Increasing the value of mushrooms as functional foods: induction of alpha and beta glucan content via novel cultivation methods
Authors: Schwartz Bertha , Vetvicka Vaclav , Danai Ofer , Hadar Yitzhak 
Published: January 2022

During the granting period, we performed the following projects: Firstly, we differentially measured glucan content in several pleurotus mushroom strains. Mushroom polysaccharides are edible polymers that have numerous reported biological functions; the most common effects are attributed to β-glucans. In recent years, it became apparent that the less abundant α-glucans also possess potent effects in various health conditions. In our first study, we explored several Pleurotus species for their total, β and α-glucan content. Pleurotuseryngii was found to have the highest total glucan concentrations and the highest α-glucans proportion. We also found that the stalks (stipe) of the fruit body contained higher glucan content then the caps (pileus). Since mushrooms respond markedly to changes in environmental and growth conditions, we developed cultivation methods aiming to increase the levels of α and β-glucans. Using olive mill solid waste (OMSW) from three-phase olive mills in the cultivation substrate. We were able to enrich the levels mainly of α-glucans. Maximal total glucan concentrations were enhanced up to twice when the growth substrate contained 80% of OMSW compared to no OMSW. Taking together this study demonstrate that Pleurotuseryngii can serve as a potential rich source of glucans for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate. We then compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophileffector functions (P. eryngiiwas more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability. In our next study, we tested the effect of glucans on lipopolysaccharide (LPS)-induced production of TNF-α. We demonstrated that glucan extracts are more effective than mill mushroom preparations. Additionally, the effectiveness of stalk-derived glucans were slightly more pronounced than of caps. Cap and stalk glucans from mill or isolated glucan competed dose-dependently with anti-Dectin-and anti-CR-3 antibodies, indicating that they contain β-glucans recognized by these receptors. Using the dextran sulfate sodium (DSS)-inflammatory bowel disease mice model, intestinal inflammatory response to the mill preparations was measured and compared to extracted glucan fractions from caps and stalks. We found that mill and glucan extracts were very effective in downregulatingIFN-γ and MIP-2 levels and that stalk-derived preparations were more effective than from caps. The tested glucans were equally effective in regulating the number of CD14/CD16 monocytes and upregulating the levels of fecal-released IgA to almost normal levels. In conclusion, the most effective glucans in ameliorating some IBD-inflammatory associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii. These spatial distinctions may be helpful in selecting more effective specific anti-inflammatory mushrooms-derived glucans. We additionally tested the effect of glucans on lipopolysaccharide-induced production of TNF-α, which demonstrated stalk-derived glucans were more effective than of caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms. Based on the findings that we could enhance glucan content in Pleurotuseryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW) and that these changes are directly related to the content of OMSW in the growing substrate we tested the extracted glucans in several models. Using dextran sulfate sodium (DSS)–inflammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans, glucans extracted from stalks cultivated at 20% OMSWdownregulated to a HDS value of 6.4 ± 0.5 and at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7 % for DSS to 6.4 ± 2.0 for DSS +glucans extracted from stalks cultivated at 50% OMSW. We finally tested glucans extracted from Pleurotuseryngii grown on a substrate containing increasing concentrations of olive mill solid waste (OMSW) contain greater glucan concentrations as a function of OMSW content. Treatment of rat Intestinal epithelial cells (IEC-6) transiently transfected with Nf-κB fused to luciferase demonstrated that glucans extracted from P. eryngii stalks grown on 80% OMSWdownregulatedTNF-α activation. Glucans from mushrooms grown on 80% OMSW exerted the most significant reducing activity of nitric oxide production in lipopolysaccharide (LPS) treated J774A.1 murine macrophages. The isolated glucans were tested in vivo using the Dextran Sodium Sulfate (DSS) induced colitis in C57Bl/6 mice and found to reduce the histology damaging score resulting from DSS treatment. Expression of various intestinal cytokines were efficiently downregulated by treatment with the fungal extracted glucans. We conclude that the stress-induced growing conditions exerted by OMSW induces production of more effective anti-inflammatory glucans in P. eryngii stalks.