High-intensity ultrasound for extraction and controlled degradation of high molecular weight polysaccharides from medicinal mushrooms : process characteristics and product properties

Abstract

Polysaccharides (PS) and polysaccharide-protein complexes (PSPs) from edible/medicinal fungi or mushrooms have attracted increasing research and commercial interests for nutraceutical and pharmaceutical uses. The aim of this research project is at a comprehensive study on the application of an innovative food processing technique, high-power ultrasound (US), for the processing and modification of bioactive polysaccharides from several important medicinal fungi. Ultrasound-assisted extraction (UAE) has been widely evaluated as a more favourable and efficient process than the traditional hot water extraction (HWE) for isolation and recovery of food and medicinal products. On the other hand, high power ultrasound (US) is an interesting mechanical means for partial and controlled degradation of high molecular weight (MW) polysaccharides to improve the solution properties for desirable clinical applications. UAE was evaluated in comparison with HWE in several important edible/medicinal fungi in mushroom fruit body and mycelial form, including Cordyceps sinensis (Chinese caterpillar fungus Dong Chong Xia Cao), Grifola frondosa (Maitake), Coriolus versicolor (Yunzhi) and Lentinus edodes (Shiitake), and Ganoderma lucidum (Linzhi) for extraction of water soluble products and isolation of PSPs. Compared with those by HWE, the yield of PSP by UAE was similar on G. frondosa, notably higher on L. edodes and lower on C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE (1) had higher protein but lower carbohydrate contents (2) exhibited an overall shift to lower MW (3) contained distinct protein bands between 10-130 kDa on SDS-PAGE than those by HWE. This suggested that UAE at low temperature can retain the protein constituents, avoiding the denaturation by HWE. The antioxidant activities of PSPs from the three mushrooms by UAE were generally higher than those by HWE. Further study was performed on the kinetics and process parameters for UAE of water-soluble components and PS from these fungal materials. At a fixed power and a temperature below 50°C, the experimental data were fitted to several kinetic models by linear regression. More detailed kinetic study was performed on UAE of the Cordyceps sinensis Cs-HK1 fungal mycelium with four process variables (factors) at different levels, ultrasound intensity (2.44 - 44.1 W/cm2), temperature (40-70 °C), solid particle size (156.5-750 μm), and solid-to-liquid ratio (1/30-1/70 g/mL). The experimental data of yields versus time in most cases were fitted closely to two empirical kinetic models for solid-liquid extraction, parabolic diffusion equation (y = y0 + y1t1/2) and power law (y = βtn) with high correlation coefficients (R2) of 0.95-0.99 for total extract yield, and 0.90-0.96 for PS yield. Reducing the particle size and increasing the extraction temperature led to a higher yield and extraction rate; increasing the solid-to-liquid ratio (or decreasing the liquid volume) increased the PS yield and extraction rate but had little influence on the total extract. Significant correlations were found between extraction rate (dy/dt) and ultrasound power density (P/V), and between extract yield (y) and energy density (Pt/V). High-intensity ultrasound (20 kHz) was applied to the degradation of a high-MW PS extracted from C. sinensis Cs-HK1. With US applied at selected power intensities (10-50 W/cm2), the intrinsic viscosity (correlated to the MW) of the polysaccharide decreased rapidly in the initial period (~10 min) and attained steady level gradually. The degradation rate was increased with US power and PS initial MW. The US treatment also shifted the particle size distribution of the PS in aqueous solution from large to small particle size range (40-50 nm). Transmission electron microscopy (TEM) of the PS particles suggested that US at a relatively low intensity (e.g. 10.6 W/cm2) was mainly effective to disperse the polysaccharide aggregates, and US at a high intensity (e.g. 48.4 W/cm2) caused the change in the particle morphology from globular to longitudinal shape. From the study on a PS of known-structure, curdlan from Alcaligenes faecalis, the degradation kinetics by US was established. The US of degradation of studied for curdlan in 0.1M and 0.3M NaOH, which molecules were in triple-helix and random coil conformation, respectively. Degradation rate constant k (mol/g·min) was determined based on the kinetic model: 1/Mt - 1/M0 = t) (with R2 values > 0.91). The value of k in 0.3M was higher than that for in 0.1M, which suggested that the random coil conformation of curdlan molecules in 0.3M NaOH was more liable to to degradation than the rigid rod conformation in 0.1M NaOH. A test was performed to detect the existence of single helix structures in both original and US-degraded curdlan, and the amount of single-helix-structured molecules was increased by US treatment. High-intensity US was also applied to facilitate the extraction of both intracellular and extracellular PS from the viscous mycelial broth of a medicinal fungus C. sinensis Cs-HK1. The results showed that high-intensity US irradiation of the Cs-HK1 mycelial broth caused mycelium fragmentation and fungal cell disintegration, and a dramatic reduction of the apparent broth viscosity and the release of intracellular products into the liquid medium. Statistic experimental design and response surface methodology were applied to evaluate and optimize the major factors affecting the ultrasonic extraction yields of total water soluble product and PS (PS), including time (5-25 min), US intensity (10-30 W/cm2) and concentration of mycelial broth (0.25-1). The kinetics of total water-soluble product release (yield y versus time t) as a result of mycelium disruption by US at various power intensities was well represented by the Elovich model y = yo + y1lnt with a high regression coefficient R2 . 0.99, while that of PS yield was close fit to the parabolic morel y = yo+ y1t1/2 (R2 > 0.85). The US treatment also appeared to improve the antioxidant activity of PS recovered from the broth with a stronger cytoprotective effect against H2O2 induced cell death. Overall the results from this project have shown that high power US is an efficient and versatile means for extraction, processing and controlled degradation of bioactive PS from edible/medicinal fungi. The kinetic models and process parameters derived from the project will be useful for design and operation of the ultrasonic processes.