In the present study, the total β-glucan (TG) content in Shiitake mushroom was measured along with its triple helical and single-double helical forms for the very first time through an indirect method.
Estimation THG by Congo red method
Three different extraction methods were used to isolate -glucan from shiitake mushrooms: enzymolysis, supercritical water, and acid-alkali, with acid-alkali extraction (KOH, HCl, NaOH) preserving the most bioactive THG. Congo red dye was used in the colorimetric assay to determine the triple helix tertiary structure of LNT because the dye and LNT have a distinct interaction that results in a significant bathochromic shift, transitioning from 488–516 nm (> 20 nm) when measured on a UV-Vis spectrophotometer. This change enabled the detection of -1,3;1,6-D-glucan, confirming the presence of THGs. Indeed, a colorimetric assay using this dye revealed a preference for branched mushroom − 1,3;1,6-D-glucan because linear − 1,3-D-glucan did not produce a bathochromic shift (Nitschke et al. 2011, Semedo et al. 2015). The quantification of bioactive -glucan from different Shiitake mushroom accessions was found to be highest in DMRO-623 (37.39%) and lowest in LE-25 (11.28%). The data (refer to supplementary material Table 2) revealed a bathochromic shift at 510 nm, with an alkaline (NaOH) extract of DMRO-623 exhibiting a prominent shift. With the exception of the LE-25 strain, the results indicated that THGs were prevalent in all glucans.
Quantification of TG by GOPOD method
The quantification of total TG involved a process of controlled acid hydrolysis with H2SO4 followed by the measurement of the glucose specifically released using the glucose oxidase peroxidase (GOPOD) reagent. This enzymatic extraction method completely breaks down the mushroom β-glucan into smaller glucose molecules and THGs will not be preserved to bind with congo red dye. The results of TG content estimated using the GOPOD method varied from 20.71–46.74%. The highest LNT content was found in DMRO-623 and the least was in LE-23, respectively (See supplementary material Table 2).
Indirect estimation method for single-double helical glucan (SDG) content in Shiitake mushroom
The quantification of SDG was carried out by comparing the TG and THG from it. A variable range of single and double helical β-glucan content was observed, from 2.089% in LE-16 to 17.25% in LE-11 among the studied Shiitake accessions.
The reported data of β-glucan content ranged from 27–40% depending upon the samples and the extraction method used. Notably, the LNT content of DMRO-623 both in total glucan and bioactive triple-helical form is a novel addition to the global Shiitake landscape. Our study sets up a novel pipeline for mushroom research and its application as this is the first conformation-dependent quantification of glucan. The exceptionally high β-glucan content in DMRO-623 could be attributed to gene expression enhancements or gain-of-function mutations in the biosynthetic pathway.