In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5x106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure-activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells, HUVEC) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10-100 µg/ml, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates - most likely due to a different mode of selenium binding and its higher degree of oxidation.

For Lentinula edodes, its characteristic flavor is the key determinant for consumer pref-erences. However, the tissue-specific volatile flavor variations of the fruiting body have been overlooked. Here, we comprehensively investigated volatile flavor profiles of different tissues including the pileus skin, context, gill and stipe of the fruiting body of two widely cultivated L. edodes strains (T2 and 0912). We show that the eight-carbon and sulfur com-pounds, which represented 43.2-78.0% and 1.4-42.9% to the total volatile emissions for strain 0912 and T2, dominated their volatiles profiles. The sulfur compounds could rep-resent 32.2% and 42.9% to the total volatile emissions for the context of the strain 0912 and T2, respectively, whereas only represented 1.4% for the stipes of the strain 0912 and 9.0% for the skin of strain T2. Proportions of the predominant C8 compounds (1-Octen-3-one, 1-Octen-3-ol and 3-Octanone) and sulfur compounds (Lenthionine, 1,2,4-Trithiolane, Di-methyl disulfide and Dimethyl trisurfide) changed depending on tissues and strains. We further reveal that the overall volatile flavor profiles varied greatly in different tissues and strains. With machine learning algorithm, we show that volatile profiles could 100% pre-dict strains 0912 and T2. The prediction accuracy for different tissues could also reach 100%. Our results highlight the tissue-specific volatile flavor variations of the L. edodes fruiting body.

This study aimed to determine the impact of a novel formulation of a supplement composed of the natural ingredients: bromelain, quercetin, and Lentinula edodes, on the gut microbiota of healthy adult dogs. Adult healthy female dogs were administered either a placebo (CTR, n = 15) or the supplement (TRT, n=15) over 28 days. Stool samples were collected for 16S rRNA sequencing before supplement administration (T0), at completion of supplement administration (T28), and one week after the end of supplement administration (T35) to characterize changes in the gut microbial communities. QIIME was used to determine both alpha- and beta-diversity and ANCOM-BC was used to identify differences in taxonomic abundances before and after supplementation. We found a significant decrease in overall diversity in CTR group, but no significant differences in overall diversity in the TRT group over time. Furthermore, we found differences in the abundance of several taxa in both CTR and TRT groups but differences in abundance of beneficial bacteria were more pronounced in TRT group. Specifically, we found increases in the abundance of sequences belonging to the genera Bifidobacterium, Lactobacillus, and Pediococcus at T28 in the TRT group with significant increases in Bifidobacterium and Lactobacillus persisting at T35 when compared to T0. Importantly, members of these genera are known to be pro anti-inflammatory and associated with a healthy gut microbiota in dogs. The results of our study show the potential of our supplement to selectively enhance specific beneficial bacterial taxa, offering a targeted approach to modulating the gut microbiome without causing disruptions to the overall equilibrium.

Lp16-PSP from Lentinula edodes strain C91-3 has been reported previously in our laboratory to have selective cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to characterize the Lp16-PSP-induced cell death using HL-60 as model cancer. The results of phase contrast microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the activation of caspase-8, -9 and -3 cascade highlighting the involvement of Fas/FasL-related pathway. Whereas, western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic pathway might be involved in Lp16-PSP-induced apoptosis either. Low doses of Lp16-PSP resulted in the anchorage-independent growth inhibition, induction of G1 phase arrest accompanied by the increased expression of p21WAF1/CIP1 along with the decreased expression of cyclin D, E, and cdk6. Our findings suggest that induction of apoptosis and p21WAF1/CIP1 mediated G1 arrest might be one of the mechanisms of the action of Lp16-PSP, however, further investigations on multiple leukemia cell lines and in vivo models are of ultimate need.