| Stevioside has the advantages of zero glycemic index effect,zero carbohydrates and zero calories.It is extracted from the natural stevia herb and meets the requirements of a healthy,green diet.It has been found that stevioside can selectively inhibit common microorganisms,but its inhibition range and inhibition mechanism are not yet clear.As a new type of sugar substitute,stevioside is gradually expanding its application in food.In this study,we conducted a more comprehensive screening of the bacterial inhibitory effect of stevioside based on the different salinities of common microbial habitats,and initially described that the biological inhibitory activity of stevioside was positively correlated with the concentration of sodium chloride in microbial habitats.Interestingly,in the archaebacterial domain,stevioside was able to inhibit archaebacteria in a broad spectrum with low inhibition concentration and strong inhibition effect.This phenomenon is obviously different from the selective inhibition of bacteria by stevioside,what is the mechanism? This provokes us to ponder.In addition,in terms of community,the current research on the effect of stevioside on intestinal flora mostly focuses on bacterial flora,but ignores its research on intestinal archaea.The intestine is rich in archaeal communities,and although their abundance is extremely low,they are rich in diversity,and changes in abundance seem to be associated with diseases,such as colorectal cancer.Therefore,does stevioside have an effect on intestinal archaea? This study is worth exploring.In this paper,the physiological effects of stevioside on bacterial and archaeal organisms were described and the changes of archaeal communities in the intestine of mice under stevioside stimulation were investigated.Based on the obtained inhibition phenomena and patterns of stevioside,the mechanism was further explored in a preliminary manner from the perspective of screening for tolerant bacteria.The main experimental results obtained are shown below:1)Through pure culture experiments with microorganisms,we studied the physiological effects of stevioside on common microorganisms,marine bacteria,salt-tolerant bacteria,and haloarchaea.We found that the inhibition effect and range of stevioside on bacteria were limited at medium to high concentrations,while low concentrations of stevioside had an extremely high inhibitory effect on haloarchaea,with a range of up to 100%! Overall,it appears that the antibacterial spectrum of stevioside on microorganisms seems to expand as the environmental salinity in which the microorganisms live increases.2)The effect of stevioside on archaea community in intestinal community was studied by in vitro fermentation experiment of mouse colon contents.Although the abundance of archaea in intestinal community was extremely low,the experimental results showed that its order of magnitude was within the measurable range.Stevioside of 0.2 m M had no effect on the species diversity of intestinal community,but increased the relative abundance of archaea in Haloarcula and methanomethylaceae,but had no effect on the abundance of Haloferax.3)In order to confirm the haloarchaea Inhibition mechanism of stevioside,a mutant strain with stevioside tolerance was obtained by UV mutagenesis with Haloferax mediterranei DF50 as the starting strain.At present,there are few experimental parameters for UV mutagenesis of archaea organisms.The successful screening of tolerant strains in this experiment provides experience for the setting of UV mutagenesis conditions.The mutation sites were analyzed by resequencing,and it was found that the specific mutation genes HFX＿2143 and HFX＿2145 of tolerant strains DF50-α1 and DF50-β1 were simultaneously regulated and participated in the same metabolic process,which was related to the synthesis of extracellular polysaccharide of haloarchaea.Moreover,UDP-glucose-6-dehydrogenase(UGDH)encoded by HFX＿2145 gene is the key enzyme of glucuronic aldehyde pathway,and UGDH participates in the synthesis of stevioside by catalyzing the conversion of UDP-glucose(UDP-Glc)to UDP-glucuronic acid(UDP-Glc A).UDP-glucuronic acid is the key precursor of steviol glycosides(SGs)biosynthesis in stevioside.It acts as a donor of UDP-Glc A in steviol glycosides biosynthesis,and forms steviol glycosides by linking with steviol.The inhibition degree of stevioside on the growth of gene knockout strain DF50-ΔEPS in laboratory was investigated by liquid culture method.It was found that the inhibition degree of DF50-ΔEPS strain was slightly lower than that of DF50 in low concentration stevioside.DF50 can grow slowly and DF50-ΔEPS growth is completely inhibited under the condition of high concentration of stevioside.The HFX＿2143-2148 gene cluster involved in extracellular polysaccharide synthesis seems to promote the growth of the strain under the condition of medium and low concentration of stevioside,which seems to have a negative impact on the resistance to high concentration of stevioside.4)The other unique mutant genes HFX＿2051 and HFX＿0818 of tolerant strains DF50-α1and DF50-β1 were studied.HFX＿2051 and HFX＿0818 encode proteasome-activated nucleotidase Pan2 and proteasome-activated nucleotidase(PAN)respectively,which are homologous genes.The research on the structure and function of PAN gene and its relationship with cell growth and resistance was summarized.It was found that the loss of PAN could affect the growth rate and cell yield of archaea,and the function of proteasome was very important to the resistance of the strain.PAN protein,as a subfamily of AAAATP,participates in the assembly and activation of 20 S proteasome in archaea.PAN binds to ATP and causes to bind to 20 S proteasome,thus opening the door for substrate to enter,which makes unfolded protein transfer to proteasome in the middle exponential period.Therefore,PAN can ensure the correct assembly and function of proteasome and reduce the accumulation of misfolded protein,which plays an important role in the cell growth and survival of archaea.This study found that the antimicrobial effect of stevioside on microorganisms is positively correlated with salt concentration.The acquisition of resistance genes for haloarchaea can provide new marker genes for the archaeal domain,which can not only reduce development costs but also make the development process safer.In addition,the mechanism of stevioside tolerance was preliminarily studied,providing a basis for further research on the high sensitivity of haloarchaea to stevioside from multiple aspects. |
PDF Full Text Request