| Lactobacillus acidophilus is an important probiotic.The ?-glucosidase produced by it can convert quercetin glycosides to release quercetin and improve the functional activity of natural products in fruit and vegetable raw materials(Rosa roxburghii Tratt).Previous studies had found that Vc had a stabilizing and activating effect on the activity of Lactobacillus acidophilus?-glucosidase,and at the same time,it improved the biotransformation efficiency of ?-glucosidase to quercetin glycosides,but the relevant performance and mechanism were still unclear.Therefore,this thesis explored the reaction conditions for the biotransformation of quercetin glycosides by Lactobacillus acidophilus ?-glucosidase,and analyzed the influence of Vc and Lactobacillus acidophilus ?-glucosidase on the stability of characteristics.At the same time,after obtaining the secondary and tertiary structure of ?-glucosidase by using circular dichroism and homology modeling,using atomic force microscopy,circular dichroism,fluorescence spectroscopy and molecular docking technology to further explore the effect of Vc on Lactobacillus acidophilus ?-glucosidase and its relationship with quercetin glycoside,the main research contents and results were as follows:1.Study on the conversion of Lactobacillus acidophilus ?-glucosidase to quercetin glycosides of Rosa roxburghii Tratt(1)Using ultrasonic extraction technology to extract flavonoids from raw materials of fruits and vegetables(Rosa roxburghii Tratt),the single factor experiment and response surface method were used to optimize the extraction process,and the optimal process conditions were determined as the material-to-liquid ratio of 10:1(m L·g-1),The ethanol concentration is 70%,and the extraction time is 93 min.Under these conditions,three parallel verification tests are carried out,and the yield of Rosa roxburghii Tratt flavonoids is 3.57%.(2)Using single factor test and Box-Behnken method to optimize and explore the biotransformation conditions of Lactobacillus acidophilus ?-glucosidase to Rosa roxburghii Tratt quercetin glycosides,the best conditions were obtained:transformation temperature44 ?,transformation time 58 min,,the enzyme concentration is 35%.Under these conditions,the conversion rates of rutin and isoquercitrin were 47.89% and 62.03%,respectively.On this basis,the agonistic effect of polyhydroxy compounds such as Vc on the conversion of quercetin glycosides by Lactobacillus acidophilus ?-glucosidase was investigated,and the study found that polyhydroxy compounds had a certain promoting effect on the conversion efficiency of quercetin glycoside The effect on the conversion of rutin into quercetin is: glucose>Vc>fructose>xylitol,and the effect on isoquercitrin is:glucose>fructose>Vc>xylitol.2.Study on the interaction characteristics of Vc and Lactobacillus acidophilus ?-glucosidase stability(1)The effect of Vc on the stability of Lactobacillus acidophilus ?-glucosidaseThe optimal reaction temperature of Lactobacillus acidophilus ?-glucosidase was 45 ?,and it had high thermal stability in the range of 35 ? to 65 ?.Vc can significantly improve the thermal stability of ?-glucosidase.In the range of 35°C to 75°C,with the increase of the amount of Vc,the relative enzyme activity of ?-glucosidase continues to increase,and at 45°C,it reached the highest when the Vc addition amount was 4%,and then the enzyme activity began to decrease.Lactobacillus acidophilus ?-glucosidase had certain acid and alkali resistance,the optimal reaction p H was 6,and it had good p H stability in the range of p H 3 to 8.Vc can improve the p H stability of ?-glucosidase.In the range of p H 2~5,the ability of Vc to activate the enzyme activity of ?-glucosidase decreases.In the range of p H 6-9,the relative enzyme activity of Lactobacillus acidophilus ?-glucosidase increases firstly and then decreased with the increase of Vc addition.At p H6 and Vc addition was 2%,it will increase Lactobacillus acidophilus ?-glucosidase had the best p H stability effect.(2)Study on the protective effect of Lactobacillus acidophilus ?-glucosidase on VcBy comparing the content of Vc in ?-glucosidase solution,it is found that Lactobacillus acidophilus ?-glucosidase can improve the stability of Vc solution.After Vc was stored in the control solution at 25°C for 72 h,the Vc content dropped to 18.33%.After the formation of a complex between Vc and ?-glucosidase,the degradation rate slowed down.After 72 hours of storage,there was still a residual content of 38.76%,which was 1.12 times higher than that of the control group.3.Study on the molecular interaction between Vc and Lactobacillus acidophilus?-glucosidase(1)AFM analysis showed that after Lactobacillus acidophilus ?-glucosidase combined with Vc,the ?-glucosidase molecule agglomerated and the diameter increased,indicating that Lactobacillus acidophilus ?-glucosidase and Vc interacted with each other.Interaction,forming a complex,there was a hydrophobic force between the two.(2)Vc maked the ?-glucosidase CD spectral signal change,the proportion of ?-helix was reduced;the content of ?-turn was increased;the irregular curling changes were not obvious,it showed that Vc and BGL had specific binding,the two were combined by hydrogen bonding and hydrophobic force.(3)Fluorescence spectrum analysis showed that the fluorescence quenching caused by the interaction between Vc and Lactobacillus acidophilus ?-glucosidase was a static quenching mechanism.The calculated binding constants and thermodynamic parameters of Vc and?-glucosidase indicate that the interaction between Vc and ?-glucosidase was mainly driven by hydrogen bonds and hydrophobic forces.(4)The Lactobacillus acidophilus ?-glucosidase protein has 8 ?-sheets and 8 ?-helixes,and the whole is cone-shaped.The ?-glucosidase model constructed by SWISS-MODEL was used for molecular docking research,the study found that Gly293 and Arg56 were the main residues of the interaction between Lactobacillus acidophilus ?-glucosidase and Vc,and Arg56 forms two hydrogens with Vc,the bond contributes a lot.The binding free energy of the two is-79.198 k Cal·mol-1,which had a medium binding strength.4.Study on the mechanism of effect of Vc on the relationship between Lactobacillus acidophilus ?-glucosidase and quercetin glycoside(1)AFM analysis showed that after Lactobacillus acidophilus ?-glucosidase was combined with rutin and isoquercitrin,?-glucosidase molecules agglomerated and increased in diameter.Vc further aggravated the phenomenon of molecular aggregation(2)When rutin and isoquercitrin were added to the ?-glucosidase system,the CD spectrum changed more obviously,and the absorption intensity of both positive and negative peaks was weakened.The proportion of ?-helix increases,and the other secondary structure elements had varying degrees of change,indicating that the interaction between rutin and isoquercitrin and BGL can cause changes in the secondary structure of the enzyme protein.In the presence of Vc,after BGL combined with rutin and isoquercitrin,the amplitude of ?-helix change was smaller than that of rutin,isoquercitrin and BGL alone,it showed that the presence of Vc kept ?-glucosidase relatively stable secondary structure,helped maintain the tight structure of ?-glucosidase peptide chain,and improved its catalytic performance on substrates.(3)The fluorescence quenching caused by the interaction of rutin and isoquercitrin with Lactobacillus acidophilus ?-glucosidase was a static quenching mechanism,and the quenching rate of isoquercitrin on ?-glucosidase was higher than that of rutin faster.The binding constants and thermodynamic parameters of rutin,isoquercitrin and ?-glucosidase were calculated separately,indicating that the binding effect of rutin and ?-glucosidase was stronger.The interaction between rutin,isoquercitrin and ?-glucosidase is mainly driven by hydrogen bonds and hydrophobic forces,and there was also electrostatic attraction.According to the fluorescence quenching rate,Vc makes ?-glucosidase and rutin and The affinity of isoquercitrin increased,among them,it had a more obvious influence on the action of rutin and ?-glucosidase.(4)The molecular docking results of ?-glucosidase and quercetin glycoside showed that the main residues that rutin interacted with Lactobacillus acidophilus ?-glucosidase were Asp159,Arg56,Ile294,Phe292,Gly25,two The free energy of binding was-184.54 k Cal·mol-1,and the main residues interacting between isoquercitrin and Lactobacillus acidophilus ?-glucosidase were Arg56,Ile294,Gln218,Trp113,and Asn115.The free energy of binding was Arg56,Ile294,Gln218,Trp113,and Asn115.It was-119.35 k Cal·mol-1;In the presence of Vc,the binding free energy of rutin and ?-glucosidase increased to-213.587 k Cal·mol-1,and the binding free energy of isoquercitrin and ?-glucosidase increased to-140.067 k Cal·mol-1,indicating that the intervention of Vc enhanced the affinity of Lactobacillus acidophilus ?-glucosidase and the two quercetin glycosides.This paper primarily explores the effect of Vc on ?-glucosidase and its conversion of quercetin glycosides,and provides a theoretical basis for improving the biotransformation efficiency of quercetin glycosides,the in-depth development of the natural components of related processing materials,and the efficient use of probiotics.And technical strategy. |
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