Preparation And Characterization Of Polyphenol Oxidase-pectin Nanoparticles Loaded With EGCG

Micro-/nano-particles form during the hot water extraction process of tea.They are the products of interactions between the original components of tea leaves,when they migrate into the water phase from the mesophyll tissues or cells driven by heat.Under the combined action of several chemical bonds,they self-assemble to form particles,constituting the major part of the colloidal phase of tea infusion,affecting tea's quality and function.Our previous study has isolated and identified the self-assembled colloidal nanoparticles(NPs)in black tea infusion,whose main components included pectin,polyphenol oxidase and catechins.Using the tea infusion NPs as prototype,this study extracted and identified pectin from Lapsang souchong black tea infusion,and fabricated the heat-induced pectin-polyphenol oxidase nanoparticles,and characterize its properties as a carrier for catechin(EGCG).The results are briefly reported as below:(1)The pectin was extracted from tea infusion by ethanol sedimentation.The protein and polyphenols were eliminated by adding 3% trichloroacetic acid to the infusion.About84% of protein were removed,while the loss of pectin were less than 14%.The contents of galacturonic acid and polyphenols in tea soup pectin samples were determined to be 554.8?g/m L and 15.26 ?g/m L by the carbazole sulfuric acid method and Folin phenol method,respectively.The esterification degree of pectin was 53% determined by FTIR.Its average molecular weight was 54.9 k Da identified by size-exclusive chromatography coupled with multi-angle laser light scattering.The tea infusion pectin was mainly RG ? type and HG type,maybe a small amount of XG type,but not RG ? type.(2)The PPO-pectin NPs(PP-NPs)were fabricated by heat-induced self-assembly of polyphenol oxidase and tea infusion pectin.The influences of p H,temperature,ratio of PPO and pectin on the formation of PP-NPs were determined.The optimum fabrication conditions were: PPO and pectin solution at 0.5 mg/m L,molecular weight ratio of PPO and pectin was1:1,incubated at 90? for 30 min.The average hydrodynamic diameter of PP-NPs was 241.3nm,and ?-potential was-34.3 m V.The PP-NPs are stable under acidic condition,susceptible to alkaline condition and resistant to high temperature.(3)The PPO-Pectin-EGCG nanoparticles(PPE-NPs)were fabricated by the same way as the PP-NPs.The EGCG loading efficiency was 32.4% in PPE-NPs determined by HPLC method.The PPE-NPs encapsulation improved the resistance of EGCG to high temperature.The PP-NPs and PPE-NPs showed no toxicity on the macrophages with the survival rate above 90%.Their antioxidant activities were determined by ORAC assay and cellular antioxidant activity(CAA)assay.Both NPs showed potent antioxidant activity,which was positively correlated with EGCG content.The encapsulated EGCG survived the oral digestion and gastric digestion,with a residual percentage about 80%.However,only 8.3% of pure EGCG was retained after 2 h of intestinal digestion,while 19.2% of the encapsulated EGCG was retained.The antioxidant activity of NPs-carried EGCG was higher than that of EGCG in free form.