Five New Species of Gymnopilus from Xizang Autonomous Region of China and Surrounding Areas.

The species of Gymnopilus (Hymenogastraceae, Agricales) are commonly recognized as wood-decaying fungi. Certain members of this genus have been identified as psilocybin-producing mushrooms. Gymnopilus exhibits a diverse range and has a global distribution. In this study, a total of seventy-eight specimens were gathered from ten provinces in China. A comprehensive molecular phylogenetic analysis was conducted, employing gene sequences including ITS, nrLSU, nrSSU, rpb1, rpb2, and tef1-α. Additionally, morphological examinations were also carried out. The phylogenetic topology of Gymnopilus from this study generally agreed with previous studies and facilitated the identification of all those specimens. As a result, eleven species, including five newly discovered ones named Gy. gyirongensis, Gy. variisporus, Gy. tomentosiceps, Gy. tenuibasidialis, and Gy. aurantipileatus, were recognized. Significantly, four of the five newly identified species are native to the Xizang Autonomous Region, emphasizing their specialization in this distinctive habitat. This research contributes to our comprehension of Gymnopilus diversity and lays the groundwork for the conservation and sustainable utilization of Gymnopilus resources.

Overexpression of cat2 restores antioxidant properties and production traits in degenerated strains of Volvariella volvacea.

Strain degeneration is an important factor hindering the development of the edible fungus industry. Strain degeneration is associated with the excessive accumulation of reactive oxygen species (ROS) in vivo. Catalase (CAT), an important antioxidant enzyme, can promote the clearance of ROS. In this study, the cat2 gene of Volvariella volvacea was first cloned into an overexpression plasmid via homologous recombination. Finally, through Agrobacterium-mediated transformation, this plasmid was inserted into degenerated strains of V. volvacea T19. The physiological properties, antioxidant properties, ROS content, matrix degradation activity, and cultivation properties of the transformants were tested. The results showed that the cloned cat2 gene was 99.94% similar to the reference sequence. Screening revealed that six positive transformants were successfully obtained. After the overexpression of cat2, the growth rate and biomass of the mycelium increased significantly in the transformant strains (versus the V. volvacea T19 degenerated strains). Moreover, the accumulation of superoxide radical (O2•-) and hydrogen peroxide (H2O2) was significantly reduced, and the activity of the enzymes CAT, superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPX) was significantly increased. Meanwhile, the expression of cat2, Mnsod1, Mnsod2, gpx, and gr was significantly upregulated, and the activity of eight matrix degradation-related enzymes was increased to varying degrees. More importantly, the overexpression of the cat2 gene promoted the regrowth of fruiting bodies in degenerated strains of V. volvacea T19. This study provides a new biotechnological strategy to control the degeneration of V. volvacea and other edible fungi.

Microbial succession and its correlation with the dynamics of flavor compounds involved in the fermentation of Longxi bacon.

Introduction: Longxi bacon is a traditional fermented meat from Gansu province, China. The ripening process of the bacon is crucial for quality and flavor. The aim of this study was to gain deeper knowledges on the bacterial and fungal community diversity and the changes of chemical components including fatty acids and volatile compounds at different time points during the ripening of the bacon and to understand the relationship between microbial profiles and the chemical components related the bacon flavor. Methods: Bacon samples were collected from days 0, 15, 30, 60 and 90. The bacterial and fungal compositions were analyzed with next generation sequencing targeting the 16S rDNA loci for bacteria and ITS loci for fungi. The fatty acids and the volatile components were analyzed by headspace solid phase micro extraction followed by gas chromatography/mass spectrometry (HS-SPME-GC/MS). Results: We found that the abundance of bacteria in bacon was higher than that of fungi, and Psychrobacter, Brochothrix, Phoma and Trichoderma was the dominant bacon's population. The largest contributors of volatiles were aldehydes, ketones and esters, and the main fatty acids were palmitic, oleic and linoleic acids. Pearson correlation analysis between microbial succession and key flavor substances showed that the production of Longxi bacon flavor is the result of a combination of bacteria and fungi. Ten bacteria genera and six fungi genera were determined as functional core microbiota for the flavor production based their dominance and functionality in microbial community. In addition, bacteria and fungi are involved in the oxidation and hydrolysis of fatty acids during the ripening of bacon, which also contributes to the formation of bacon flavor. Discussion: This study provides a comprehensive analysis of the key microbiota involved in shaping bacon's distinctive flavor. Here, the results presented should provide insight into the influence of the microenvironment on the microbial community in bacon and lay a foundation for further investigations into the food ecology of bacon.

Oxidative damage from repeated tissue isolation for subculturing causes degeneration in Volvariella volvacea.

The fungal fruiting body is the organized mycelium. Tissue isolation and mycelium succession are common methods of fungal species purification and rejuvenation in the production of edible mushrooms. However, repeated succession increases strain degeneration. In this study, we examined the effect of repeated tissue isolation from Volvariella volvacea fruitbodies on the occurrence of degeneration. The results showed that less than four times in succession improved production capacity, however, after 12 successions, the traits indicating strain degeneration were apparent. For instance, the density of aerophytic hyphae, hyphal growth rate and hyphal biomass were gradually reduced, while the hyphae branching was increased. Also, other degenerative traits such as prolonged production cycles and decreased biological efficiency became evident. In particular, after 19 successions, the strain degeneration became so severe no fruiting bodies were produces anymore. Meanwhile, with the increase in successions, the antioxidant enzyme activity decreased, reactive oxygen species (ROS) increased, the number of nuclei decreased, and the mitochondrial membrane potential decreased along with morphological changes in the mitochondria. This study showed that repeated tissue isolation increased oxidative damage in the succession strain due to the accumulation of ROS, causing cellular senescence, in turn, degeneration in V. volvacea strain.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1-S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13-S20, obtained after 13-20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Optimisation and Characterisation of Novel Angiotensin-Converting Enzyme Inhibitory Peptides Prepared by Double Enzymatic Hydrolysis from Agaricus bisporus Scraps.

Food-derived hypotensive peptides have attracted attention in the field of active peptide research in recent years. In this study, based on ACE inhibition rate and using the Box-Behnken central combination design principle to optimise the process of ACE inhibitor peptides prepared by double-enzyme hydrolysis. The amino acid sequences of ACE inhibitor peptides were determined by liquid chromatography mass spectrometry (LC-MS/MS), and their binding to ACE was studied by molecular docking. The optimal processing conditions were 1:1 alkaline protease: compound protease, pH was 8.43, enzymolysis temperature was 44.32 °C, and enzymolysis time was 3.52 h. Under these conditions, the ACE inhibition rate reached 65.12%, and the inhibition rate after separation and purification was 80.68% (IC50 = 0.9 mg/mL). Three novel peptides with ACE inhibitory activity were detected by LC-MS/MS, with sequences LVYP (Leu-Val-Tyr-Pro), VYPW(Val-Tyr-Pro-Trp) and YPWT(Tyr-Pro-Trp-Thr). Molecular docking revealed that the three novel peptides all established hydrogen bonds with the S1(Tyr523, Glu384, Ala354) and S2 (His353) pockets of ACE. Among them, LVYP, VYPW and YPWT, respectively, formed eleven hydrogen bonds, six hydrogen bonds and nine hydrogen bonds with ACE. The study revealed that these peptides have the potential for the development of novel ACE inhibitor drugs and provide a new avenue for high-value utilisation of mushrooms scraps.

Novel colorimetric aptasensor based on unmodified gold nanoparticle and ssDNA for rapid and sensitive detection of T-2 toxin.

A novel colorimetric aptasensor based on unmodified gold nanoparticle (AuNPs) and single-strand DNA (ssDNA) aptamer was developed for the rapid and sensitive detection of T-2 toxin. In the absence of T-2, the AuNPs were wrapped by the aptamer to avoid the salt-induced aggregation and the solution remains red. In the presence of T-2, the aptamer was bound with T-2 and released from the surface of AuNPs, resulting in the aggregation of AuNPs under proper salt solution and the color change from red to purple-blue. The aptasensor exhibited a high sensitivity and selectivity for the detection of T-2. The range of linearity and detection limit were 0.1 ng/mL-5000 ng/mL (0.21435 nM-10717.5 nM) and 57.8 pg/mL (0.124 nM), respectively. The aptasensor developed here was applicable to assay T-2 in wheat and corn samples. These results implied that the colorimetric aptasensor was potentially useful in food detection.

Two novel cationic antifungal peptides isolated from Bacillus pumilus HN-10 and their inhibitory activity against Trichothecium roseum.

Public concern for food safety and environmental issues and the increase in fungicide-resistant pathogen have enhanced the interest in developing alternative methods to fungicides to control postharvest fruit decay. In this study, a bacterial strain isolated from stale potato vermicelli was identified as Bacillus pumilus HN-10 based on morphological characteristics and 16S rRNA gene sequence analysis. Furthermore, two novel cationic antifungal peptides named P-1 and P-2 were purified from B. pumilus HN-10 using macroporous adsorbent resin AB-8, Sephadex G-100 chromatography, and reversed-phase high-performance liquid chromatography. The primary structure of P-1 and P-2, which were proved to be novel antifungal peptides by BLAST search in NCBI database, was PLSSPATLNSR and GGSGGGSSGGSIGGR with a molecular weight of 1142.28 and 1149.14 Da, respectively, as indicated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Both P-1 and P-2 exhibited strong antifungal activity against Trichothecium roseum with minimum inhibitory concentrations starting from 1 µg/mL. The two novel antifungal peptides were stable below 80 °C for 2 h, but lost their activity in 15 min at 121 °C. In addition, they were resistant to the proteolytic action of pepsin, trypsin, and papain, and stable within a wide range of pH (2.0-12.0). These results showed that P-1 and P-2 are novel cationic antifungal peptides with specific activity against T. roseum.