Optimization Of Agarases Fermentation By Vibrio Sp. And Agar Enzymolysis For Agarooligosaccharides Production

Agar was consisted of agarose and agaropectin,and it was used for the biochemical,clinical,pharmaceutical,food and other fields.Agaro-oligosaccharides,derived from agarose,had plenty of important biological activities,such as antioxidant,slow down the starch hydrolysis,in favor of absorption by human etc.The agarase-producing Vibrio sp.NTi was isolated from coastal sediment in Xiamen using agar as the sole carbon source.The aim of the present work was to investigate the effects of medium composition and culture condition on agarases produced by Vibrio sp.NTi so as to enhance the agarases productivity.Also,main content were focused on studing fermentation process in 250 mL flask and 7 L bioreactor,as well as procedure scale-up.Furthermore,agar enzymolysis process was optimized by means of agarase produceded by Vibrio sp.NTi.With the aim to increase agarase activity,optimization of medium composition and culture conditions in shaking flask fermentation was carried out by single factor experiment and response surface analysis.It was found that agarase synthesis was repressed when glucose was added into culture medium and the existence of NH₄⁺also negatively affected cell growth and agarase production.Maximum agarase production was obtained when the fermentation condition was carried out at 27oC,pH 6.5,agar concentration 3.3 g/L,yeast extract concentration 6.33 g/L,NaCl concentration 20 g/L,inoculum size 2%and loading volume 32 mL.Under optimum conditions,agarase activity reached 2.81 U/mL after 24 h of fermentation and increased by 230%compared with the activity of initial conditon.It was shown that agarase was mostly synthesized during the mid and late logarithmic growth phases of bacteria when agarase fermentation was carried out in 7 L bioreactor.Under the optimal condition:agar as the sole carbon source at a concentration of 3.4 g/L,pH 7.5 or natural pH,agitation speed 700 r/min,and culture temperature 27oC,agarase activity reached 3.37U/mL after 36 h of fermentation,which resulted in an increased yield of agarase activity by21.3%over the shaking fermentation.Furthermore,pilot scale tests were conducted based on the result of 7 L bioreactor.The maximum agarase activitives harvested in 20 L and 200 L bioreactors were 3.61 U/mL and 3.73 U/mL,increasing by 8.5%and 10.5%over that of the 7 L bioreactor respectively.This result indicated that the fermentation process in laboratory scale was easy to scale up.Taking the amount of reducing sugar as an indicator to optimize agar enzymolysis condition by the single factor experiment and response surface analysis.Maximum yield of reducing sugar reached 1386.2 mg/L after 30 min of enzymolysis and 1762.3mg/L after 40 min of enzymolysis respectively,which was obtained under the optimum condition of 40oC,pH 7.04,the substrate concentration of agar 11 g/L,enzyme dosage 14.4 U,and NaCl concentration 15 g/L under static state.Under this optimal condition,an increased yield of reducing sugar was got by three-fold over the initial condition,meanwhile the conversion rate of agar reached 30%.The components of hydrolytic products were analyzed by thin-layer chromatography,and it was showed that agar was hydrolyzed to produce the final product neoagarobiose via neoagarohexaose and neoagarotetraose.