The Optimization Of Extracting And Separating Process Of α-Acids And β-Acids In Hops And The Analysis Of Their Antimicrobial Activity

This study carried out the basic research on the extracting and separating process ofα-acid and β-acid in Tsingdao flower hops which were grown in Yumen area of Gansuprovince. Through single factor test, orthogonal test and Center rotating combination test,the optimal extraction process parameters of organic solvent, microwave extraction andultrasound assisted extraction of α-acid and β-acid were determined. Based on the singlefactor test and regression orthogonal experiment, the acid and alkali separation process ofα-acid and β-acid were optimized. In addition, the purity of α-acid and β-acid was analysedby HLPC. On the basis of above, the antimicrobial activity of α-acid and β-acid were tested.This paper aims to provide a basis and reference for related business. The main resultsobtained are as follows:1. The results of organic solvent extraction showed that: the order of α-acid contentwas petroleum ether> methylene chloride> toluene> benzene> ethanol> acetone, and theeffect of petroleum ether was the best, the content of α-acid was40.97%; The results ofβ-acid indicated that the order of β-acid content was petroleum ether> methylene chloride>ethanol> toluene> benzene>acetone, the petroleum ether had the most significant effectand the β-acid content was26.82%.2. The optimum parameters of microwave-assisted extraction of α-acid and β-acidwere confirmed by the single factor and orthogonal experiments, the results showed that:the best combination of α-acid was A1B1C2D1, that was soaking time30min, microwavetime15s, microwave power267W,the ratio of solid to liquid1:15（g: mL）,and the order offactors were microwave time> the ratio of solid to liquid> microwave power> soaking time.Under that condition, the content of α-acid was53.13%. The optimal parameter of β-acidwas A1B2C3D3, that was soaking time25min, microwave time35s, microwave power373W, the ratio of solid to liquid1:30（g: mL）,and the order of factors were soaking time>the ratio of solid to liquid> microwave time>microwave power. Under that condition, thecontent of α-acid was35.03%.3. Through single factor test and central composite rotatable test the ultrasonic assisted extraction technologic condition of α-acid and β-acid were determined, the resultsshowed that: the optimal conditions of α-acid were for the ratio of liquid to solid1:18,ultrasonic time15min, ultrasonic power203W, soaking time59min. Under optimalcondition, the predicted value of the content of α-acid was43.91%, the testing value was43.65%, and its relative error was0.59%. The regression equation was determined byDesign-Expert7.1for: Y=-104.73646+10.33525X₃₁+4.36234X₃₂-0.29762X₃₁²-0.14882X₃₂2-5.52196×10-4X₃₃²-1.09107×10-3X₃42. The best technology conditions of β-acid were the ratioof liquid to solid1:18, ultrasonic time17min, ultrasonic power228W, soaking time68min.Under optimal condition, the predicted value of the content of α-acid was16.22%, thetesting value was16.12%, and its relative error was0.62%. The regression equation wasdetermined by Design-Expert7.1for: Y=-181.73796+14.23562X₃₁+2.81392X₃₂+0.31502X₃3-0.081552X₃₁X₃₂-2.99542E-003X₃₁X₃3+0.018411X₃₁X₃4+4.16242E-003X₃₂X₃3+3.02428E-004X₃3X₃4-0.39475X₃₁²-0.077761X₃₂2-8.51607E-004X₃₃²-9.70674E-003X₃42。4. By single factor experiment and a regression orthogonal test, the optimization ofα-acid and β-acid separation process were confirmed, the study showed that: the optimalparameters of alpha-acids were the dosage of potassium hydroxide8.5%,the consumptionanhydrous alcohol1mL/g, the ratio of material liquid1:60, and the primary order forfactors was the consumption anhydrous alcohol than the ratio of material liquid than thedosage of potassium hydroxide. The multiple regression equation, which was obtainedthrough the method of regression, was Y=82.667+1.515X₃₁-2.950X₃₂+2.037X₃3; The optimalparameters of beta-acids were the dosage of potassium hydroxide6.5%，the consumptionanhydrous alcohol1mL/g, the ratio of material liquid1:60, and the primary order forfactors was the dosage of potassium hydroxide than the ratio of material liquid than theconsumption anhydrous alcohol. The multiple regression equation, which was obtainedthrough the method of regression, was Y=84.375-2.843X₃₁-1.995X₃₂+2.360X₃3.5. Through HPLC external standard method, α-acid and β-acid were analysed,which obtained by petroleum ether as the extraction solvent, microwave Extraction Methodand the Separation Method of acid and alkali precipitation, the results showed that: Thesubstances, which obtained by this separating process, were really α-acid and β-acid, andtheir purity were86.43%and87.12%. These Results demonstrated that the process was anideal separation purification of hop α-acid and β-acid method.6. The antimicrobial activity of α-acid and β-acid was determined by inhibitory zone,the results showed that: the tested concentration of α-acid and β-acid100mg/mL- 10mg/mL range for Sacchromuces cerevisiae, Asperyillus nigar van, Trichotheciumroseum, Penicillium sp., Geotrichum candidum were no inhibitory effect, no inhibitionzone; against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, the three kinds ofbacteria had significant antibacterial activity, and along with the increase of concentrationthe more obvious bacteriostatic effect, the greater the bacteriostatic circle. Staphylococcusaureus, Escherichia coli and Bacillus subtilis, concentrations in the10-100mg/mL α-acidand β-acid solution, the minimum inhibitory concentration （MIC） of α-acid were:5.0mg/mL,2.5mg/mL,2.5mg/mL; the minimum inhibitory concentration （MIC） of β-acidwere:1.25mg/mL,1.25mg/mL,2.5mg/mL.In sum, it is believed that the extracting and separating process of α-acids and β-acidscould be improved and optimized through orthogonal experiments, central compositerotatable test and a regression orthogonal test. And they have significant antibacterialactivity against Staphylococcus aureus, Escherichia coli and Bacillus subtilis.