Study On Saponin Production By Rutin Addition In The Liquid Fermentation Of Tuber Melanosporum

Truffle is a kind of large edible fungi with good medicinal value.Wild truffles are rare and they cannot meet the current market demand.Therefore,liquid fermentation is an efficient strategy to obtain truffle mycelium with high production and low cost.Truffle saponins are the secondary metabolites synthesized in truffle liquid fermentation,which possess different bioactivities,such as antioxidant,anti-tumor,anti-inflammatory and so on.However,there were few studies about truffle saponins and the related knowledge was limited at present.In this study,the liquid fermentation of Tuber melanosporum was used as the research system and a strategy was developed to increase the production of extracellular truffle saponin by rutin addition.For this purpose,the medium composition and culture condition were optimized to improve saponin production and its antioxidant activities in the liquid fermentation of T.melanosporum.The regulation mechanism of rutin in the saponin synthesis of T.melanosporum was investigated by using transcriptome technology.The scaling-up of truffle liquid fermentation was also studied in stirring reactor.The main conclusions are as follows:(1)The optimal medium composition and the optimal culture condition in shake flask were achieved for the saponin production by the liquid fermentation of T.melanosporum after the optimization using orthogonal design and experiment.The optimal medium composition consisted of rutin 4.8 g/L,glucose 43.5 g/L,peptone 6g/L,NH₂PO₄ 1.15 g/L,Na Cl 0.2 g/L,VB₂ 0.082 g/L,VB₆ 0.1 g/L,VC 0.02 g/L.The optimal culture condition included p H of 5.7,inoculation amount of 12.5%(v/v),culture temperature of 24?,rotation speed of 190 rpm and culture period of 4 days.Under the optimal fermentation condition,the truffle saponin production reached the peak value of 0.413 g/L,which was 134.7%higher than that before optimization.The addition of rutin increased the truffle saponin production by 50.2%compared to that without rutin addition.Both truffle saponins,RTS(with rutin)and TS(without rutin),exhibited antioxidant activities.The maximal DPPH scavenging activities of RTS and TS were 93.98%and 76.88%,respectively.The maximal hydroxyl radical scavenging activities of RTS and TS was 79.26%and 68.83%,respectively.The maximal ferric reducing antioxidant power of RTS and TS was 42.22 m M and 38.57 m M,respectively.After adding rutin to the medium,the increase in membrane permeability and the decrease in cell wall thickness happened to truffle mycelium.It could be one of the reasons that rutin improved the saponin production of T.melanosporum.(2)According to the GO functional enrichment of differential genes,the differential genes in rutin group were mainly enriched in molecular function compared to the control without rutin.It indicated that rutin could affect molecular function significantly and exhibited less influence on the gene expression related to biological process and cell component in T.melanosporum.The KEGG enrichment of differential genes showed that the differential genes in rutin group were mainly enriched in Ribosome biogenesis in eukaryotes,Pyrimidine metabolism,Purine metabolism and metabolism in specific amino acids.Most of the genes of the enzymes involved in the biosynthesis pathway of polysaccharides and saponins were up-regulated in rutin group,which indicated that rutin could promote the anabolism of polysaccharides and saponins in T.melanosporum.(3)The scaling-up of T.melanosporum liquid fermentation was carried out in 5 L mechanical stirring reactor and the best production of polysaccharide and saponin was obtained at 150 rpm and 1.4 lpm,48 g/L and 14.44 g/L,respectively.The relationship between truffle pellet morphology and its production of polysaccharide and saponin was investigated and compared under different stirring speeds with fixed aeration.It can be found that production of polysaccharide and saponin in truffle liquid fermentation was low when the large pellets with smooth surface occurred.On the contrary,the high production of polysaccharide and saponin was achieved when the small and evenly-dispersed pellets with long hypha on surface were formed.According to the relationship between pellet morphology and production of polysaccharide and saponin under different aeration with fixed stirring speed,there were less pellets under the aeration of 1.4 lpm compared to that under better aeration.There was no difference in the diameter of pellet under different aeration.However,the highest production of saponin and polysaccharide was obtained in truffle liquid fermentation under lower aeration.Under high stirring speed or low aeration,the permeability of cell membrane increased and the thickness of cell wall decreased during the liquid fermentation of T.melanosporum,which can be due to the environmental stress resulted from high shear force or low dissolved oxygen.