Facultad de Ciencias

Eva Tejedor Calvo

Directores: Dra. Cristina Soler Rivas y Dr. Pedro Marco Montori

Nowadays, the market for food products containing back truffles (Tuber melanosporum) is increasing as response to restaurants and supermarkets demands. However, truffle availability is scarcely due to is a seasonal product together with short shelf-life, so the food industry follows two strategies: to supplement food matrices with cheaper truffles species (with similar morphological characters) and/or to add artificial aroma to simulate that they include truffles or to compensate the aromatic loss. However, the flavoring enhancers offered at markets do not resemble the fresh black truffle aroma mainly because of its complexity. On the other hand, apart from their aromatic compounds, truffles, as well as mushrooms, contain several bioactive compounds (fungal sterols, b-D-glucans, among others) with potential bioactivities that are still scarcely investigated. Therefore, the aim of this PhD thesis was to design different extraction methods using environmentally friendly technologies to obtain flavoring and bioactive enriched fractions from truffles species. Since the fresh and good quality truffles have already their business place in the market, this work was focus on the val orization of low quality
truffles to generate natural aromatic extracts and bioactive fractions that can be used to design novel functional foods. Thus, the developed experiments were divided in two sections, one to achieve natural aromatic extracts from t ruffles (Part 1) and the other to obtain bioactive ingredients to functionalize foodstuff (Part 2).

In the first part of this research, a previous screening through forty-one truffled products purchased at supermarkets was performed to detect the type of aroma and truffles species added. A sensory evaluation was carried out where consumers indicated the main attributes that were for them considered as good quality truffled products. The non-frequent truffle consumer group associated negative attributes with real black truffle products. On the contrary, frequent fresh truffle consumers valued positively products without artificial aromas added. Results indicated that consumers need to be informed about differences between truffle species and product labelling, and that it was essential to deep into black truffle aromatic compounds.

Thus, aromatization of different food material (milk, egg yolk, grape seed oil and sunflower oil) was followed by olfactometry to select the more adequate food matrix to trap aromatic compounds. After a 24 h odorization, the key truffle aroma could be detected in all the products, but grape seed oil was selected as a potential trapping material because it retained many flavoring compounds and was odorless.

Afterwards, truffles were submitted to supercritical CO2 extractions to obtain fractions containing volatile organic compounds (VOCs) that were identified and quantified by head space gas chromatography and olfactometry. Other molecules related with truffle aroma such as fatty acids and fungal sterols were also evaluated in the extracts using supercritical fluid chromatography system coupled with electrospray ionization quadruple time-of-flight mass spectrometry (UHPSFC/ESI-QTOF-MS). The aroma released during depressurization could also be trapped with grape seed oils, gelatin, or agar-agar materials indicating that supercritical fluid extraction (SFE) was an interesting method to produce natural flavoring fractions.

In the second part of the work, the bioactive compounds present in black (T. melanosporum) and summer (Tuber aestivum) truffles -glucans, chitins, sterols and phenolic compounds were evaluated together with other nutritive constituents (carbohydrates, proteins). The effect on the mentioned compounds of preservative treatments such as electron-beam or gamma irradiations was followed as well as their evolution after modified atmosphere packaging (MAP) storage. Results indicated that bioactive compounds levels were maintained stable during longer period (35 and 42 days for T. melanosporum and T. aestivum respectively) than control truffles without the treatments. The biosynthesis of phenolic compounds was stimulated by irradiation because a higher level of these compounds was noticed after 7 storage days.

Truffles were also submitted to pressurized liquid extractions (PLE) to obtain fractions with high content of bioactive compounds. Firstly, extraction methods were optimized by testing different parameters such as extraction time, temperature and solvent following a response surface methodology (RSM). The conditions were adjusted using three different truffle species as model, two from the Tuber genus (T. melanosporum, T. aestivum) and a third one that was a desert truffle (Terfezia claveryi) and afterwards, the optimal conditions were applied to several other species including other desert truffles. Extractions using water at 180ºC and 16.7 MPa were more suited to obtain b-glucan enriched fractions while ethanol with same parameters was a better solvent to extract fungal sterols and phenolic compounds.

According to nuclear magnetic resonance (NMR) studies, T. melanosporum contained mainly - -glucans, other constituents (detected by GC-MS) were ergosterol and brassicasterol as main sterols, particular interesting fatty acids (oleic and linoleic acids), tocopherols, organic (oxalic and fumaric acid) and phenolic acids (gallic aid, protocatechuic acid, p-Hydroxybenzoic acid, among others) as well as certain soluble sugars that were also noticed by HPLC. Similarly, the other truffle species showed similar compositions, except for T. magnatum that showed interesting higher levels of phenolic compounds. Polysaccharides from T. aestivum and T. claveryi were able to reduce secretion of two pro-inflammatory cytokines (40% of IL- -6) suggesting a potential immunomodulatory activity. Moreover, T. claveryi extracts were more effective than T. aestivum on inhibiting secretion on macrophages cultures. Other truffle species also showed interesting bioactivities, i.e Tuber magnatum showed a high radical scavenger capacity (using DPPH, FRAP, ORAC, reducing power tests) while only Tuber magnusii showed a slightly antimicrobial activity against gram+ and gram- bacteria. The cytotoxic potential against four tumour cell lines of dessert truffles (Terfezia genus) was higher than Tuber spp. suggesting potential as anti-proliferative agents.

When PLE extracts were obtained from those truffles, the bioactive compounds were extracted and concentrated, and they did not lose some of the noticed bioactivities. Extracts obtained using water as solvent contained (1-3),(1-6)-b-D-glucans, chitins and other heteropolymers with galactose and mannose in their structures. Those obtained with ethanol showed beside ergosterol and brassicasterol, other sterols such as stigmasterol and ergosta7,22-dienol. The latter compounds were described for mushrooms as hypochelesterolemic compounds, and those b-glucans positively modulated human intestinal microbioma. Moreover, the ethanol extracts (more than water or water:ethanol extracts) were able to inhibit enzymes related with diabetes type-2 (a-amylase and a-glucosidase). Thus, PLE could be an interesting tool to obtain truffle
extracts with potential to design novel functional foods.