Preparation Of β-1,3-glucooligosaccharides From Curdlan And The Elicitation Effect Against Fungi

Curdlan, a water-insoluble, unbranched microbial β-1,3-glucan, is mainly produced byAgrobacterium sp. fermentation under nitrogen-limited conditions. Besides the massiveapplication in the food and cosmetic industries, curdlan and its derivative oligosaccharideshave drawn attention in the field of life science owing to the extinguished biological activities.Potato has been cultivated worldwide as an important economic plant, whereas the infectionof deleterious microorganisms causes the yield reduction of potato every year. To activate theinnate defense responses of potato cells is a promising way to protect the plant from microbialinfections while reducing the use of agricultural chemicals. In the current study the productionof curdlan, preparation of curdlan oligosaccharides, and the elicitation effect of curdlanproducts on the potato defense responses were comprehensively investigated and the mainconclusions are as follows:(1) The effect of dissolved oxygen (DO) level on the fermentation kinetics, rheologicalproperty and molecular weight (Mw) distribution of curdlan was investigated in theAgrobacterium sp. ATCC31749fermentation process. The results showed that the conversionrate of carbon source kept stable at the DO level from5%to75%. However, the productionintensity at DO5%was much lower than that at higher DO levels. The specific productionrate of curdlan (qP) linearly decreased along with the accumulation of exocellular curdlan(R2=0.939). This correlation can be removed by heightening the DO level. Furthermore, therheological results indicated that the curdlan-alkali solution exhibited distinct pseudoplasticcharacters (η=Kγn, n<1). The apparent viscosity (μa) of curdlan solution increased asfermentation time prolonging and showed the highest values at DO25%. In addition, the Mwof curdlan was determined by size exclusion chromatography (SEC) method. The resultsshowed that the correlation between the average Mw and μaof curdlan existed to a certaindegree. The moderate DO (25%) facilitated the increase of both the average Mw and μaofcurdlan in A. sp. ATCC31749fermentation process.(2) To improve the stability and hydrolysis efficiency of curdlan suspension, theapproaches for curdlan treatment and hydrolysis were consecutively investigated. The resultsshowed that the stability of curdlan suspension significantly improved after thealkali-neutralization (AN) treatment. The AN treated suspension kept stable for more than20d, whereas the untreated sample completely precipitated within5min at the same conditions.Moreover, the double-layer structure model was proposed to describe the AN treated curdlanparticle which comprises an undissolved compact core and a hydrated colloidal shell. Thecolloidal shell plays a vital role on stabilizing the particle and is susceptible to hydrolysisagents, whereas the compact core is resistant to degradation at moderate temperatures.Furthermore, based on the double-layer structure model, the two-step sulfuric acid hydrolysisstrategy was set up to prepare a series of curdlan β-1,3-oligosaccharides with degree ofpolymerization (DP) from2to10purified by HPLC method. In comparison with theconventional approaches, the yield of low Mw oligosaccharides (Mw<1000Da, DP2-6) andhigh Mw oligosaccharides (DP7-10) increased by49.4%and58.8%, respectively. (3) An endo-β-1,3-glucanase (Endo23) which exhibited high hydrolysis activity oncurdlan was purified from the fermentation broth of Trichoderma reesei GIM3.498by meansof the combination of ultrafiltration-anion exchange chromatography-SEC method. Theresults showed that Endo23specifically hydrolyzed the β-1,3-glucoside bond. The optimal pHand temperature for Endo23were pH5.0and50°C. The activity of Endo23was remarkablyinhibited by Ag+, Al3+, Cu2+, Hg2+, Fe2+, Fe3+, and Sn2+, whereas K+can activate the activityboth at5and50mmol·L-1levels. Endo23was classified on account of its properties into EC3.2.1.39. The Mw and isoelectric point of Endo23are23.6kDa and pI3.85. The percentagecontent of α-helix, β-sheet, β-turn, and random coil are21.2%,24.1%,19.1%, and39.2%,respectively. In addition, partial of the amino acid sequences of Endo23were identical withthe conserved sequence of catalytic domain in glycoside hydrolase family16. The hydrolysispattern indicated that Endo23is suitable for preparing low Mw curdlan oligosaccharides fromDP2to DP4.(4) The effect of curdlan oligosaccharides (CurdOs) on inducing the defense responses ofpotato (Solanum tuberosum L. cv. McCain G1) leaf cells was investigated. After treated withCurdOs, the intracellular H2O2accumulated promptly and the level of salicylic acid reachedthe peak value at1h. Besides, the enzymatic activities of phenylalanine ammonia-lyase andpathogeneses-related protein (β-1,3-glucanase and chitinase) were also remarkably increasedwithin4-24h after treatment, which indicates that CurdOs are capable of activating thedefense responses of potato cells. However, the response level decreased with elicitation timeand dropped to the level of control set at20d. In addition, the defense results proved thatcurdlan glucopentaose was the shortest linear β-1,3-glucooligosaccharide with completeelicitor activity. Moreover, ten up-regulated proteins in leaf cells at12h after CurdOstreatment were further identified. The function analysis showed that a systemic response wasperformed in potato cells, including the recognition of elicitor, transportation of signals,enhancement of metabolic pathways, and the expression and transportation of proteins.(5) The resistance of micro-potato plants treated with CurdOs against Phytophthorainfestans was investigated. After2-week’s infection by P. infestans zoospores, the ratio of leaflesion reached up to15.82%±5.44%, and the whole plant yield was6.82±2.80g after3-monthcultivation. When the plants were treated with250mg·L-1CurdOs at1d before P. infestansinfection, the ratio of leaf lesion significantly decreased to7.79%±3.03%, and the whole plantyield reached10.37±3.58g, which was similar to the control level (10.01±3.75g). However,the improvement of resistance was not significant when the plants were treated with CurdOsat5d before P. infestans infection. In addition, the variation of single potato weight was notsignificant under above treatments. The results indicated that the resistance of potato plantsagainst P. infestans was enhanced when treated with CurdOs in short period before infection.