Effect of Antibacterial Peptide Microsphere Coating on the Microbial and Physicochemical Characteristics of Tricholoma matsutake during Cold Storage

The effect of novel antimicrobial peptides (AMPs) and antimicrobial peptide microspheres (AMS) on the physicochemical and microbial quality of Tricholoma matsutake wild edible mushrooms was investigated. In the experiments, 1.0 g/L, 0.5 g/L of AMS, and 1.0 g/L AMPs were used as preservatives. Mushrooms coated with 1.0 g/L and 0.5 g/L of AMS as a preservative had better physicochemical and sensory qualities than did mushrooms coated with 1.0 g/L of AMPs. In the experiment, 1.0 g/L of blank microspheres without cathelicidin-BF-30 (PLGA-1.0) and distilled water was used as the control. Samples with these two treatments had minimal changes in texture, weight loss, total bacteria count, and sensory attributes. Research results suggests that the use of AMS can maintain the quality of Tricholoma matsutake wild edible mushrooms and could extend the postharvest life to 20 d.

Tricholoma matsutake-Derived Peptides Ameliorate Inflammation and Mitochondrial Dysfunction in RAW264.7 Macrophages by Modulating the NF-κB/COX-2 Pathway

Tricholoma matsutake is an edible fungus that contains various bioactive substances, some of them with immunostimulatory properties. Presently, there is limited knowledge about the functional components of T. matsutake. Our aim was to evaluate the protective effects and molecular mechanisms of two T. matsutake-derived peptides, SDLKHFPF and SDIKHFPF, on lipopolysaccharide (LPS)-induced mitochondrial dysfunction and inflammation in RAW264.7 macrophages. Tricholoma matsutake peptides significantly ameliorated the production of inflammatory cytokines and inhibited the expression of COX-2, iNOS, IKKβ, p-IκB-α, and p-NF-κB. Immunofluorescence assays confirmed the inhibitory effect of T. matsutake peptides on NF-κB/p65 nuclear translocation. Furthermore, the treatment with T. matsutake peptides prevented the accumulation of reactive oxygen species, increased the Bcl-2/Bax ratio, reversed the loss of mitochondrial membrane potential, and rescued abnormalities in cellular energy metabolism. These findings indicate that T. matsutake peptides can effectively inhibit the activation of NF-κB/COX-2 and may confer an overall protective effect against LPS-induced cell damage.

Fungal Interactions Matter: Tricholoma matsutake Domination Affect Fungal Diversity and Function in Temperate Deciduous Forest Soils

Tricholoma matsutake forms a symbiotic association with coniferous trees, developing mycelial aggregations, called ‘shiro’, which are characterized by distinct chemical and physical properties from nearby forest bulk soil. The fungal diversity living in shiro soil play key roles in nutrient cycles for this economically important mushroom, but have not been profiled across large spatial and environmental gradients. Samples of shiro and non-shiro (nearby bulk soil) were taken from five field sites where sporocarps naturally formed. Phospholipid fatty acids (PLFA) and Illumina MiSeq sequencing were combined to identify fungal biomass and community structure. Matsutake dominated in the shiro, which had a significantly reduced saprotrophic fungi biomass compared to non-shiro soil. Fungal diversity was negatively correlated with the relative abundance of T. matsutake in the shiro soil. The fungal community in the shiro was characterized by similar fungal species composition in most samples regardless of forest types. Matsutake coexisted with a specific fungal community due to competition or nutrient interactions. Oidiodendron was positively correlated with the abundance of T. matsutake, commonly cohabitant in the shiro. In contrast, Helotiales and Mortierella were negatively correlated with T. matsutake, both of which commonly inhabit the non-shiro soil but do not occur in shiro soils. We conclude that T. matsutake generate a dominance effect to shape the fungal community and diversity in shiro soil across distinctive forest types.

Structure, Antioxidant Activity and In Vitro Hypoglycemic Activity of a Polysaccharide Purified from Tricholoma matsutake

The structure, antioxidant activity and hypoglycemic activity in vitro of a novel homogeneous polysaccharide from Tricholoma matsutake (Tmp) were investigated. Structural features suggested that Tmp was consisted of arabinose (Ara), mannose (Man), glucose (Glc) and galactose (Gal) with a molar ratio of 1.9:13.6:42.7:28.3, respectively, with a molecular weight of 72.14 kDa. The structural chain of Tmp was confirmed to contain →2,5)-α-l-Arabinofuranose (Araf)-(1→, →3,5)-α-l-Araf-(1→, β-d-Glucopyranose (Glcp)-(1→, α-d-Mannopyranose (Manp)-(1→, α-d-Galacopyranose (Galp)-(1→, →4)-β-d-Galp-(1→, →3)-β-d-Glcp-(1→, →3)-α-d-Manp-(1→, →6)-3-O-Methyl (Me)-α-d-Manp-(1→, →6)-α-d-Galp-(1→, →3,6)-β-d-Glcp-(1→, →6)-α-d-Manp-(1→ residues. Furthermore, Tmp possessed strong antioxidant activity and showed the strong inhibitory effect on α-glucosidase and α-amylase activities. Then, a further evaluation found that there was a dramatic improvement in the glucose consumption, glycogen synthesis and the activities of pyruvate kinase and hexokinase when the insulin-resistant-human hepatoma cell line (IR-HepG2) was treated with Tmp. The above results indicated that Tmp had good hypoglycemic activity and also exhibited great potentials in in terms of dealing with type 2 diabetes mellitus.

A Practical Method for Determination of Nine Nucleosides in Tricholoma matsutake by UPLC/MS and Quantitative Analysis of Multicomponents Using Single Marker Method

Nucleosides can be used as quality evaluation indicators of Tricholoma matsutake. In this work, a new ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) strategy for quantitative analysis of multiple components using a single marker (QAMS) was proposed to determine nine nucleosides (adenosine, cytidine, guanosine, inosine, uridine, 2′-deoxyadenosine, 2′-deoxycytidine, 2′-deoxyguanosine, and 2′-deoxyuridine) in T. matsutake. Guanosine was set as the internal reference substance, whose content in T. matsutake was determined using the conventional external standard method. Relative correction factors (RCFs) between guanosine and eight other nucleosides were measured. The concentrations of the eight components were calculated with the obtained RCFs by QAMS. An ultrasonic extraction method is used for sample preparation. This method was validated to be sensitive, precise, and accurate with the LODs of 0.31–1.9 ng, the overall intraday and interday variations less than 4.08%, and the overall recovery over 89.0%. The correlation coefficients (r2) of the calibration curves were higher than 0.9918. The values of vector angle analysis were above 0.99845, which indicates no significant differences between the conventional external standard method and the present QAMS method. As far as we know, this is also the first report of UPLC/MS analysis of nucleoside compounds by QAMS, providing an efficient and feasible quality assessment method for other natural products containing nucleosides.

Scaled-up ethyl formate fumigation to replace methyl bromide on traded mushroom to disinfest mushroom fly (Lycoriella mali)

Mushroom fly, Lycoriella mali (Diptera: Sciaridae), is the primary pest in imported mushrooms. The amount of Tricholoma matsutake imported from China increases every fall when it is harvested. When importing T. matsutake, disinfestation using methyl bromide (MB) or phosphine (PH3) is performed to prevent the introduction of L. mali. However, MB will be phased out due to ozone-depletion, chronic toxicity to workers, and residual issues. PH3 fumigation in mushroom disinfestation requires a long exposure time (24 h). In this study, we used ethyl formate (EF), which can replace MB and reduce exposure time. The efficacy of EF, PH3 and EF + PH3 on L. mali was evaluated. Using 4-h EF fumigation at 5 °C, the 3rd and 4th instar was the most tolerant stage in terms of 99% killed lethal concentration × time products (LCt99%). When 4-h EF fumigation at 5 °C was applied on all stages of L. mali, the LCt99% values of EF were 73.1 g h/m3 to the 1st and 2nd instar, 112.9 g h/m3 to the 3rd and 4th instar, 68.9 g h/m3 to pupae, and 20.1 g h/m3 to adult. It was confirmed that combination treatment with EF + PH3 had a synergistic effect on L. mali. The LCt99% of EF + 0.5 g/m3 of PH3 to the 3rd and 4th instar was 48.3 g h/m3. When only 140 g/m3 of EF was applied for 4 h at > 5 °C and 35 g/m3 of EF + 0.5 g/m3 of PH3 for 4 h at > 5 °C in commercial trials containing T. matsutake, proven efficacy (100%) on L. mali was confirmed. In the case of EF treatment only, phytotoxic damage occurred due to high Ct products, and there was no phytotoxic damage in combination treatment with EF + PH3. This study provides a new guideline for EF + PH3 combination treatment within a shorter exposure time (4 h) than existing PH3 treatment (24 h) and replacement of MB use.

Distinct Compartmentalization of Microbial Community and Potential Metabolic Function in the Fruiting Body of Tricholoma matsutake

The uniquely compartmentalized fruiting body structure of the ectomycorrhizal fungus (EMF) Tricholoma matsutake, is a hotspot of microbial habitation and interaction. However, microbial diversity within this microniche structure of the EMF is rarely investigated. Furthermore, there is limited information concerning microbiomes associated with sporomes belonging to the ubiquitous fungal phylum Basidiomycota, particularly with respect to fungus-EMF interactions. In this study, we conducted high throughput sequencing, using ITS (fungal) and 16S rRNA (bacterial) marker genes to characterize and compare fruiting body microbiomes in the outer (pileipellis and stipitipellis) and inner layers (pileum context, stipe context, and lamellae) of the fruiting body of T. matsutake. Our results show the number of unique bacterial operational taxonomic units (OTUs) among the different compartments ranged from 410 to 499 and was more than double that of the shared/common OTUs (235). Micrococcales, Bacillales, Caulobacter, and Sphingomonas were the primary significant bacterial taxa within the different compartments of the dissected T. matsutake fruiting body. Non-parametric multivariate analysis of variance showed significant compartmental differences for both the bacterial and the fungal community structure within the T. matsutake fruiting body. The metabolic profiling revealed putative metabolisms (of amino acids, carbohydrates, and nucleotides) and the biosynthesis of secondary metabolites to be highly enriched in outer layers; in the inner parts, the metabolisms of energy, cofactors, vitamins, and lipids were significantly higher. This study demonstrates for the first time the distinct compartmentalization of microbial communities and potential metabolic function profiles in the fruiting body of an economically important EMF T. matsutake.