Pectic Oligosaccharides From Mango Peel Hydrolyzed By Immobilized Polygalacturonase And Assessment Of Their Antibacterial Activities

China is the second largest producer of mangoes,which can produce plenty of discarded mango peel every year due to the deep processing of mangoes.Mango peel contains 15% to20% pectin.Pectic oligosaccharides(POS)generally deriving from pectin by enzymatic hydrolysis are the most promising functional oligosaccharides with special antibacterial properties,which can be used as a new natural preservative in the food industry.In order to achieve the effective utilization of mango peel by-products and the efficient preparation of POS,this project first successfully immobilized polygalacturonase by sol-gel encapsulation,and then packed them into a packed-bed column reactor to continuously hydrolyze mango peel pectin.What's more,the bacteriostatic performance and mechanism of POS were preliminarily studied,which provided a rationale for the successive industrial production of pectic oligosaccharides as natural preservatives in future.The main conclusions were as follows:Firstly,polygalacturonase was immobilized by sol-gel entrapment.The immobilized enzymes were characterized by scanning electron microscope(SEM),thermo gravimetric analysis(TGA),fourier transform infrared spectroscopy(FT-IR)and Raman spectra,and their optimal temperature and p H,heat resistance and reusability were also measured.It was found that the polygalacturonase was successfully immobilized by the sol-gel encapsulation,which retained 94.6% of its initial activity,indicating that this method is a mild and effective immobilization method.After incubated at 55°C for 2 h,the immobilized polygalacturonase kept 57% of its original activity,whereas the free enzyme kept only 17% of its original activity,indicating that the immobilization improved the heat resistance of the enzyme.Furthermore,the immobilized enzyme displayed good recyclability,which reserved 65% of its primary activity after 6 batch reactions.Next,the mango peel pectin was hydrolyzed by immobilized enzyme to obtain POS,and then the antibacterial activity of POS on foodborne pathogens was evaluated via Oxford cup method.In addition,the products were quantified and qualitative by high performance gel filtration chromatography(HPGFC),high performance anion exchange chromatography(HPAEC)and nuclear magnetic resonance(NMR).It was found that pectic oligosaccharides with a degree of polymerization of 2 to 8 has the best antibacterial activity,accounting for 88%of the pectin hydrolysate.According to the NMR results,the prepared products were mainly polygalacturonic acid formed by galacturonic acid(Galp A)through ?-1,4 glycosidic bonds,namely POS.Thirdly,the immobilized enzyme was used to prepare a packed-bed column reactor.The effects of key factors such as the flow rate,substrate concentration and enzyme amount on the extraction rate of POS were measured,and the conditions for extraction of POS were optimized through response surface methodology.It was found that the prediction model of the regression equation simulated by the BOX-Behnken method had high credibility.It was found that the influence on extraction rate of POS was followed as the order: substrate concentration > enzyme amount > flow rate.The best extraction conditions obtained from the analysis were as follows:enzyme amount of 47.77 U,substrate concentration of 0.12%,flow rate of 2.2 m L/min.The predicted extraction yield of POS was 96.52%,while according to the actual situation it reached94.56%.Finally,the bacteriostatic performance of POS was measured.The bacteriostatic activity of POS obtained by continuous hydrolysis were compared with the commercially available preservatives such as sodium benzoate,potassium sorbate,and nisin against the foodborne pathogens: Escherichia coli(E.coli),Staphylococcus aureus(S.aureus),Salmonella and Bacillus subtilis(B.subtilis).The antibacterial mechanism of POS was preliminary studied through SEM and nucleic acid release experiment.It was found that the antimicrobial activity of POS was equivalent to commercially available preservatives and the minimum inhibitory concentrations were 0.8,0.6,1.0 and 0.8 mg/m L,respectively.In addition,POS destroyed the integrity of the bacterial membrane,leading to the liberating of nucleic acid,which effected the normal metabolism of the pathogens,thus resulting in the death of the bacteria.