Modified Yeast Glucan Preparation And Its Application As Immunostimulant For Two Crustaceans

Yeast β-glucan is a widely used immunostimulant in aquaculture. However, theinsolubility of β-glucan is against its immunoenhancing effects. In the present study,carboxymethylglucan (CMG) and sulfoethylglcuan (SEG) were made to increase thesolubility. Each kind of glucans had four derivatives with different degree ofsubstitution (DS). The β-glucan and its eight derivatives were added to the primarycultured haemocytes of white shrimp Litopenaeus vannamei under the concentrationof5,25and100μg/mL, respectively. After incubated with immunostimulants for6,12and24hours respectively, the haemocytes were sampled and their phenoloxidase(PO) activity and respiratory burst (RB) were assayed. After6hours, the PO activityof the haemoctyes incubated with all the β-glucan derivatives was significantly higherthan those with the same concentration of β-glucan (P<0.05). The RB of thehaemocytes incubated with25μg/mL of β-glucan derivatives was significantly higherthan those with25μg/mL of β-glucan (P<0.05). After12hours, PO activity and RBof the haemoctyes incubated with all the β-glucan derivatives were significantlyhigher than those with β-glucan (P<0.05). However, at these two time points, therewas no significant difference in PO activity and RB among the haemoctyes incubatedwith β-glucan derivatives under the same concentration (P>0.05). The present workrevealed that the immunoenhancing effects of β-glucan derivatives were higher thanβ-glucan. However, there was no significant difference in immunoenhancing effectsbetween CMG and SEG. The concentration of β-glucan derivatives couldsignificantly influence their immunoenhancing effects, but DS did not showsignificant influence on the immunoenhancing effects of β-glucan derivatives.In the in vivo tests, β-glucan and its eight derivatives were added to the shrimpfeed at the contents of1g kg-1or2g kg-1, respectively, to prepare18experimentaldiets. Shrimps (initial average weight0.65g) were fed with experimental diets for35days, and then were sampled for immune parameters analyses and WSSV challenging. The results showed that the types of derivatives and their DS, as well as contents indiet significantly influenced the immunity and resistance against WSSV infection of L.vannamei. For the shrimps fed with dietary CMGs, the total haemocyt count (THC),phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD)activity and resistance against WSSV challenge significantly decreased with theincreasing of DS. However, SEGs could not increase THC. The DS of SEGssignificantly influenced the RB, SOD activity and resistance of L. vannamei. Dietswith1g kg-1β-glucan derivative resulted in higher immunity and resistance thanthose with2g kg-1β-glucan derivative, regardless of the derivative type. Moreover,the shrimps fed with1g kg-1dietary CMG with DS0.352showed the highestimmunity and WSSV resistance. This CMG could be a better immunostimulant for L.vannamei.Some yeast isogenic mutants have been proved to have better immunoenhancingeffects compared with the wild type yeast. However, the optimal time to apply theseyeast isogenic mutants is still unknown and the optimal time is very important for theyeast application. In the present work, the yeast mutants Mnn9, Chs3and wild type(WT) yeast were added to the gnotobiotic Artemia franciscana48,24and12hoursbefore challenging with Vibrio campbellii, respectively. After challenge, the survivaland proPO gene expression of Artemia were investigated. The results showed that WTyeast could not protect Artemia against the challenge of Vibrio campbellii, regardlessof the adding time point. The yeast mutants could protect Artemia when added12hours before the challenge. Moreover, prolonging the time could not significantlyenhance the protection. And there is no significant difference of the protectionbetween Mnn9and Chs3, regardless of the adding time point. The results of geneexpression showed that without challenge, no yeasts could influence the proPO geneexpression. However,12and24hours after challenge, yeast mutants could increasethe proPO gene expression. The present work proves that12hours before challenge isenough for the yeast mutants to prevent disease.Based our previous work, it could be inferred that the difference of protectionprovided by the different yeast isogenic mutants is related with the quality of its cell wall glucan. In order to test this inference and provide information on the relationshipbetween conformation and bioactivity of glcuan, four different glucans were extractedfrom yeast mutants Mnn9, Chs3, Gas1and wild type (WT) yeast. The glucan fromSigma-Aldrich was also used as control. All glucans were added to the gnotobioticArtemia franciscana at the same available amount. After12hours, the Artemia werechallenged with Vibrio campbellii. After the challenging, the survival and the proPOgene expression of Artemia were investigated. All the glucan could protect Artemiafrom the infection of Vibrio campbellii. The glucans from yeast Mnn9and Gas1couldprovide full protection while the other three glucans could provide half protection.The results of gene expression revealed the glucans from yeast Mnn9and Gas1couldkeep the gene expression at higher level for longer time than the other three ones. Thisis one possible reason for the protection difference. The present work proves that theglucans from different yeasts have difference protective and immunenhancing effects.