Study On Preparation And The Potential Prebiotic Activities Of Apple Pectin Oligosaccharides

Nowadays,with the increasing market demand for prebiotic products such as inulin,fructooligosaccharides and galactooligosaccharides,people have a strong interest in finding new prebiotic candidates.Pectin oligosaccharides,as one of the important prebiotic candidates,have the potential of sustainability,economy and scale,which attracted extensive attention.In this paper,apple pectin was selected as the research object.Pectinase and high hydrostatic pressure(HHP)assisted metal-free Fenton reaction were used to hydrolyze pectin,the appropriate separation and purification processes were constructed to obtain pectin oligosaccharides with potential prebiotic activity and identify their structures;Molecular docking technology was used to explore the interaction mechanism between pectin oligosaccharides and hydrolases from healthy intestinal flora.The effects of different types of pectin oligosaccharides on the community distribution and metabolism of fecal flora were explored.The important results and conclusions obtained were as follows:(1)The commercial apple pectin was degraded by the pectinase.The optimum conditions were 30 mg enzyme dosage/100 mL,60 min reactive time and 40℃reactive temperature.Membrane separation,anion exchange chromatography column,graphite carbon column and C18 were used for separation and purification,and obtained the purified pectin oligosaccharide.The purified pectin oligosaccharide mainly contained glucose,galactose,xylose and arabinose,without galacturonic acid,and the structure was mainly Gall→4Gal1→4Gal1→4Gal1→3Ara.The prebiotic scores of oligosaccharides on Lactobacillus plantarum CICC20261(L.plantarum CICC20261,0.50± 0.05)and Lactobacillus reuteri DPC16(L.reuteri DPC16,0.46 ±0.04)were not significantly different from those of commercial inulin(L.plantarum CICC20261(0.49±0.02)and L.reuteri DPC 16(0.48±0.03)).The binding energy of Gal4Ara and β-galactosidase from Lactobacillus delbrueckii subsp.bulgaricus and Bacillus subtilis were-9.64 and-9.69 kcal/mol,respectively,and that of Klebsiella pneumoniae was-6.90 kcal/mol.The affinity of probiotics was higher than Klebsiella pneumoniae(harmful bacteria).(2)The commercial apple pectin was degraded by HHP assisted metal-free Fenton reaction.The optimum conditions were 600 MPa(30 min)and pectin concentration of 0.5 mg/mL.Membrane separation and anion exchange chromatography column were used for separation and purification,the obtained the pectin oligosaccharide mainly contained oligoxyloglucan and oligoxyloglycuronic acid.The prebiotic scores of the purity pectin oligosaccharide on the three selected lactobacillus were L.plantarum CICC20261(0.80±0.01),L.reuteri DPC16(0.66 ±0.01)and L.rhamnosus CICC24233(0.70± 0.01),repectively.The results of molecular docking indicated that the binding energy gap between Butyrivibrio fibrisolvens and Escherichia coli was the largest when the number of xylose substitutions was four in the molecular docking of Xyloglucan oligosaccharides,and the largest gap was existed in the molecular docking of Xylo-galacturonic acid oligosaccharides when the number of xylose substitutions was zero.Polar amino acids played a key role in the interaction between oligosaccharides and glycosidases,especially the positively and negatively charged polar amino acids.(3)The different types pectin oligosaccharides were fermented by fecal flora.The results showed that pectin oligosaccharides could significantly reduce the the ratio of Firmicutes/Bacteroidetes compared to commercial inulin.Galacturonic acid and Bifidobacterium presented a significant positive correlation(0.423,P=0.04).The increase of total acid content was mainly positively correlated with the abundance of Streptococcus(0.765,P=0.004).The addition of pectin oligosaccharides could significantly increase the content of taurine,L-proline and pantothenic acid and reduce the content of uric acid.The difference metabolites were mainly negatively correlated with the abundance of Veillonella,and positively correlated with the abundance of Bifidobacterium,Megamonas,Phascolarctobacterim and Paraclostridum.