Analysis And Utilization Of Solid-state Fermentation Products By Monascus Pilosus MS-1 Using Fuling As Substrate

Fuling,the sclerotium of Wolfiporia cocos,which has been listed in the directory of both food and drug by The Department of Health and Family Planning Commission of China,is a kind of traditional Chinese medicine.Pachyman(PM),the main components of Fuling,contribute to 70%-90%of Fuling dry weight.PM mainly consists of β-1,3-glucan.Due to its high molecular weight,most of PM is water-insoluble,while water-soluble PM accounts for merely about 0.5%.Since ancient times,Fuling as a traditional Chinese medicine has been extracted and used its water-soluble constituents by water-boiling method.Water-insoluble PM,however,is often discarded as herb residue.In China,more than 30 billion tons of Fuling herb residues per year make waste and cause huge environmental damage.Expanding the application of Fuling residue is extremely urgent.Until now,researchers have made use of modification methods such as chemical modification to increase solubility of water-insoluble PM.Nevertheless,chemical modification give rise to chemical residues and secondary pollution.Using microbial fermentation to modify water-insoluble PM is not only safe but also can avoid environmental pollution,which is a new way to increase the utilization ratio of Fuling.This research analysed the potential ability of Monascus pilosus(MS-1)on degrading water-insoluble PM by bioinfomatic and then optimized the fermentation condition of MS-1 on fermentating Fuling to increase the yield of water soluble polysaccharides(WSP).On the basis of this,the fermentation products were applied to manufacture steamed bread which contains Fuling and Hongqu afterwards quality properties such as sensory evaluation were proceed.The research contents and results are as follows:1 Potential abilities of M.pilosus MS-1 on water-insoluble PM hydrolysisIn order to investigate whether M.pilosus MS-1 was able to hydrolyze water-insoluble PM from large molecules into small molecules or not,bioinformatics methods were utilized to analyze potential abilities of M.pilosus MS-1 on water-insoluble PM hydrolysis.Putative β-1,3-glucanase genes from 4 strains of Monascus whose whole genome has been sequenced were identified and analyzed by HMMER 3.0 and Blast database respectively.The results showed that there are 5 endo-β-1,3-glucanase and 5 exo-β-1,3-glucanase genes in Monascus.Endo-glucanase belong to GH16(3),GH17(1)and GH81(1)family and exo-glucanase belong to GH5(2)and GH55(3)family.At the same time,endo-β-1,3-glucanase(GH81)gene were obtained through amplification by RT-PCR using RNA of M.pilosus MS-1 as a template.This gene’s length and name is 2101 bp and MEngll(M.pilosus endo-1,3-β-glucanase 1 gene),respectively.Comparation of homologous sequence was made between MEngll and sequences from GenBank.MEngll sequence encoded by M.pilosus MS-1 shared 69%identity with endo-β-1,3-glucanase MEngll gene of Byssochlamys spectabilis.Further study indicated that the expression of MEngll gene increased 20.33 times on the solid medium with water-insoluble PM as carbon source cultivated at 28 C after 3 d by qRT-PCR,comparing with the one on the medium with glucose as carbon source.M.pilosus MS-1 was inoculated on solid-state medium of Fuling andβ-1,3-glucanase activity in the medium was determined using laminarin as substrate.The glucanase activity was firstly increased and then decreased overtime.The maximum ofβ-1,3-glucanase activity was 85.42 U/g after 4 d.These results suggested that M.pilosus MS-1 has a good potential ability on water-insoluble PM hydrolysis.2 Optimization of M.pilosus MS-1 solid-state fermentation using Fuling as substrateIn order to improve the WSP yield,solid-state fermentation parameters were optimized by single-factor experiments,Plackett-Burman design,steepest ascent experiments,and response surface methodology.Fermentation parameters were as follow:the dry substrate,including 15 g Fuling(without WSP)powder(20 sieve mesh),5.13%sucrose(percentage of medium weight,similarly hereinafter),0.25%NH4C1,0.63%acetic acid,73%water,0.15%corn steep solids,and 0.15%CaCI2·2H2O,was put into a 250-ml conical flask.Then 3%inoculum was added and incubated at 28℃ for 14 d.In the above conditions,the production of fermentation product WSP is 14.40 mg/g,which is 3.91 times compared with the production before fermentation(approximately 3.68mg/g).3 Development of functional steamed bread with Fuling and HongquFuling and Hongqu were ground into powders(60 sieve mesh and 100 sieve mesh)before being added with different proportion of wheat flour during steamed bread process.The optimal conditions were determined through orthogonal experiment and the color,texture and sensory evaluation properties of steamed bread were analyzed.The optimal steamed bread formula was determined as follows:0.3%Hongqu powder(percentage of raw material powder,similarly hereinafter),10%Fuling powder addition and 95%water(in proportion to ral material powder).The specific volume Was 1.8×10-3 m3/kg,and the steamed bread got high scores of appearance,color,structure,springiness and toughness and adhesiveness with special fragrance of Fuling.The maximum total score was 94.30.Subsequently,Fuling was replaced with Monascus-fermented Fuling.According to the above process conditions,high WSP yield and bright-coloured steamed bread with Monascus-fermented Fuling and Hongqu was produced.Compared with steamed bread with non-fermented Fuling and Hongqu,brightness and whiteness of steamed bread were enhanced.While the springiness,toughness,resilience,cohesion,springiness,and chewiness were slightly reduced.The total score of sensory evaluation was 78.34.Besides,there were no significant difference in sensory evaluation between steamed bread with Monascus-fermented Fuling and with wheat flour.