Whitish truffles found in Finland: soil characteristics, and identification based on morphological and molecular properties.

The growing demand for truffles has led to a global pursuit for their occurrence in the wild, and studies concerning their cultivation. Although European countries such as Italy, France and Spain have been known for truffle production, truffle hunting is new to Finland. The present study reports for the first time, the findings of Tuber maculatum in Finland on the basis of morphological and molecular analysis. The chemical characteristics of soil samples collected from the truffle sites have also been discussed. The species of the Tuber samples were identified primarily using morphological analysis. Molecular analysis was carried out for the confirmation of the identity of the species. Two phylogenetic trees were constructed based on internal transcribed spacer (ITS) sequences produced in this study and including representative sequences of whitish truffles available in GenBank. The truffles were identified as T. maculatum and T. anniae. This study could be considered as a basis for encouraging research on findings and identification of truffles in Finland.

Anti-Angiogenic Effect of Cantharellus cibarius Extracts, its Correlation with Lipoxygenase Inhibition, and Role of the Bioactives Therein.

Angiogenesis is a complex physiological process that cannot be treated with single agent therapy. Several edible fungi have been known to encompass bioactive compounds, and are promising sources of multi-component drugs. One such widely consumed edible fungi is Cantharellus cibarius, which has been explored for its biological activities. The present study focused on assessing the anti-angiogenic activity of petroleum ether and ethanol extracts of C. cibarius using chick chorioallantoic membrane (CAM) assay. Both the extracts showed a dose-dependent response which was compared with the anti-angiogenic activity of the positive controls silibinin, and lenalidomide. The extracts were also studied for their lipoxygenase (LOX) inhibitory potential and compared to ascorbic acid as the positive control. The IC50 values of the petroleum ether extract, ethanol extract, and ascorbic acid for LOX inhibition assay were 135.4, 113.1, and 41.5 µg/mL, respectively. Although both the extracts showed similar responses in CAM assay, ethanol extract proved to be more potent in LOX inhibition assay. Finally, the extracts were investigated for their chemical composition using GC-MS. A correlation between LOX inhibition and anti-angiogenic potential was established at the molecular level. A meticulous literature search was carried out to correlate the biochemical composition of the extracts to their anti-angiogenic activity.

Investigation on mycelial growth requirements of Cantharellus cibarius under laboratory conditions.

The golden chanterelle represents one of the commonly found, edible mushrooms that is highly valued in various cuisines. The present study focused on assessing the requirements of Cantharellus cibarius such as pH, temperature, as well as the carbon and nitrogen sources for mycelial growth. Optimization of the growth parameters was carried out by one-factor-at-a-time method. The optimal pH and temperature were determined to be 6.0 and 22.5 °C, respectively. Among the various carbon sources studied, sucrose at a concentration of 2% gave maximum mycelial growth and proved to be the most suitable one. Amongst the nitrogen sources studied, peptone, ammonium sulphate, and sodium nitrate, gave the maximum mycelial growth at an optimized concentration of 0.5%. In the presence of beef extract and yeast extract, a change in colony pigmentation from yellow to dark grey was observed. Finally, the carbon to nitrogen ratio of 2:0.5 proved to be optimal for mycelial growth. This study is the first report on the optimisation of in vitro growth requirements of C. cibarius.

An investigation of the antioxidant potential and bioaccumulated minerals in Tuber borchii and Tuber maculatum mycelia obtained by submerged fermentation.

Submerged fermentation of Tuber borchii and T. maculatum was performed to produce mycelia towards evaluating their bioactive potential and probable toxicity. Truffle mycelia had been mostly explored for biopolymers previously. Methanolic extraction yield, polyphenol content, and flavonoid content varied with species and period of fermentation. In vitro DPPH, FRAP, ABTS and ORAC radical scavenging activity of mycelial extracts of T. maculatum and T. borchii at 7 and 10 days of bioreactor fermentation corroborated with phenolics content. Absence of toxicity and antimutagenicity in lag and log phases in Saccharomyces cerevisiae D7 cells supported possibly safe nutraceutical use of mycelia of both truffles. Mineral content showed variation with species and fermentation time for macro- (calcium, potassium, phosphorus, sodium, iron, magnesium, and manganese) and trace elements (zinc, selenium, copper, chromium, and nickel). Mineral bioaccumulation can be explored for food-fortification applications. The demonstrated activities and detected constituents show potential applications in food, pharmaceutical, cosmetics and nutraceuticals.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization.

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Statistical optimization of media components for production of extracellular lipase from edible mushroom Cantharellus cibarius.

Cantharellus cibarius is a wild edible mushrooms and considered as a plethora of compounds with potential biotechnological applications. This study highlighted the utilization of C. cibarius mushroom in the production of extracellular lipase under submerged fermentation, representing the first report on lipase production by this mushroom. Various physicochemical factors were optimized via one-factor-at-a time (OFAT) method. Maximum enzyme production was recorded when the mushroom mycelium was grown at 30 °C on pH 6.0 for 96 h in the medium supplemented with 1% [(v/v)] olive oil. Productivity of enzyme was affected by variation in the nitrogen sources, carbon sources, metal ions and NaCl salt. Glucose and peptone significantly enhanced enzyme production as carbon and nitrogen sources, respectively. Stimulatory and inhibitory effects were found by Ca2+ and Zn2+ ions, respectively. Furthermore, Box-Behnken Design (BBD) of Response Surface Methodology (RSM) was employed to optimize the interactive effects of specific media components like glucose, olive oil and CaCl2. The regression model was significant with a coefficient of determination (R2) value of 0.9483. Statistically optimized design (RSM) resulted approximately two-fold increase (23.5-42.283 UmL-1) of lipase production than classical optimization method (OFAT), confirmed the validation of model. The kinetic parameters for p-nitrophenyl palmitate hydrolysis, Km and Vmax were 5.24 mM and 0.768 mmol/min/mg respectively, established a high affinity for the substrate.

In silico characterization of bacterial chitinase: illuminating its relationship with archaeal and eukaryotic cousins.

BACKGROUND: Chitin is one of the most abundant biopolymers on Earth, only trailing second after cellulose. The enzyme chitinase is responsible for the degradation of chitin. Chitinases are found to be produced by wide range of organisms ranging from archaea to higher plants. Though chitin is a major component of fungal cell walls and invertebrate exoskeletons, bacterial chitinase can be industrially generated at low cost, in facile downstream processes at high production rate. Microbial chitinases are more stable, active, and economically practicable compared to the plant- and animal-derived enzymes. RESULTS: In the present study, computationally obtained results showed functional characteristics of chitinase with particular emphasis on bacterial chitinase which is fulfilling all the required qualities needed for commercial production. Sixty-two chitinase sequences from four different groups of organisms were collected from the RCSB Protein Data Bank. Considering one suitable exemplary sequence from each group is being compared with others. Primary, secondary, and tertiary structures are determined by in silico models. Different physical parameters, viz., pI, molecular weight, instability index, aliphatic index, GRAVY, and presence of functional motifs, are determined, and a phylogenetic tree has been constructed to elucidate relationships with other groups of organisms. CONCLUSIONS: This study provides novel insights into distribution of chitinase among four groups and their characterization. The results represent valuable information toward bacterial chitinase in terms of the catalytic properties and structural features, can be exploited to produce a range of chitin-derived products.

First report of European truffle ectomycorrhiza in the semi-arid climate of Saudi Arabia.

Tuber melanosporum Vittad. (Black or Périgord truffle) is a truffle native to the Mediterranean Southern Europe, popular for its unique flavor, and has great economic importance. The present work focused on assessing the possibility of cultivating T. melanosporum associated with Quercus robur L. in the desert climate of Saudi Arabia. The plantation was initiated in November 2018 by planting 271 oak seedlings in the Al-Qassim desert area and checked for survival and ectomycorrhiza development after 1.5 years of plantation maintenance. Amongst the 271 seedlings planted, 243 plants survived two harsh seasons (2019 and 2020), and the randomly selected and tested seedlings were still mycorrhized with T. melanosporum. The mycorrhization level with T. melanosporum was between 5 and 35% of all fine roots, and the share of contaminant ectomycorrhiza was low. In comparison to other areas where T. melanosporum is successfully cultivated, the Al-Qassim desert area has 10-15 °C higher average summer temperatures and a low total annual precipitation, which necessitates regular irrigation of the plantation. This work opens the avenue for an adapted, yet sustainable cultivation of T. melanosporum-inoculated oak tree in a desert climatic condition and introduces new opportunities of the agro-forest business in Saudi Arabia and GCC region.

A study on the fatty acid composition of lipids in truffles selected from Europe and Africa.

The gas chromatography of hexane extracts from ascocarps of Tuber maculatum (mostly the first report), T. aestivum/unicantum, T. borchii, T. melanosporum and Tirmania nivea dominantly showed palmitic, stearic, oleic and linoleic acids followed by traces of polyunsaturated fatty acids. The fatty acid content varied from ca. 8-61 mg g- 1, dry-weight-basis with species with highest for T. maculatum. Polyunsaturated fatty acids contributions varied from ca. 42-59%. The dominant fatty acid varied with the species. A comparison with existing reports on same species cultivated in different regions showed differences in contributions by saturated, monounsaturated and polyunsaturated fatty acids as well as dominant fatty acids detected. Lesser explored species such as T. borchii, T. maculatum call for further research. This is a preliminary study that indicates fatty acid composition as a potential tool for distinction like aroma between truffle species and geographies of cultivation. This forms the basis for further studies in different species and regions.

Anti-angiogenic and anti-inflammatory activity of the summer truffle (Tuber aestivum Vittad.) extracts and a correlation with the chemical constituents identified therein.

Fungi are a huge source of unexplored bioactive compounds. Owing to their biological activities, several fungi have shown commercial application in the health industry. Tuber aestivum Vittad. is one such edible fungi with an immense scope for practical biological applications. In the present study, the anti-angiogenic activity of petroleum ether and ethanol extracts of T. aestivum was investigated using the chick chorioallantoic membrane assay and compared to the positive controls silibinin and lenalidomide. Both the extracts showed a dose-dependent anti-angiogenic response. The extracts were also assessed for their anti-inflammatory potential by lipoxygenase-inhibition assay. The IC50 values for LOX inhibition assay, computed by the Boltzmann plot, were 368.5, 147.3 and 40.2 µg/mL, for the petroleum ether extract, ethanol extract, and the positive control ascorbic acid, respectively. The ethanol extract of T. aestivum showed superior anti-angiogenic and anti-inflammatory activity than the petroleum ether extract. Compositional investigation of the extracts by GC-MS revealed the presence of various bioactive compounds. The compounds were correlated to their anti-angiogenic and anti-inflammatory activity based on a meticulous literature search.

Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation.

Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH 7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5 % w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70 U/mL of cellulase in the cell-free supernatant after 7 days of incubation time. The optimum of the obtained cellulase's activity was at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 99 % of its maximal activity over an incubation time of 100 min. The cellulase activity was inhibited by Fe2+ and slightly activated by Mn2+ and Cu2+ at 1 mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

Characterization of natural habitats and diversity of Libyan desert truffles.

Desert truffles have traditionally been used as food in Libya. Desert truffle grows and gives fruit sporadically when adequate and properly distributed rainfall occurs with existence of suitable soil and mycorrhizal host plant. The present study aimed to identify and characterize two kinds of wild desert truffles from ecological and nutritional points that were collected from the studied area. The truffle samples were identified as Terfezia (known as red or black truffle) and Tirmania (known as white truffle). The nutritional values (protein, lipid and carbohydrate) of both Libyan wild truffle (Terfezia and Tirmania) were determined on a dry weight basis and result showed that Tirmania and Terfezia contained 16.3 and 18.5% protein, 6.2 and 5.9% lipid, 67.2 and 65% carbohydrate, respectively, in ascocarp biomass. The soil pH of the upper and lower regions of the Hamada Al-Hamra ranged between 8.2 and 8.5 giving suitable conditions for fructification. The plants, Helianthemum kahiricum and Helianthemum lippii were the dominant plants in Hamada Al-Hamra region found to form a mycorrhiza with desert truffles. The phylogenetic analysis of the genomic rDNA ITS region showed that, out of five collections three represented Tirmania pinoyi (Maire) Malencon, one Tirmania nivea (Desf.) Trappe, and one Terfezia boudieri Chatin.