Study On Production Of Hydroxytyrosol From Olive Polyphenols By Enzymatic Catalysis

Olive is a ver y important wood y oil crop,which has been planted on a large scale.Because of pruning and olive processing,a large number of olive leaf wastes are produced ever y year.Clean and efficient utilization of olive leaves has become an urgent problem to be solved.H ydrox yt yrosol has strong antioxidant activit y,which can prevent cardiovascular disease,diabetes mellitus,cancer and inflammation.Therefore,in this dissertation,the methods of producing h ydrox yt yrosol with high yield were studied through the screening of free enz ymes and the optimization of technological conditions,in order to reach the goal of resource utilization of olive leaves and to solve the bottleneck of the low yield of h ydrox yt yrosol.At the same time,several immobilization methods were used to stud y the production of h ydrox yt yrosol b y immobilized enz yme in the packed bed reactor with continuous multi-batch process,which provided theoretical basis and technical guidance for industrialized production of h ydrox yt yrosol.Separation and purification of h ydrox yt yrosol,the stability and antioxidant activit y of oleuropein and h ydrox yt yrosol were further studied.The main results were shown as follows:1.The optimum extraction technology o f oleuropein b y ethanol solvent extraction was as below:ethanol volume fraction was 90%,extraction temperature was 80 ^oC,extraction time was 1.5 h,solid-liquid ratio was 1:40,olive leaf diameter was 20-40 mesh.This technology was determined b y single factor,orthogonal experiment and variance anal ysis.Comparing ethanol extraction with Sox hlet extraction and microwave-assisted extraction,the yields of oleuropein have a little difference.The yields of oleuropein b y ethanol extraction,Sox hlet extraction and microwave-assisted extraction were 8.44%,8.34%and 8.16%,respectivel y.Each of the three extraction methods has its advantages and disadvantages.Solvent extraction is feasible,but it consumes a lot of solvents.Soxhlet extraction is simple but time-consuming.Microwave extraction has the advantages of high efficiency,short time,less reagent dosage,wide application range and simple equipment,but it has less processing capacit y.It is onl y suitable for extraction of polar substances.2.According to the static adsorption and desorption ex periment,AB-8 was determined to be the best macroporous resin for oleuropein.After treatment with AB-8 macroporous resin,the purit y of oleuropein increased from 18.8%to 40.1%.Oleuropein extract can be enriched b y AB-8 macroporous resin to obtain a relatively high content.This operation is simple to operate and cheap,and can be used repeatedl y.It is suitable for industrial production.3.The optimum h ydrol ysis conditions for oleuropein extract to produce h ydrox yt yrosol with high added value were as follows:substrate concentration 20g/L,cellulase CTec2 5 FPU per gram of dried oleuropein extract,enz ymatic h ydrol ysis 12 hours at 50 ^oC,and then the temperature was raised to 90 ^oC to continue h ydrol ysis.The concentration of oleuropein decreased from 9.68 g/L to2.02 g/L after 12 hours at 50 ^oC,and the degradation rate of oleuropein was 79.58%.At 24 h,oleuropein was completel y degr aded.The yield of hydrox yt yrosol was24.83%,73.32%and 84.03%at 12 h,24 h and 36 h,respectivel y.The highest yield of h ydrox yt yrosol was 88.90%at 48 h.Cellulase CTec2 was h ydr ol yzed at 50 ^oC for12 hours,and then the temperature was raised to 90 ^oC for chemical h ydrol ysis.The degradation rate of oleuropein was 100%and the yield of h ydrox yt yrosol was84%-88%after 36-48 hours.The enz ym atic h ydrol ysis of 50 ^oC and chemical h ydrol ysis of 90 ^oC are needed for high yi eld of h ydrox yt yrosol.In the first stage,glucose is released from oleuropein,and in the second stage,h ydrox yt yrosol is formed b y cleavage of ester bonds.4.In a packed bed reactor,cellulase CTec2 was immobilized on calcium alginate beads,chitosan beads with deacet ylated?-chitin nanofibers and porous ceramic beads for 20 batches of continuous enz ymatic h ydrol ysis of oleuropein extract to produce h ydrox yt yrosol.The initial cellulase activit y was 10 FPU.The concentration of oleuropein extract was 20 g/L.The degradation rate of oleuropein was higher than 90%,the yield of h ydr ox yt yrosol was⁷0%,and the average concentration of h ydrox yt yrosol was¹.9 g/L.B y comparing the three carriers,it is found that porous ceramic beads are cheaper and better than the two organic materials.They are ideal carriers for immobilizing cellulase in industrial production and are suitable for industrial scale production.This technology proves that it is feasible to produce h ydrox yt yrosol from olive processing waste olive leaf extract b y long-term continuous bioconversion using immobilized cellulase.5.The stud y on static adsorption and desorption of h ydrox yt yrosol enz ymatic h ydrol ysate b y six different macroporous resins showed that NKA-II was the best macroporous resin for separation and purification of h ydrox yt yrosol.The total phenol contents of the original enz ymatic h ydrol ysate,the sample after separation with macroporous resin,and the sample after purification with preparative liquid chromatograph y C₁₈ column were 8.05%,35.72%and 91.14%,respectivel y.The figues of h ydrox yt yro sol purit y in the samples were 4.76%,21.17%and 82.25%,respectivel y.After two-step separation and purification with macroporous resin and preparative liquid chromatograph y C₁₈ column,the purit y of hydrox yt yrosol was17.28 times higher than that of the original enz ymatic h ydrol ysate.6.Oleuropein and h ydrox yt yrosol were relativel y stable under acidic and weak alkali conditions.Oleuropein and h ydrox yt yrosol did not change significantl y at the commonl y used processing temperature of 50 ^oC⁹0 ^oC.Oleuropein and h ydrox yt yrosol had little effect on fluorescent lamp?2000 Lux?and ultraviolet lamp of 254 nm,and were relativel y stable.Oleuropein and h ydrox yt yrosol are better preserved at low temperature.When the concentration was 0.03 mg/m L,the total antioxidant capacities of oleuropein ex tract,h ydrox yt yrosol solution and V_C were40.28%,71%and 36.63%,respectivel y.The total antioxidant capacit y,total reduction capacit y and DPPH free radical scavenging capacit y of h ydrox yt yrosol solution showed obviousl y higher than oleuropein extract and V_C,and stronger antioxidant capacit y was obtained b y h ydrox yt yrosol solution.The antioxidant activit y of oleuropein extract was similar to that of Vc.