Oligo-curdlan Production By Co-cultivation Fermentation And Its Biological Activity

Curdlan is a kind of linear glucan composed ofβ-1,3-linked glucose residues.Due to its unique thermal gelling properties,curdlan has wide applications in food,medicine,and life science fields.Curdlan is also an important source for the preparation of oligo-curdlan.Compared with curdlan,oligo-curdlan has a broader application prospect in the fields of life science and medicine.Therefore,effective production of curdlan and oligo-curdlan is critical to meet the increasing market demand.Nowadays,the prebiotics reportedly have a quick fermentation process,large gas production and obvious side effects,thereby severely reducing the recognition of prebiotics.Therefore,exploring the potential of oligo-curdlan used as prebiotics could further excavate the biological activities of oligo-curdlan and contribute to discovering new prebiotics.Against this background,in this study,a co-cultivation fermentation system,composed of Agrobacterium sp.ATCC 31749 and Trichoderma harzianum GIM 3.442（CFS-AT）,was established for the preparation of oligo-curdlan;the effect of Tween-80 on the production of oligo-curdlan in the CFS-AT and its possible mechanism were investigated;and the feasibility of oligo-curdlan used as prebiotics was evaluated by in vitro experiments.The main results are as follows:（1）A CFS-AT was established after the screening of a suitable nutrient,and the effects of the CFS-AT on the production of curdlan produced by Agrobacterium sp.were investigated.Wheat bran was the optimal nutrient for the CFS-AT.Because of wheat bran,both curdlan and endo-β-1,3-glucanase could be simultaneously synthesized in the CFS-AT;When the initial inoculation ratio of Agrobacterium sp.to Trichoderma harzianum,pH,and fermentation time in shaker flasks were 1:2.8,6.5,and 144 h,respectively,the maximum production of curdlan was achieved(51.10 g?L^-1)along with the molecular weight of 69.27 kDa;In a 7 L fermentor,compared with the single fermentation system of Agrobacterium sp.（SFS-A）,the maximum production of curdlan from the CFS-AT increased by 119.93%;The structure of curdlan produced by the CFS-AT was the same as that of SFS-A;The effective curdlan productivity of the CFS-AT was related to the enhanced aerobic respiration intensity of Agrobacterium sp.and curdlan synthetase expression levels.（2）The effects of surfactants on the production of curdlan in the SFS-A were investigated.The maximum production of curdlan was achieved(51.94 g?L^-1)when 16 g?L^-1 Tween-80 was added at the beginning of Agrobacterium sp.cell growth stage;The structure of curdlan was not altered by Tween-80,however,the addition of Tween-80 reduced the density of curdlan layer and effectively weakened the envelopment of curdlan on Agrobacterium sp.;The addition of Tween-80 changed cell membrane morphology and increased cell membrane permeability;The results of intracellular nucleotides and transcription levels of related genes indicate that the addition of Tween-80 enhanced UTP regeneration,ATP regeneration,metabolism,the capacity of stress response,and energy supply of Agrobacterium sp.,provided more substances and evergy for curdlan biosynthesis,thereby enhancing curdlan production.（3）A direct microbial fermentation method of preparing oligo-curdlan was established using the CFS-AT.Following this,the CFS-AT was optimized for the effective production of oligo-curdlan and pilot-scale experiments were carried out.When the initial inoculation ratio,pH,and Tween-80 concentration in shaker flasks were 1:10,6.0,and 10 g?L^-1,respectively,the maximum production of oligo-curdlan was achieved(8.59 g?L^-1);The effects of glucose flow rate and the use of a dissolved oxygen（DO）control strategy on oligo-curdlan production were assessed,and a two-stage glucose feeding and DO control strategy was proposed:At the Trichoderma harzianum cell growth stage,the optimal glucose flow rate and agitation speed were2.0 g?L^-1?h^-1 and 600 rpm,respectively,and at the oligo-curdlan production stage,they were 0.5g?L^-1?h^-1 and 400 rpm,respectively.The oligo-curdlan production reached 17.31 g?L^-1,with a degree of polymerization of 19-25;The pilot-scale experiments in a 3 t fermentor,with the oligo-curdlan production of 9.68 g?L^-1,proved the feasibility of industrial production of oligo-curdlan by the CFS-AT.The production of oligo-curdlan by the CFS-AT allows for omission of the extraction,purification,and drying procedures used for both curdlan and endo-β-1,3-glucanase.This novel,cost-effective,and efficient microbial fermentation method has great application potential in the industrial production of oligo-curdlan.（4）The potential of oligo-curdlan used as prebiotics were evaluated.The in vitro digestion of oligo-curdlan indicate that oligo-curdlan,which was hardly digested by saliva,simulated gastric fluid,and simulated intestinal fluid,exhibited the basic characteristic of prebiotics.The in vitro fermentation experiments of specific probiotics indicate:During the early fermentation stage（0-24 h）,the promoting effects of oligo-curdlan on the growth,the decrease in pH,and short-chain fatty acids content of Bifidobaterium adolescentis,Bifidobacterium longum,Bifidobacterium bifidum,Lactobacillus plantarum,Lactobacillus brevis,and Lactobacillus acidophilus were lower than those of inulin;However,during the later fermentation stage（24-48 h）,except for Lactobacillus plantarum,the promoting effects of oligo-curdlan on the growth and short-chain fatty acids content of the other five probiotics were stronger than inulin.The in vitro fermentation experiments of fecal samples indicate:Oligo-curdlan promoted the growth of intestinal probiotics（Bifidobacterium,Lactobacillus,Faecalibacterium prausnitzii,etc.）,and the content of various short-chain fatty acids increased;Compared with inulin,when oligo-curdlan was used as the carbon source,the fermentation process of fecal samples lasted longer and the gas production decreased by 78.68%,thereby leading to less side effects such as meteorism and loose stools.The concentration-dependence of prebiotic role of oligo-curdlan decreased with increasing oligo-curdlan concentration(10-40 g?L^-1).In summary,oligo-curdlan which had a growth-promoting effect on intestinal probiotics,could be potentially used as new prebiotics.