The aroma active compounds of three Tuber fruiting bodies (i.e., Tuber himalayense, Tuber indicum, and Tuber sinense) were firstly systematically evaluated by instrumental gas chromatography-olfactometry combining with quantitative analysis, aroma reconstitution, and omission tests. Twelve aroma active compounds were characterized by aroma extract dilution analysis, and 3-(methylthio) propanal, 3-methylbutanal, and 1-octen-3-ol with the highest flavor dilution (FD) factor (i.e., 1,024-2,048) were suggested as key contributors to the aroma. Odor activity value (OAV) was employed to determine the relative contribution of each compound to the aroma, and the compound with the highest FD factor also had the highest OAV (i.e., 10,234-242,951). Then, the synthetic blends of odorants (aroma reconstitution) were prepared with OAV larger than 15, and their aromas were very similar to the originals. Omission tests were carried out to verify the significance of 3-(methylthio) propanal, 1-octen-3-ol, and 3-methylbutanal as key compounds in the aroma of tested Tuber fruiting bodies.

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Truffle (Tuber spp.), also known as "underground gold," is popular in various cuisines because of its unique and characteristic aroma. Currently, truffle fruiting bodies are mostly obtained from nature and semi-artificial cultivation. However, the former source is scarce, and the latter is time-consuming, usually taking 4 to 12 years before harvest of the fruiting body. The truffle submerged fermentation process was first developed in Tang's lab as an alternative to its fruiting bodies. To the best of our knowledge, most reports of truffle submerged fermentation come from Tang's group. This review examines the current state of the truffle submerged fermentation process. First, the strategy to optimize the truffle submerged fermentation process is summarized; the final conditions yielded not only the highest reported truffle biomass but also the highest production of extracellular and intracellular polysaccharides. Second, the comparison of metabolites produced by truffle fermentation and fruiting bodies is presented, and the former were superior to the latter. Third, metabolites (i.e., volatile organic compounds, equivalent umami concentration, and sterol) derived from truffle fermentation could be regulated by fermentation process optimization. These findings indicated that submerged fermentation of truffles can be used for commercial production of biomass and metabolites as a promising alternative to generating its fruiting bodies in bioreactor.

Tuber melanosporum, known as the black diamond of cuisine, is highly appreciated for its unique and characteristic aroma, which is mainly due to its volatile organic sulfur-containing compounds (VOSCs). In this work, by adding 5 g/L L-methionine to the fermentation medium, the activities of aminotransferase and α-ketoacid decarboxylase were significantly enhanced by 103 and 250%, respectively, while the activities of alcohol dehydrogenase and demethiolase were decreased by 277 and 39%. Then, the six VOSCs, i.e., methanethiol (MTL), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 3-(methylthio)propanal (methional), and 3-(methylthio)-1-propanol (methionol), were first detected in the submerged fermentation of T. melanosporum. These results indicated that the biosynthesis of VOSCs was triggered by aminotransferase and α-ketoacid decarboxylase. The production of methional and methionol increased with the increased concentrations of L-methionine (i.e., 5, 10, 15, and 20 g/L) before day 4 of the culture protocol, and methionol was the major product in the Ehrlich pathway. The production of MTL was significantly decreased after day 4 with a significantly increased DMDS, and DMDS was the major product of the demethiolation pathway. Compared with the demethiolation pathway with a total flux of sulfur of 11.33-24.32 μM, the Ehrlich pathway with a total flux of sulfur of 6,149-10,330 μM was considered the major pathway for the biosynthesis of VOSCs. This is the first report linking the metabolism of L-methionine to the biosynthesis of VOSCs by the Ehrlich and demethiolation pathways during the submerged fermentation of T. melanosporum.

The aroma attributes of sulfurous, mushroom and earthy are the most important characteristics of the aroma of Tuber melanosporum. However, these three aroma attributes are absent in the T. melanosporum fermentation system. To improve the quality of the aroma, repeated freeze-thaw treatment (RFTT) was adopted to affect the interplay of volatile organic compounds (VOCs). Using RFTT, not only was the score on the hedonic scale of the aroma increased from the “liked slightly” to the “liked moderately” grade, but the aroma attributes of sulfurous, mushroom and earthy could also be smelled in the T. melanosporum fermentation system for the first time. A total of 29 VOCs were identified, and 9 compounds were identified as the key discriminative volatiles affected by RFTT. Amino acid analysis revealed that methionine, valine, serine, phenylalanine, isoleucine and threonine were the key substrates associated with the biosynthesis of the 9 key discriminative VOCs. This study noted that amino acid metabolism played an important role in the regulation of the aroma of the T. melanosporum fermentation system.

As a potential intermediary feedstock, succinic acid takes an important place in bulk chemical productions. For the first time, a method combining Plackett-Burman design (PBD), steepest ascent method (SA), and Box-Behnken design (BBD) was developed to optimize Actinobacillus succinogenes ATCC 55618 fermentation medium. First, glucose, yeast extract, and MgCO3 were identified to be key medium components by PBD. Second, preliminary optimization was run by SA method to access the optimal region of the key medium components. Finally, the responses, that is, the production of succinic acid, were optimized simultaneously by using BBD, and the optimal concentration was located to be 84.6 g L−1 of glucose, 14.5 g L−1 of yeast extract, and 64.7 g L−1 of MgCO3. Verification experiment indicated that the maximal succinic acid production of 52.7 ± 0.8 g L−1 was obtained under the identified optimal conditions. The result agreed with the predicted value well. Compared with that of the basic medium, the production of succinic acid and yield of succinic acid against glucose were enhanced by 67.3% and 111.1%, respectively. The results obtained in this study may be useful for the industrial commercial production of succinic acid.