Chinese Truffle Research Articles

Extraction of water-soluble polysaccharides (WSPS) from Chinese truffle and its application in frozen yogurt

WSPS from truffles are very valuable source which can be used in the areas of foods as natural functional ingredient. In this work, WSPS were extracted from Chinese truffle by RSM combined with single-factor screening, and applied into milk to make a frozen yogurt. Optimum conditions were: temperature 98.96–100°C, time 4.94–5.37h, particle size 155.91–164.77 mesh, water to truffle ratio 46.66–49.3:1 and pH value at 7.0. These optimum conditions yielded WSPS of 12.89–13.03%, which is well matched with the predictive yield. WSPS prepared contained 86.37% carbohydrates, 3.15% proteins, 7.54% moisture and 2.88% ash. WSPS consisted of glucose, mannose, galactose, and lyxopyranose. Based on the experiments of the addition, the presence of WSPS reduced significantly the syneresis, improved the viscosity and titratable acidity and contributed better flavour in the FYP yogurt in comparison to the FYC yogurt, which provided important insight in the potential commercialization of WSPS as a functional component of yogurt.

A review of research on Chinese Tuber species

Truffles are abundant in some regions of China. Nevertheless, it was not until the 1980s that Tuber species were discovered by Chinese mycologists. In recent years, international truffle markets have shown an increasing interest in the import of Chinese Tuber. These truffles serve as a complement to European truffles due to their lower prices and their greater availability in a deficit market. However, Chinese truffles have been the subject of fraudulent commercial practices, and these could have a negative effect on truffle culture. These concerns have been behind numerous recent studies designed to obtain detailed information about Chinese Tuber species. Unfortunately, many of these studies are not published in English, and are dispersed throughout the specific local or national bibliography and proceedings of specialized truffle conferences. In view of the need to expand current knowledge of Chinese Tuber species, we present a comprehensive summary of the taxonomy, ecology, mycorrhizae, genetics, biochemistry, and cultivation of Chinese Tuber species. We also provide a synthetic taxonomy and morphological characterization of 16 Chinese Tuber species in order to assist in their verification and monitoring.

Truffle Wars

Chinese truffles, close relatives of the French black truffle, arrived on world markets in the middle of the 1990s. Available in quantity at low prices, they were eagerly snapped up by traders facing a steep decline in European production. Despite having less intense flavour, they have captured a large part of world trade in truffles, and their harvest has brought significant economic benefits to rural communities in Sichuan and Yunnan provinces of southern China. In parallel, the development of artificial truffle flavouring has led to an explosion in ersatz ““truffle”” products, and the practice in many restaurants of boosting the flavour of inferior truffles with artificial truffle oils. However, recent expansion of truffle growing in North America and the arrival of southern hemisphere producers from New Zealand and Australia is extending both the quantity of truffle available and the season.

New data on ectomycorrhizae and soils of the Chinese truffles Tuber pseudoexcavatum and Tuber indicum, and their impact on truffle cultivation

Chinese truffles serve as a good complement to the market for Tuber melanosporum (Périgord black truffle). However, Chinese truffles could be introduced accidentally or fraudulently into the plantations of Mediterranean truffles, and they could have a negative effect on truffle production and natural ecosystems. The study of Tuber species from China which are commercialized in Europe began 14 years ago. Tuber pseudoexcavatum was proposed as a new species, and this has been validated by some authors based on molecular and phylogenetic studies. We synthesize their ectomycorrhizae using samples from the type collection, and we compare T. pseudoexcavatum and Tuber indicum ectomycorrhizae. The ectomycorrhizae of these species have a morphology which is related to the ectomycorrhizae of T. melanosporum. We provide useful information for the rapid screening of the above-mentioned Chinese truffles ectomycorrhizae, for the quality control of commercial plants mycorrhized with Tuber. Moreover, we analyze the soil tolerance and the host plant affinity of T. pseudoexcavatum and T. indicum, in order to assess the capacity of both Chinese truffles to penetrate T. melanosporum plantations and habitats.

Response surface modeling the significance of nitrogen source on the cell growth and Tuber polysaccharides production by submerged cultivation of Chinese truffle Tuber sinense

Truffles are hypogeous ascomycetes and ectomycorrhizal fungi with unique organoleptic properties. Effects of nitrogen source and its concentration were studied during the submerged fermentation of Chinese truffle Tuber sinense. Yeast extract was identified to be the most favorable for the cell growth, and the next was peptone. The maximal dry cell weight of 19.53 ± 0.13 g/L obtained at an initial yeast extract concentration of 25 g/L was higher by 40.9% compared to that at 10 g/L. Within the range of initial peptone concentration (i.e., 10, 20, 30 and 40 g/L) as investigated, the maximal biomass and the production of extracellular polysaccharide (EPS) were obtained at 30 g/L, while 20 g/L was favorable for the production of intracellular polysaccharide (IPS). A mathematical model, which was constructed by response surface methodology combination with Box-Behnken design (BBD), was applied to study the synergic effect of glucose, yeast extract and peptone. According to the model prediction, the optimal initial concentration of glucose, yeast extract and peptone was 60, 30 and 10 g/L, respectively. The validation experiment showed the experimental values agreed with the predicted values well. The highest biomass (i.e., 23.94 ± 0.49 g/L), the production of EPS (i.e., 1.59 ± 0.06 g/L) and IPS (i.e., 1.48 ± 0.26 g/L) were obtained under the predicted optimal conditions, which were higher by 22.6%, 42.0% and 16.5% compared with the best results of one-variable-at-a-time experiments, respectively.

Quantitative response of cell growth and Tuber polysaccharides biosynthesis by medicinal mushroom Chinese truffle Tuber sinense to metal ion in culture medium

During the submerged fermentation of medicinal mushroom Chinese truffle Tuber sinense, there was no significant effect of metal ion on the cell growth and the production of intracellular polysaccharides, while metal ion and its concentration significantly affected the production of extracellular polysaccharides (EPS). By using the approach of “one-variable-at-a-time”, 50mM Mg2+ was identified to be the most favorable for EPS production, and the next was 10mM K+. A mathematical model, constructed by response surface methodology combination with full factorial design, was applied to study the synergic effect of Mg2+ and K+. EPS production reached its peak value of 5.86g/L under their optimal combination of 30mM Mg2+ and 5mM K+ predicted by the model, which was higher by 130.7% compared with the basal fermentation medium without metal ion. The validation experiment showed the experimental values agreed with the predicted values well. EPS production obtained in this work was the highest reported in the culture of T. sinense.

Significance of inoculation density and carbon source on the mycelial growth and Tuber polysaccharides production by submerged fermentation of Chinese truffle Tuber sinense

Truffles are among the most valuable gourmet mushrooms on the market. By taking Chinese truffle Tuber sinense as a typical example, a submerged fermentation process for the production of mycelia and Tuber polysaccharides was developed for the first time. Significances of inoculation density, carbon source and its initial concentration were studied in details. For inoculation density within the range of 160–653 mg dry weight (DW)/L, a maximal biomass of 15.59 ± 0.59 g DW/L was obtained at its lowest level of 160 mg DW/L, while the maximal extracellular polysaccharides (EPSs) production of 1.97 ± 0.08 g/L was attained at its highest level of 653 mg DW/L. The maximal intracellular polysaccharides (IPSs) production of 1.40 ± 0.10 g/L was obtained at its level of 487 mg DW/L. The carbon sources examined were glucose, maltose, sucrose and lactose, and sucrose was suitable for the cell growth and IPS production. Lactose was beneficial for EPS production although the cell could not grow well. There was no β-galactosidase activity when T. sinense grew in lactose, which was the reason why lactose was not favorable for the cell growth. Initial sucrose concentration within the range of 20–125 g/L significantly affected the process. At sucrose 125 g/L, both biomass (i.e., 24.07 ± 1.94 g/L) and EPS production (i.e., 2.85 ± 0.04 g/L) reached their peak values. The maximal IPS production of 2.92 ± 0.20 g/L was obtained at sucrose 80 g/L. This work demonstrated the submerged fermentation of Chinese truffle T. sinense is a potential alternative for the efficient production of mycelia and Tuber polysaccharides.

Phylogenetic relationships betweenTuber pseudoexcavatum, a Chinese truffle, and otherTuberspecies based on parsimony and distance analysis of four different gene sequences

Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, beta-tubulin, protein kinase C and elongation factor 1alpha. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.

Typing Tuber melanosporum and Chinese black truffle species by molecular markers.

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.

A phylogenetic study of commercial Chinese truffles and their allies: Taxonomic implications

Phylogenetic relationships of commercial Chinese truffles and their allies were investigated mainly by morphological studies and analyses of the sequences of ITS regions of nuclear ribosomal DNA. Two species, Tuber indicum and T. himalayense, closely related to the European T. melanosporum (the Perigord Truffle), are recognized among commercial Chinese black truffles. Both T. pseudohimalayense and T. sinense should be regarded as synonyms of T. indicum. Tuber species producing excavated ascomata are not monophyletic, suggesting that excavation of ascomata may have evolved more than once, or evolved once during the evolution of truffle species and then was lost once during the evolution of Tuber species.

A new trihydroxy fatty acid from the ascomycete, Chinese truffle Tuber indicum.

From the chloroform/methanol extract of the fruiting bodies of the ascomycete Chinese truffle Tuber indicum Cooke et Massee, a new trihydroxylated monounsaturated fatty acid (1) has been isolated. The structure of this new linoleic acid-derived metabolite was established as 9,10,11-trihydroxy-(12Z)-12-octadecenoic acid by means of spectroscopic and chemical methods. The fatty acid composition of the chloroform-soluble fraction of this fungus was analyzed by gas chromatography-mass spectrometry. The content of the predominant unsaturated fatty acids (oleic and linoleic acids) is as high as 68%. The use of dimethyl disulfide adduct was effective in the determination of the position of the double bond, and the glycol oxidation fission reaction with sodium metaperiodate supported on silica gel was helpful in establishing the location of the trihydroxylic groups in the new fatty acid.

A novel sterol from Chinese truffles Tuber indicum.

From the fruiting bodies of Ascomycetes Tuber indicum, a new steroidal glucoside with polyhydroxy ergosterol nucleus, tuberoside (2), has been isolated along with additional four known ergosterol derivatives, (22E, 24R)-ergosta-7, 22-dien-3beta, 5alpha, 6beta-triol (1), 5alpha, 8alpha-epidioxy-(22E, 24R)-ergosta-6, 22-dien-3beta-ol (3), (22E, 24R)-ergosta-5, 22-dien-3beta-ol (4), and (22E, 24R)-ergosta-4, 6, 8(14), 22-tetraen-3-one (5). The structure of new compound was established as 3-O-beta-D-glucopyranosyl-(22E, 24R)-ergosta-7, 22-dien-5alpha, 6beta-diol (2) on the basis of chemical and spectroscopic means ((1)H NMR, (13)C NMR, HMQC, HMBC, MS, and IR). This is the first example of isolation of a polyhydroxylated ergosterol glucoside from higher fungi in nature.

Molecular tools for the identification of Tuber melanosporum in agroindustry.

Tuber melanosporum Vitt., Tuber magnatum Pico, and Tuber uncinatum Chat. can be differentiated by their morphological characters. Fraud problems have arisen recently with the importation to Europe of truffles from China. T. melanosporum is morphologically very close, but distinct from the Chinese species [Tuber indicum (Cooke and Massee) and T. himalayense BC (Zhang and Winter)]. We have optimized molecular tools to unequivocally identify T. melanosporum. DNA extraction from ascocarps of black truffles is not straightforward. Problems to obtain pure DNA are due to high contents of phenolic compounds, melanine, and various polymers (proteins, polysaccharides, etc). These compounds coprecipitate with the DNA during extraction and strongly inhibit the PCR reaction. We have developed an efficient and reliable protocol for DNA extraction from truffle ascocarps. It was used successfully for DNA extraction from mycorrhizal root tips as well as from canned preparations of T. melanosporum. Several approaches to identify T. melanosporum by PCR were developed. Two specific primers for T. melanosporum were designed after comparison of the ITS region of this species with those of three Chinese fungi. They proved to be efficient to specifically detect the presence of T. melanosporum by PCR. The mycorrhizal status of trees inoculated with T. melanosporum but unable to produce truffles was confirmed in a single-step PCR reaction. A multiplex PCR approach was also developed with three sets of primers (including a specific one for Chinese truffles) to detect, in one PCR reaction, the presence of any other Tuber species mixed with T. melanosporum ascocarps. This optimized protocol, in association with the specific primers we designed, is applicable to quality control in the truffle industry from the production stages to final commercial products.

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species ( T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences.

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.

Tuber pseudoexcavatum sp. nov. A new species from China commercialised in Spain, France and Italy with additional comments on Chinese truffles

Tuber pseudoexcavatum sp. nov. A new species from China commercialised in Spain, France and Italy with additional comments on Chinese truffles

Typing Tuber melanosporum and Chinese black truffle species by molecular markers

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.