle sourced from Bredbo in NSW. It was present in a significant amount accounting for 33% of the top 20 flavour volatiles. Interestingly, this compound is a common descriptor used in ripening of bananas. Finally 1,3-Pentadiene, which has an odour similar to kerosene or acrylic paint, was 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. Prior to this study, it has not been observed in the aroma profile of T. melanosporum grown overseas. Interestingly, when diced, 1,3-pentadiene is released as a major component of the aroma profile (present 27.3% of total volatiles). Final Word As truffle aroma is generally regarded as the truffles primary attribute, it makes sense that a grading system underpinned by a chemical profile or fingerprint of the bouquet of aroma emitted by the truffle is a necessity. As the sustainability of the truffle industry is highly dependent on exports, poor quality truffles have the potential to severely harm the industry and cause irreversible damage to its reputation. There are several reasons as to why there is no agreed truffle grading system based on aroma profiles. This is in spite of the rather substantial volume of work produced by researchers in Europe over the past 30 years which has seen over 300 volatile organic
compounds identified, two formulations for artificial truffle aromas (one used to train dogs) and claims that the main chemical compounds involved in the chemical bouquet emitted by the black truffle have been identified. The main reason is that most if not all of these studies relate to developing a system for locating truffles or for flavour recreation purposes and not truffle grading standards. Moreover, most of these studies were flawed in one or more of the following ways: (i) the quality of the truffles used was not documented; (ii) the truffles were purchased or donated without knowledge of its region of origin and/or time since harvest; (iii) the studies did not account for seasonal variation, (iv) the sample size was small and not representative of the industry, (v) storage and processing of the truffles were not consistent or were unknown and (vi) the sensory evaluations, if any, were conducted separate from spectrometric analysis and thus any correlation between odour and chemical is implied or based on prior experience. Most importantly, none of the previous studies employed a focus group which comprised of the main users and thus the primary customers of the truffle industry, the Chefs, to provide a description of the aroma of a high grade truffle. In order to develop reliable aroma grading models, it is necessary first to understand the relationship between the volatile profiles of truffles and the aroma quality (determined by sensory panels trained using descriptors set by the main group of truffle users). This research accounted for these variables and thus the proposed characteristic aroma profile could be used to standardise all Extra Class graded truffles produced in Australia.