Magnetic Separation And Characteristics Of Active Ingredients Related To Alcohol Dehydrogenase In Tea Saponin

Tea saponins(TS)are a kind of by-products extracted from tea seed meal.Their basic skeleton is mainly composed of ligands,glycosides and organic acids.The diversity of composition methods endows TS with abundant biological activities,such as hemolytic activity,anti-inflammatory activity,antibacterial activity,liver and stomach protection,influence on alcohol metabolism,etc.In recent years,researchers have begun to concentrate on the separation and extraction of active components in tea saponin.Due to the complexity of the composition of tea saponin,the separation of active monomers is still a challenge.Magnetic Nanoparticles(MNP)have been widely used in various fields due to their unique properties such as excellent magnetic responsiveness and high biocompatibility.In food-related applications,it is often used for enzyme immobilization,protein purification and food analysis.Immobilization of the enzyme on the magnetic carrier can not only improve the stability,but also facilitate the recovery and utilization of the enzyme.In this study,Alcohol dehydrogenase(ADH)which is directly related to Alcohol metabolism was immobilized on amino functionalized magnetic nanoparticles to prepare magnetic immobilized ADH.MIADH was used as a tool to isolate the ADH active ingredient(AAI)in TS,and an in vitro detoxification model was established to investigate the mechanism of AAI's influence on alcohol metabolism.It provides theoretical basis and related technical support for magnetic targeted separation technology and the application and development of tea saponin.The main research results are as follows:1.Fe₃O₄ magnetic nanoparticles were prepared by chemical co-precipitation method and the preparation conditions were optimized by single factor test.A rapid synthesis method of magnetic nanoparticles was established and composite magnetic nanoparticles with appropriate size and good performance were obtained.The results showed that when FeSO₄ and Fe Cl₃ were used as substrates,the reaction temperature was 60?and the reaction time was 30 min,the particle size was the smallest and most uniform.Under these conditions,the particle size was controlled between 90 nm and100nm,which was verified by transmission electron microscopy and scanning electron microscopy.And the saturation magnetization was 80.2 emu/g,with super paramagnetism and good performance.The results of X-ray diffraction show that the prepared Fe₃O₄ accord with the spinel-type Fe₃O₄ crystal surface.The Fe₃O₄ was coated with Si O₂ on the surface and modified with amino functionalization.The modified Fe₃O₄ particles were more uniformly dispersed in the aqueous solution.It was shown by electron microscopy that the modified Fe₃O₄ particles were still spherical,and the outer layer of Si O₂ was about 16 nm thick.According to the VSM curve,the saturated magnetization intensity was reduced due to the uniform density of the silicon layer after modification,but the particles still had good geomagnetic response before and after the modification of amino functionalization.2.Magnetic immobilization of ADH was studied using composite magnetic nanoparticles as the carrier.TGA,FT-IR and VSM were used to characterize MIADH,and its enzymatic properties were analyzed.The results showed that the-CO-NH-characteristic peak contained in the immobilized enzyme appeared in the FT-IR spectrum,indicating that ADH was successfully immobilized on the functionalized magnetic nanoparticles and the MIADH was successfully prepared.This conclusion was confirmed by the TGA analysis results.With a saturation magnetization value of21.6 emu/g,MIADH still had good magnetic response.When the initial concentration of ADH was 50?g/mL,the immobilized cross-linking time was 2.5 h,and the concentration of cross-linking agent was 5%,the optimal MIADH was obtained and the enzyme activity retention rate was 73.13%.The immobilized ADH significantly improved its storage stability and reuse stability.The relative enzyme activity of MIADH was 10 times higher than that of free ADH when stored at 4?to the 4th day.The initial enzyme activity of MIADH was still more than 70%after the 15 times of use,showing strong reuse stability.After magnetic immobilization,MIADH enzyme activity retention rate was high,which can realize the rapid magnetic separation and recycling of ADH.3.The purification of TS was studied,and AAI in TS was separated by MIADH.TS was purified by two-stage precipitation method.The UV and FT-IR spectra showed that there was a UV absorption peak at 270 nm before and after purification.After purification,the infrared transmittance at 1047.15 cm^-1 and 1049.36 cm^-1 was enhanced.AAI was isolated by MIADH magnetic targeting,and detected by UPLC-QTOF-MS.Compared with the database and references,AAI contained TS compounds and non-TS compounds.According to the ionic fragments and references,Oleiferasaponin A1(C₅₉H₉₂O₂₆),Camelliasaponin B1(C₅₈H₉₀O₂₆)and Theasaponin A₂(C₅₉H₉₂O₂₇)can be identified as active substances.Their biological activities related to ethanol absorption had been reported.It was speculated that they were the main components of AAI separated by MIADH magnetic separation.4.The liver-protecting properties and hangover mechanism of AAI was explored from the aspects of physiological and biochemical indexes and gene expression.The results showed that in L02 and Hep G2 cells,AAI can reduce ethanol-induced liver cell toxicity and apoptosis,reduce the secretion of AST and ALT,reduce the production and leakage of LDH and MDA,increase the activity of GSH and SOD,and at the same time promote the enzyme activities of ADH and ALDH,which were concentration-dependent.Furthermore,the results of RT-PCR showed that AAI had different mechanisms on L02 and Hep G2 cells,and its effects on the expression of related genes were opposite:for L02 cells,high-dose(10?g/mL)of AAI up-regulated the expression of ADH1B,ALDH2,and CYP2E1.Inflammation-related factors IL-6 and TNF-?were inhibited by low-dose(5?g/mL)AAI,while for Hep G2 cells,the expression of ADH1B,ALDH2 and CYP2E1 was promoted by low-dose AAI,and inflammation-related factors require high dose intervene.It showed that AAI can indeed reduce the inflammatory response in liver cells,regulate the expression of hangover-related genes and slow down the pathogenesis of liver injury.5.Based on the physicochemical properties of AAI,the effect of AAI addition on the quality of sponge cake was investigated.Combined with the foaming and liver protecting properties of AAI and the soft and delicate texture of sponge cake,a sponge cake containing AAI was developed.According to the physicochemical properties and sensory characteristics of the cake,it was proved that the addition of AAI can improve the properties of sponge cake.The cake body had a fine and uniform internal structure and a suitable flavor.When the amount of AAI added is 1‰,the sponge cake not only has a brighter color,but also has the best antiseptic effect,and can maintain the longest shelf life without adding other preservatives.