Preparation Of Methoxy-?-benzoquinone And 2,6-dimethoxy-?-benzoquinone By Wheat Germ Fermentation And Mimic Enzymes

Wheat germ is one of byproduct of wheat processing and contains a variety of bioactive ingredients which are beneficial to the human body.Fermented wheat germ extract?FWGE?has significant antitumor property and has been developed as a therapeutic for cancer patients.In [present work,the wheat germ were used as raw materials,and the most important antitumore components,i.e.methoxy-?-benzoquinone?MBQ?and 2,6-dimethoxy-?-benzoquinone?DMBQ?in fermented wheat germ were regard as the objects in the present research.By applying ultrasonic-assisted fermentation and adding macro elements,trace elements and vitamins,related problems were systematically studied to increase the contents of MBQ and DMBQ in fermented wheat germ.Then the magnetic iron oxide nanoparticles were used as separation carried,MBQ and DMBQ were separated from wheat germ fermentation broth.Finally,a novel nano-material mimetic enzyme was constructed to study the non-enzymatic preparation of MBQ and DMBQ.The main contents of the paper include the following four aspects:The hydroquinone glucosides of the precursors of MBQ and DMBQ were isolated by preparative high performance liquid chromatography.The components were identified by time-of-flight mass spectrometry.The three hydroquinone glucosides were: 4-hydroxy-3-methoxyphenyl-?-D-triglucopyranoside,4-hydroxy-3-methoxyphenyl-?-D-diglucopyranoside and 4-hydroxy-3,5-dimethoxyphenyl-?-D-diglucopyranoside.The contents of MBQ and DMBQ in fermented wheat germ reported in the previous literature were low and the basic fermentation conditions were not optimized.In addition,the optimum conditions for the preparation of MBQ and DMBQ by ultrasonic-assisted fermentation were obtained by artificial neural network?ANN?combined with genetic algorithm?GA?on account of the difficulty in modeling microbial fermentation.The optimum conditions for fermentation were: shaking speed 142 r/min,initial pH 6.09,temperature 31.6 ° C.The ?-D-glucosidase was mainly located in intracellular and the content of hydrogen peroxide in the fermentation broth was low.To increase the production of target components,ultrasonic-assisted method was applied.The optimal conditions were as follows: the power was 74 W,the duration was 70 s,and the starting time was 25.2 h.Under these conditions,the yield of MBQ and DMBQ was 65.2% higher than that of the control group,and the fermentation time was greatly shortened.It was found that intermittent ultrasound promoted the release of key enzymes in the cell by breaking the wall and increased the level of hydrogen peroxide in the fermentation broth,which promoted the conversion of hydroquinone glycosides to MBQ and DMBQ.Macro elements?Ca,Mg?,trace elements?Fe,Cu,Zn,Mn,Se?and vitamins(B₁,B₂,B₃,B₅,B₆,B₁₂,K₃,K₅,C).were optimized by artificial neural network and genetic algorithm.The maximum predicted value of MBQ + DMBQ was 0.939 mg/g,and the actual yield of MBQ + DMBQ was 0.913 ± 0.021 mg/g,which was 117% higher than MBQ + DMBQ in the control group.On the other hand,the relationship between the single nutrient and the target value was described by probability.In addition,based on the previous literature,there was no report on evaluation of the factors' second-order interaction based on artificial neural network.Based on the concept of evaluating second-order interaction in fractional factorial design,this paper proposeed a new method for evaluating factors' second order interaction based on an artificial neural network model as the framework,combined with the Garson algorithm and partial derivative and preliminary validation was performed.The results show that the relative importance of the additive was sorted by: Mg×B₆ > Mg×B₂ >Fe×B₅ > Ca×B₃ > Zn×B₃ > Mg×B₁ > Ca×B₂ > Ca×B₁₂ > Cu×B₆ > Cu× K₅ > Mg×B₁₂ > Ca×K₃ > Mg×K₃ > C > Zn×B₃.To avoid the traditional separation methods which caused solvent consumption,high cost and complicated operation,The magnetic adsorption of MBQ and DMBQ in wheat germ fermentation broth was studied by preparing magnetic Fe₃O₄ nanoparticles with high biocompatibility.The effects of the preparation conditions of Fe₃O₄ nanoparticles on the adsorption capacity of MBQ and DMBQ in wheat germ fermentation broth were studied.The results of adsorption experiments showed that the maximum adsorption capacity of MBQ and DMBQ is higher than 93.0%,while a variety of phenolic acids also have been adsorpted by nanoparticles.The adsorption capacities of amino acids depended on surface charge amino acid.Desorption experiments showed that by use of water or ethanol solution,desorption rate of ferulic acid,vanillic acid,coumaric acid reached 84% or more.Meanwhile desorption rate of MBQ and DMBQ were less than 4%.The results also showed that the desorption rate of MBQ and DMBQ reached 97.5% and 98.1% under 20% acetic acid or ultrasound enhanced desorption.The adsorption behavior of MBQ and DMBQ on magnetic Fe₃O₄ nanoparticles was in accordance with Langmuir isothermal adsorption equation.Considering the low yields of MBQ and DMBQ,the acidic ionic liquid and iron oxide nanoparticles were used as mimic enzymes and the MCM-41 were used as a carrier to form composite catalysts which could catalyze hydroquinone glucosides to MBQ and DMBQ.The hydrolysis of hydroquinone glucosides by four Br?nsted acidic ionic liquids with sulfonic acid groups and different anion types was investigated.The results showed that under the same conditions,the catalytic activity sequence was: [MIMSO₃H][HSO₄]>[MIMSO₃H][Cl]>[MIMSO₃H][H₂PO₄]> [MIMSO₃H][BF₄].Catalytic activity of [MIMSO₃H][Cl] and [MIMSO₃H][HSO₄] were almost same.The order of the catalytic activity was basically the same as acidity order of the ionic liquid.[MIMSO₃H] [Cl] and [MIMSO₃H] [HSO₄] were immolobilized on MCM-41-magnetic Fe₃O₄ nanoparticles,respectively.The optimum conditions for the preparation of MBQ were as follows: reaction temperature was 60?,the molar ratio of composite catalyst to glucoside was 1:4,hydrogen peroxide concentration was 20 mmolol/L.The yield of MBQ was 88.1%?Fe₃O₄-MCM-41-[MIMSO₃H][HSO₄]?and 84.4%?Fe₃O₄-MCM-41-[MIMSO₃H][Cl]?under optimal conditions.The yield of MBQ was not reduced after repeated use of the two composite catalysts by 7 times.