Optimization of medium components for the production of exopolysaccharide by Lycoperdon pyriforme Schaeff.: Pers. using response surface methodology

Abstract

Lycoperdon pyriforme Schaeff.:Pers.is one of medicinal macro-fungi,which has become an attractive topic as a functional food and as a source for the development of drugs and nutraceuticals. Pharmaceutically active compounds including polysaccharide,steroids,polysaccharides-peptides,nucleosides and triterpenoids were reported to be found in its fruiting body,in which polysaccharides isolated from its fruiting body were reported to possess various important biological functions such as antitumor activity,immunomodulation and antioxidant activity with low toxicity and good safety profile.However,it is known that the time required for obtaining either naturally harvested or farmed fruiting bodies of most medicinal fungal species is relatively long,while its product quality is also difficult to control in mushroom farming.In addition,even mushroom farming techniques for L.pyriforme.have not been developed adequately to meet the current and promising market for bio- based antioxidants.The submerged culture is thought as an alternative approach to produce a large amount of mycelia biomass and polysaccharides by medicinal fungi.Currently,although many studies about exopolysaccharides(EPS)bioproduction by macro-fungi have been reported,there is a little information available in literature about the optimization of medium components for enhanced EPS production by L.pyriforme.in submerged culture to date. The objectives of this study were to screen the factors significantly affecting EPS production by L.pyriforme. and subsequently to determine the optimal medium components for enhanced EPS yield through response surface methodology(RSM),which is a collection of statistical techniques for experimental design,model developing, evaluation factors and searching for optimum conditions,and which consists of a group of mathematical and statistical procedures.The concentration of EPS was measured by phenol-sulfuric acid method.For the optimization of medium components for enhanced EPS production,RSM was subsequently applied in this study.At last,the validation tests were performed under the optimal conditions at shake flask scale. Firstly,Plackett-Burman experimental design(PB)was applied to implore the relative effect of several medium components on EPS yield and vitamin B1(VB1)was determined to affect EPS yield significantly.And combining the results of one-factor-one-time method(data not shown),three factors,namely,maltose,glutamic acid and VB1 were further optimized for enhanced EPS yield using Box-Behnken design(BBD).A total of 15experimental runs(with three factors at three levels)were conducted to determine the optimum combination of these medium components by ANOVA analysis of experimental data.A second-order polynomial models with EPS yield as response variable was then obtained and confirmed to adequately represent the actual relationship between the independent variables and responses through validation tests.The optimal medium compositions were acquired as follows:maltose 6.11%, glutamic acid 0.70%,VB1 2.8×10-4%,yeast extract 0.50%,KH2PO4 0.15%,MgSO4·7H2O 0.10%,initial pH 5.5.The result of validation tests in triplicate was(3.71±0.16)g/L under the above optimum culture conditions, and in which EPS yield was enhanced by 4.6times compared with that produced in the basal medium in which(0.82±0.09)g/L was just obtained. In conclusion,RSM procedure is convenient,feasible and highly efficient for the optimization of culture conditions for enhanced EPS production by fungi.