Black Truffle Aroma Evaluation: SPME-GC-MS vs. Sensory Experts.

Nowadays, the truffle aroma attribute is not included as a quality parameter in the current recommendation that explains the truffle quality (UNECE standard 53 FFV3) and establishes the truffle commercial categories. However, the aroma is the main reason why truffles are worldwide appreciated. Indeed, more than 30 aromatic molecules compose it, and this is the reason why the human evaluation and identification of these odorants, without previous training, is quite subjective. Analytical techniques such as gas chromatography techniques, however, can establish an aromatic profile and detect potential aromatic markers. In this study, 16 tasting experts were trained to make more objective the truffle aroma evaluation and odorants identification. For this, a comparison between solid-phase microextraction gas chromatography coupled with mass spectrometry (SPME-GC-MS) and sensory expert evaluation was carried out in six sessions during different harvesting times in the black truffle season (December, January, and February). Both techniques were able to separate truffles depending on the harvesting time. Also, a list of volatile organic compounds related to the aromatic attributes was reported. This information will help to provide a more objective T. melanosporum truffle sensory evaluation.

Aromatic profile of black truffle grown in Argentina: Characterization of commercial categories and alterations associated to maturation, harvesting date and orchard management practices.

Black truffle (Tuber melanosporum) is one of the most appreciated fungi in the world mainly due to its aromatic properties. In the emerging markets such as Argentina, the aroma of locally produced truffles has not been described yet. The volatile organic compounds (VOCs) from 102 black truffles from Argentina were analyzed using solid phase microextraction gas chromatography coupled with mass spectrometer detector (SPME-GC-MS). Several factors such as commercial category, maturity stage, host tree, geographical origin, and aromatic defects detected during classification were also registered and considered. As a result, 79 VOCs were detected, among which 2-methyl-propanal, 2-butanone, 2-methyl-1-propanol, butanal-3-methyl, 3-methyl-1-butanol, 2-methyl-1-butanol were present in high percentage in fresh mature truffles, whereas immature truffles were associated with 3,5-dimethoxytoluene, 2-phenyl-2-butenal, 2,3-dimethoxytoluene. The Argentine black truffles showed significant similarities in their aromatic profile when compared with their Australian and European counterparts, but with some distinctive notes.

Interannual dynamics of Tuber melanosporum and fungal communities in productive black truffle orchards amended with truffle nests.

Truffle growers devote great efforts to improve black truffle productivity, developing agronomic practices such as 'truffle nests' (peat amendments that are supplemented with truffle spore inoculum). It has been hypothesized that improved fruiting associated with nests is linked to stimulation of truffle mycelia previously established in soil or to changes generated in soil fungal community. To assess this, we used real-time PCR to quantify black truffle extraradical mycelium during 2 years after nests installation. We also characterized the fungal community via high-throughput amplicon sequencing of the ITS region of rRNA genes. We found that neither the abundance of truffle mycelium in nests nor in the soil-nest interphase was higher than in the bulk soil, which indicates that nests do not improve mycelial growth. The fungal community in nests showed lower richness and Shannon index and was compositionally different from that of soil, which suggests that nests may act as an open niche for fungal colonization that facilitates truffle fruiting. The ectomycorrhizal fungal community showed lower richness in nests. However, no negative relationships between amount of truffle mycelium and reads of other ectomycorrhizal fungi were found, thus countering the hypothesis that ectomycorrhizal competition plays a role in the nest effect.

Pressurized Liquid (PLE) Truffle Extracts Have Inhibitory Activity on Key Enzymes Related to Type 2 Diabetes (α-Glucosidase and α-Amylase).

An optimized PLE method was applied to several truffle species using three different solvent mixtures to obtain bioactive enriched fractions. The pressurized water extracts contained mainly (1 → 3),(1 → 6)-ß-D-glucans, chitins, and heteropolymers with galactose and mannose in their structures. The ethanol extracts included fatty acids and fungal sterols and others such as brassicasterol and stigmasterol, depending on the species. They also showed a different fatty acid lipid profile depending on the solvent utilized and species considered. Ethanol:water extracts showed interesting lipids and many phenolic compounds; however, no synergic extraction of compounds was noticed. Some of the truffle extracts were able to inhibit enzymes related to type 2 diabetes; pressurized water extracts mainly inhibited the α-amylase enzyme, while ethanolic extracts were more able to inhibit α-glucosidase. Tuber brumale var. moschatum and T. aestivum var. uncinatum extracts showed an IC50 of 29.22 mg/mL towards α-amylase and 7.93 mg/mL towards α-glucosidase. Thus, use of the PLE method allows o bioactive enriched fractions to be obtained from truffles with antidiabetic properties.

Black truffle aroma transfer kinetics to food matrices.

Nowadays black truffles are so highly valued that truffled products are available in supermarkets whereas fresh truffle is mainly used in the restaurants. It is known that truffle aroma can change because heat treatments, but there is no scientific evidence about what molecules are transferred, in which concentration, and how much time is needed to aromatize products with truffle. In this study, four different fat-based food products (milk, sunflower oil, grapeseed oil and egg's yolk), were used to study black truffle (Tuber melanosporum) aroma transference for 14 days. Gas chromatography and olfactometry results showed different volatile organic compounds profile depending on the matrix used. After 24 h, some key truffle aromatic compounds were detected in all the food matrices. Among them, grape seed oil was the most aromatized product probably because of its odorless properties. According to our results, dimethyl disulphide, 3-methyl-1-butanol and 1-octen-3-one odorants showed the highest aromatization power.

Aromatic changes in home-made truffle products after heat treatments.

Truffles are highly valued by their aromatic properties and can aromatize food products. However, the truffle aroma could be reduced or lost with heat treatments (pasteurization and sterilization) necessary for products security and safety. In this study, sunflower oil and honey were aromatized with black truffle (lyophilized and fresh) using two different concentrations (5 and 10 %) for 24 h and then heat treatments (pasteurization and sterilization) were carried out. Truffle organic volatile compounds from products were investigated by SPME-GC-MS and sensory analysis by trained panel. More than 80 compounds were detected. Some of them were affected differently by heat process depending on the food matrix. Professional tasters scored higher key aromatic attributes such as sulphurous and olive oil in fresh truffle products, regardless the heat treatment applied.

Extraction and trapping of truffle flavoring compounds into food matrices using supercritical CO2.

A supercritical fluid extraction methodology was used to extract flavoring and bioactive compounds from truffles. Some parameters such as CO2 flow rate (1-3 mg/mL), extraction time (15-90 min) and different trapping food matrices (grape seed oil, gelatin, agar agar and water) were optimized using response surface methodology to enhance extraction and trapping yields. The optimal conditions (2.27 mg/mL CO2 flow rate, 82.5 min when using 40 °C and 30 MPa, with 1 mL grape seed oil as trapping matrix) obtained with Tuber melanosporum were applied to three different truffle species: Terfezia claveryi, Tuber aestivum and Tuber indicum. A total of 32 metabolites were profiled in the extracts using ultra-high-performance supercritical fluid chromatography coupled to quadrupole time-of-flight mass spectrometry. Compounds such as brassicasterol ergosta-7,22-dienol, oleic and linoleic acid were found at similar amounts in all the extracts but other molecules (e.g. fungal sterols) showed a particular distribution depending on the specie studied and whether a trapping matrix was used at the SFE outlet.

Application of Pressurized Liquid Extractions to Obtain Bioactive Compounds from Tuber aestivum and Terfezia claveryi.

A PLE (pressurized liquid extraction) method was adjusted following a full-factorial experimental design to obtain bioactive-enriched fractions from Tuber aestivum and Terfezia claveryi. Temperature, time and solvent (water, ethanol and ethanol-water 1:1) parameters were investigated. The response variables investigated were: obtained yield and the levels of total carbohydrate (compounds, ß-glucans, chitin, proteins, phenolic compounds and sterols). Principal component analysis indicated water solvent and high temperatures as more adequate parameters to extract polysaccharide-rich fractions (up to 68% of content), whereas ethanol was more suitable to extract fungal sterols (up to 12.5% of content). The fractions obtained at optimal conditions (16.7 MPa, 180 °C, 30 min) were able to protect Caco2 cells from free radical exposure, acting as antioxidants, and were able to reduce secretion of pro-inflammatory cytokines in vitro: IL-6 (50%), and TNFα (80% only T. claveryi ethanol extract), as well as reduce high inhibitory activity (T. aestivum IC50: 9.44 mG/mL).

Chemical composition and evaluation of antioxidant, antimicrobial and antiproliferative activities of Tuber and Terfezia truffles.

Ten truffle species of Tuber and Terfezia genera were chemical characterized, assessing their proximate composition, individual nutrient compounds and some bioactive molecules. The bioactive properties of these species were also evaluated, namely their antioxidant, antimicrobial and cytotoxic potential. Carbohydrates were the main macronutrients present in truffles, followed by proteins. Furthermore, the levels of polyunsaturated fatty acids (PUFA), subsequently presented as a percentage, were higher in truffles (38.2-79.3%) except in Tuber magnatum and Terfezia arenaria, which have a more saturated fatty acids (SFA) profile (70.7% and 53.7%, respectively). Comparing the species, T. magnatum revealed the highest levels of total phenolic compounds (TPC) (290 mg GAE/100 g truffle), as also the best results in the four methods used to evaluate the antioxidant activity. On the other hand, only five extracts obtained from some studied truffle species (Terfezia magnusii, Tuber aestivum, Tuber gennadii, and Tuber melanosporum) showed a slight inhibition of microbial growth, tested against different bacteria. Terfezia and T. gennadii extracts, showed potential to inhibit the cellular growth of NCI-H460, HeLa, HepG2, and MCF-7 cell lines (GI50 concentrations range: 19-78, 33-301, 83-321 and 102-321 µg/mL, respectively), indicating anti-proliferative activity. Nevertheless, T. arenaria revealed some potential hepatotoxicity, inhibiting the growth of PLP2 cells (GI50 concentration of 220 µg/mL), a primary cell culture obtained from porcine liver.

Lack of Linkages among Fruiting Depth, Weight, and Maturity in Irrigated Truffle Fungi Marks the Complexity of Relationships among Morphogenetic Stages.

The highly prized black truffle (Tuber melanosporum) has become a model species for ectomycorrhizal fungi biology. However, several questions concerning its reproductive phase remain unanswered. To provide new hypotheses on the fruitbody formation process, we have explored the causal links among development characters of black truffle fruitbodies that are primarily linked to either the mating process, fruitbody growing stage, or maturation. Path analysis was applied to test causal models outlining the relationships among fruitbody development characters such as fruiting depth, weight, shape, and spore maturity. These characters were investigated over a two-season survey and three soil typologies (plus peat-based substrate) under irrigated conditions. We found a clear and generalized relationship between fruitbody weight and shape. Among clusters of fruitbodies we found a positive relationship between the weight of the largest fruitbody and the weight of the remaining fruitbodies. However, no generalized relationships among characters linked to different development stages appeared. Our results were noticeably consistent across soil typologies, both for fruitbodies growing singly and in clusters, indicating that early-developing fruitbody characters did not influence characters linked to subsequent morphogenetic stages. The lack of links among stages opens new perspectives for pre-harvest quality management with stage-specific cultivation practices.

Glyphosate treatments for weed control affect early stages of root colonization by Tuber melanosporum but not secondary colonization.

The cultivation of the ectomycorrhizal fungus Tuber melanosporum has considerably spread in recent years throughout the world. During the first years of truffle cultivation, weed control is a key practice to improve the establishment of host trees and the proliferation of the fungus in the soil. Glyphosate is nowadays the most commonly used herbicide in Spanish truffle orchards. We explored the effect of glyphosate on the proliferation of T. melanosporum mycorrhizae, on extraradical mycelium and on the inoculum potential of T. melanosporum spores in greenhouse experiments using Quercus ilex seedlings as host plants. No detrimental effect on the secondary infection of T. melanosporum was found after three sequential glyphosate applications in young seedlings during one vegetative period. Instead, a change in the distribution of fine roots and T. melanosporum mycorrhizae along soil depth was observed. On the other hand, results indicate that high application rates of glyphosate hinder the infectivity of T. melanosporum spore inoculum, without apparent impact on the host performance. Our results suggest that glyphosate has the potential to jeopardise the role of the soil spore bank as inoculum source for the colonisation of new roots, also raising the question of whether glyphosate could hinder the presumed role of spores in sexual mating.

Forest bioeconomy in the media discourse in Spain.

Media can play a key role in shaping public opinion and setting a policy agenda by conveying and influencing public discourse. This article evaluates how the Spanish media has covered the topic of the forest bioeconomy and what kind of discourse it has produced and reproduced around it. For this purpose, we analysed the content of 204 national and regional newspaper articles. The results reveal the scarce penetration of the forest bioeconomy in media and some weaknesses in the narrative production and communicative dimension. The discourse is mainly constructed by governments with a limited presence of multiple stakeholders and an absence of conflict and divergent or alternative views. In addition, the discourse only addresses regional or local problems within the framework of an extended and dominant paradigm of economic growth considering the forest bioeconomy as an opportunity to combat fire, rural abandonment, smallholdings, and poor forest management. We conclude that media is not using its strategic potential and capacity as a public space. To become agents of change, the media should reflect a forest bioeconomy based on successful experiences of innovation and valorization, and adopt a transformative social vision that gives relevance to the interconnection between multiple stakeholders, forest multifunctionality and rural development.

Effects of gamma irradiation on the shelf-life and bioactive compounds of Tuber aestivum truffles packaged in passive modified atmosphere.

The effects of gamma irradiation (0.5, 1.0, 1.5 and 2.5 kGy doses) on Tuber aestivum packaged under modified atmosphere was evaluated. The respiration rate, microbial populations, sensory characteristics and content of bioactive compounds (total carbohydrates, chitins, ß-glucans, proteins, total phenols and sterols) were monitored from immediately after treatment up to day 42 of storage at 4 °C. All the irradiation treatments tested reduced the microbial groups studied by more than 3 log cfu/g. Increasing irradiation doses slowed down the subsequent microbial development throughout the conservation period for all the groups studied. The irradiation treatments did not negatively affect truffle sensory characteristics. Only a slight visible superficial yeast growth was detected at the end of the shelf-life in all doses applied. Total carbohydrate content, chitins, ß-glucans and proteins levels were not affected after irradiation. However, sterols, particularly stigmasterol, slightly decreased after irradiation, while levels of phenolic compounds doubled during storage. Gamma irradiation (2.5 kGy) could be used to extend the shelf-life of summer truffles packaged under modified atmosphere, since no remarkable reduction of bioactive compounds were noticed after 42 days of storage, and their sensory and microbial parameters were of higher quality than those of non-irradiated controls.

Screening of bioactive compounds in truffles and evaluation of pressurized liquid extractions (PLE) to obtain fractions with biological activities.

Truffles, besides the appreciated aromatic compounds, contain other molecules with interesting bioactive properties. A screening of fungal sterols and ß-glucans within different truffle species and locations was carried out. These compounds were extracted with pressurized liquids (PLE) generating enriched fractions. Extraction efficiency was studied with a full-factorial experimental design (Response surface methodology, RSM), using water and ethanol as extraction solvents. Polysaccharides from truffle powder (TP) and the optimal PLE extract (EP) obtained were precipitated and analysed by NMR and GC-MS. THP-1 cell cultures were utilized to test immunomodulatory properties. With the optimal PLE conditions (16.7 MPa, 180 °C, 30 min) 64 and 22.5% yields were obtained respect, with water and ethanol, generating fractions containing respect, 9.1% ß-glucan and 4.5% ergosterol. NMR analyses detected (1 â†’ 3)-ß-glucan structures in truffle. The EP induced a reduction of 40% IL-1ß and 60% IL-6 pro-inflammatory cytokines secretion suggesting potential immunomodulatory activity.

Multi-platform metabolomic approach to discriminate ripening markers of black truffles (Tuber melanosporum).

Black truffle is characterized by a black ascocarp and white veins. This hypogeous fruit body is known for its aroma. Understanding metabolic variation during ripening can shed light on truffle biology. In this work, the comprehensive polar metabolome and the volatile organic compounds of T. melanosporum were studied at different ripening stages by means of a metabolomic approach using GC-MS. Multivariate statistical data analysis indicated that the metabolic profile changed during ripening and that the metabolites that mostly discriminated truffles in the early ripening stages belonged to the classes of carbohydrates, while free fatty acids and amino acids, among which precursors of VOCs, characterized the late stages of ripening. Principal component analysis of the volatilome indicated that dimethylsulfide and dimethyldisulfide characterized most of the samples collected in December-January, while 1-octen-3-ol samples collected in February-March.

Edaphic and temporal patterns of Tuber melanosporum fruitbody traits and effect of localised peat-based amendment.

In Tuber melanosporum cultivation, fruitbody traits are gaining relevance due to their increasing prominence on prices. We investigated the edaphic and temporal patterns of fruitbody traits and characterised the effect of truffle nests (localised peat-based amendment supplemented with T. melanosporum spores) on traits. We monitored fruitbody traits throughout two fruiting seasons in three blocks along a soil gradient. Each trait followed specific edaphic and temporal patterns. The number of fruitbodies per dig and spore maturity followed characteristic within-season trends, whereas fruitbody weight and infestation by truffle beetles were subject to complex interactions among edaphic and temporal variables, suggesting a relevant influence of annual environmental conditions. The application of truffle nests increased fruitbody depth, improved its shape and decreased infestation by truffle beetles. Nests increased the number of fruitbodies per dig, but only in two of the soils, suggesting a relevant role of the bulk soil/substrate interface in fruiting initiation. These results outline a complex scenario, with each trait being differently affected by environmental factors. In this scenario, nests proved to effectively modify several traits, although not always in the desired direction.

Draft Genome Sequence of the Ectomycorrhizal Ascomycete Sphaerosporella brunnea.

Sphaerosporella brunnea is a pioneer ectomycorrhizal fungus with facultative saprophytic capacities. Here, we sequenced the genome of S. brunnea strain Sb_GMNB300, which is estimated at 51.6 Mb in size with 872 assembled contigs accounting for 12,597 predicted coding genes. This genome will be useful for comparative studies of Pezizales ectomycorrhizal symbioses.

Intensive Care to Facilitate Organ Donation: A Report on the Experience of 2 Spanish Centers With a Common Protocol.

BACKGROUND: The aim of this study is to report the experience with a program of Intensive Care to facilitate Organ Donation (ICOD) in 2 Spanish centers based on a common protocol. METHODS: Retrospective review of clinical charts of patients with a devastating brain injury whose families were approached to discuss the possibility of ICOD once further treatment was deemed futile by the treating team. Study period is from January 1, 2011, to December 31, 2015. RESULTS: ICOD was discussed with families of 131 patients. Mean age of possible donors was 75 years (SD = 11 years). The main cause of brain injury was an intracranial hemorrhage (72%). Interviews with families were held after the decision had been made not to intubate/ventilate in 50% of cases, and after the decision not to continue with invasive ventilation in the remaining cases. Most interviews (66%) took place in the emergency department. The majority of families (95%) consented to ICOD. Of the 125 consented cases, 101 (81%) developed brain death (BD), most in 72 hours or less. Ninety-nine (98%) patients transitioned to actual donation after BD, with 1.2 organs transplanted per donor. Of patients who did not evolve to BD, 4 died after an unexpected cardiac arrest and 18 after the withdrawal of life-sustaining measures. ICOD contributed to 33% of actual donors registered at both centers. CONCLUSIONS: ICOD is well accepted by families. Most patients evolve to BD within a short period of time. The practice substantially contributes to increasing organ donation and offers more patients the chance of donating their organs after death.

Black Truffle Harvesting in Spanish Forests: Trends, Current Policies and Practices, and Implications on its Sustainability.

The European black truffle is a mycorrhizal fungus native to Spanish Mediterranean forests. In most Spanish regions it was originally commercially harvested in the second half of the 20th century. Experts agree that wild truffle yields suffered a sharp decline during the 1970s and 1980s. However, official statistics for Spanish harvest are scarce and seemingly conflicting, and little attention has been paid to the regime for the exploitation of truffle-producing forests and its implications on the sustainability of this resource. Trends in harvest from 1969 to 2013 and current harvesting practices were analyzed as a case study, taking into account that Spain is a major truffle producer worldwide, but at the same time truffles have only recently been exploited. The available statistical sources, which include an increasing proportion of cultivated truffles since the mid-1990s, were explored, with estimates from Truffle Harvesters Federation showing higher consistency. Statistical sources were then compared with proxies for wild harvest (rents from truffle leases in public forests) to corroborate time trends in wild harvesting. Results suggest that black truffle production is recovering in recent years thanks to plantations, whereas wild harvest is still declining. The implications of Spanish legal and institutional framework on sustainability of wild truffle use are reviewed. In the current scenario, the decline of wild harvest is likely to continue and eventually make commercial harvesting economically unattractive, thus aggravating sustainability issues. Strengthening of property rights, rationalization of harvesting pressure, forest planning and involvement of public stakeholders are proposed as corrective measures.

Potential aromatic compounds as markers to differentiate between Tuber melanosporum and Tuber indicum truffles.

The Tuber indicum (Chinese truffle) and Tuber melanosporum (Black truffle) species are morphologically very similar but their aromas are very different. The black truffle aroma is much more intense and complex, and it is consequently appreciated more gastronomically. This work tries to determine whether the differences between the aromatic compounds of both species are sufficiently significant so as to apply them to fraud detection. An olfactometric evaluation (GC-O) of T. indicum was carried out for the first time. Eight important odorants were identified. In order of aromatic significance, these were: 1-octen-3-one and 1-octen-3-ol, followed by two ethyl esters (ethyl isobutyrate and ethyl 2-methylbutyrate), 3-methyl-1-butanol, isopropyl acetate, and finally the two sulfides dimethyldisulfide (DMDS) and dimethylsulfide (DMS). A comparison of this aromatic profile with that of T. melanosporum revealed the following differences: T. indicum stood out for the significant aromatic contribution of 1-octen-3-one and 1-octen-3-ol (with modified frequencies (MF%) of 82% and 69%, respectively), while in the case of T. melanosporum both had modified frequencies of less than 30%. Ethyl isobutyrate, ethyl 2-methylbutyrate and isopropyl acetate were also significantly higher, while DMS and DMDS had low MF (30-40%) compared to T. melanosporum (>70%). The volatile profiles of both species were also studied by means of headspace solid-phase microextraction (HS-SPME-GC-MS). This showed that the family of C8 compounds (3-octanone, octanal, 1-octen-3-one, 3-octanol and 1-octen-3-ol) is present in T. indicum at much higher levels. The presence of 1-octen-3-ol was higher by a factor of about 100, while 1-octen-3-one was detected in T. indicum only (there was no chromatographic signal in T. melanosporum). As well as showing the greatest chromatographic differences, these two compounds were also the most powerful from the aromatic viewpoint in the T. indicum olfactometry. Therefore, either of the two chromatographic methods (GC-O or HS-SPME-GC-MS), together or separately, could be used as a screening technique to distinguish between T. indicum and T. melanosporum and thus avoid possible fraud.