| Hydroxytyrosol(HT) has great antioxidant, antibacterial activities and other biological activities. However, with three phenolic hydroxyl, HT has strong hydrophilicity and is unstable.In addition, the chemical synthesis of HT has high cost and serious environmental pollution. At present, there is not a batch preparation technology internationally. In this paper, oleuropein(OL) was hydrolyzed to HT with olive leaf extract(OLE) by hemicellulase, and liposome properties and thermal stability of HT were checked. Moreover, polyphenols chemical composition of enzymatic hydrolysate(EH) were qualitative and quantitative analysis by pyrolysis-gas chromatography-mass spectrometry(Py-GC-MS) and HPLC methods, and the enzymolysis mechanism was preliminary discussed. The structure-activity relationships between key polyphenols and antioxidant, antibacterial activities were assessed. It would provide theoretical basis for efficient preparation and deep-processing utilization of olive leaf extract and HT. Main researches were summarized as followed:(1)Biological enzymolysis of OL and change of key polyphenolsIt was suggested that HT preparation from OL enzymolysis in the OLE was feasible. The optimal technology of enzymatic process was obtained by single factor test and orthogonal test.The results showed that hemicellulase was the best enzyme to hydrolyze OL to HT in the same enzymatic activity from 10 enzymes, and the cellulase and β-glucosidase were second. The optimal condition was A2B3C3D1( temperature, 60℃; pH value, 5.5; enzyme quantity, 40 mg;time, 6 h). The degradation rate of OL was 85.28%, and HT content of EH-1 was 2.81% by the best enzymatic process from OLE-1(OL content was 38.6%). Furthermore, The degradation rate of OL was 98.54%, and HT content of EH-2 was 8.64% by the best enzymatic process from OLE-2(OL content was 81.04%). Based on the enzymolysis results of different OLE, OL content was decreased and HT content was increased. It was indicated that the OL content higher, the HT content higher.(2)Enzymolysis mechanism of OLE and polyphenols chemical composition of EHChemical composition of OLE-2, EH-2 and EEEH-2 were analyzed by Py-GC-MS,respectively. Pyrolysis products had mainly phenolic acids(4-hydroxyphenylacetic acid,salicylic acid, 3,4-dihydroxyphenylacetic acid, vanillic acid, 3,4-dihydroxybenzoic acid, caffeic acid, etc.). 14 key phenols in EEEH-2 were qualitative and quantitative analysis by HPLC. HT content was highest in EEEH-2 and OL was degraded fully in the enzymatic process.The polyphenol acids of pyrolysis products mainly derived from high temperature pyrolysis of compounds with flavone or phenolic hydroxyl structure on OLE, as well as the substances with structure similar to glycoside and flavonoid glycosides. It was deduced that pyrolysis products of OL, verbacoside, ligstroside, luteolin(-glucose) and apigenin(-glucose)were HT, tyrosol, caffeic acid, 4-hydroxycinnamic acid, respectively. Of course, OL still played a important role in HT preparation. Suitable enzyme and temperature had a great influence on OL enzymolysis. Related data showed that OL was mainly hydrolyzed to OL aglycone by hemicellulase, however, HT obtained with fracture of ester bond. At the same time, it was also explained that fracture of glycosidic bond was easier relatively than ester bond.(3)Separation and purification of HT from EHEH-1 was extracted by ethyl acetate(EEEH-1) and HT content was from 2.81% to10.23%. Then, EEEH-1 were purified by macroporous resin, and desorption substance by macroporous resin(MRDS) was obtained and the HT content was 40.78%. Finally, MRDS was separated by high-speed counter-current chromatography(HSCCC) and HT content was increased from 40.78% to 85.7%.(4)Thermal stability of EEEH-1 and HTThermal stability and decomposition kinetics of HT and EEEH-1 were studied by TG-DTG techniques and kinetic methods. TG curve showed the thermal decomposition of HT was finished by one step, and the temperature between 260℃ to 409℃ was main thermal decomposition stage.. As the heating rate increased, thermal decomposing feature temperature of HT also rose. The probable kinetic mechanisms of thermal decomposition was one-dimensional diffusion D1. The theoretical shelflife of HT at room temperature was about4~5 years in the nitrogen atmosphere. In addition, TG curve showed the thermal decompositionof EEEH-1 was finished by two steps mainly. The first step was from 190.3℃ to 276.9℃, and the second step was from 276.9℃ to 559.1℃. The probable kinetic mechanisms of thermal decomposition was three-dimensional diffusion Jander equation D3 and three level chemical reaction F3. With the improvement of heating rate, the thermal decomposition feature temperature presented lag phenomenon. From the data of Ea and A, the first step thermal decomposition of EEEH-1 was easier than HT, but the second step was more difficult. HT content of EEEH-1 might affect its thermal stability a certain extent, and low content of HT would improve Ea of the second step thermal decomposition and reinforce its thermal stability when other content was the same.(5)The structure-activity relationships between key polyphenols and antioxidant and antibacterial activitiesThe current study was made to assess the antioxidant activity(DPPH radical scavenging capacity, ABTS radical scavenging capacity, total reducing power, FRAP and Fe2+ chelating ability) and antibacterial activity(E. coli, S. aureus, E. aerogenes, S. epidermidis, K.pneumoniae bacterium) of 14 key phenols. The comparison of the structural difference provided insight into the phenolic structure-antioxidant, antibacterial activities relationships.Results showed that antioxidant activities were all added with the concentration of 16 samples increased, and dose-effect relationships were existed among samples concentration and antioxidant activities. In addition, HT, OL, caffeic acid, 3,4-dihydroxybenzoic acid,3,4-dihydroxyphenylacetic acid and eugenol all had better activities relatively. EEEH had the best DPPH radical scavenging capacity in the EH and different extracts, and it was demonstrated that HT, OL and polyphenols content in EEEH had obvious influence on its antioxidant activities.14 keys phenols all possessed certain inhibition effect against 5 selected bacterium, and had better inhibition effect against tested gram-positive bacterium(S. aureus, S. epidermidis)than gram-negative bacterium(E. coli, S. epidermidis and K. pneumoniae). The effect of S.epidermidis was the best, and the effect of S. aureus, E. aerogenes and K. pneumoniae was second, and the effect of E. coli was the last. Compared to positive control, HT, OE, caffeicacid, 3,4-dihydroxybenzoic acid all presented better antibacterial effect. EAE, mixed with different proportion of 14 compounds, showed moderate antibacterial activities against selected bacterium. It resulted from a synergistic antibacterial activity against tested strains.Finally, the antioxidant and antibacterial activities mainly depended on the number and location of hydroxyl groups, the catechin moiety and electron-donating groups conjugated to the 4-position on the aromatic ring, and more depended on the interaction among various factors. Furthermore, the EH and phenols might be suitable as a source of powerful antioxidants and bacteriostat in functional foods due to their stronger antioxidant and antibacterial activities.(6)Preparation and properties and HT liposomeOptimum process of HT liposome was obtained by single factor and response surface methodology: temperature was 63℃, mass ratio of phospholipid(PL) and cholesterin(CH)was 4.5, HT mass was 5 mg, tween-80 volume was 6 mL, and encapsulation of HT was45.08%. Relative to the HT solution, HT liposomes had certain slow-releasing potential function, and its DPPH radical scavenging capacity was not very different. |
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