| Citrus pectin is a kind of pectin extracted from the peel of citrus fruit.It has a large relatively molecular mass and cannot be absorbed by the intestinal tract after consumption.Low molecular mass citrus pectin,which is considered as a new bioactive polysaccharide,is a kind of pectin modified from natural citrus pectin.To increase the value of citrus pectin,subcritical water at 100℃-130℃ was used to degrade natural pectin into low molecular mass pectin.Changes of pectin composition and structure before and after modification were studied,and the mechanism was analyzed with the study of rheology and degradation kinetics.Moreover,the effects of subcritical water treatment on pectin biological activity were studied from four aspects: antioxidant activity,anti-tumor activity,immunoregulatory activity and effect on intestinal flora of mice.The main conclusions are as follows:(1)To study the effect of different time of subcritical water treatment at different temperatures on the relative molecular mass of citrus pectin,citrus pectin solution was treated at 100℃,110℃,120℃ and 130℃ for 5 min to 100 min respectively in a autoclave sterilizer.It was found that with the increase of subcritical water temperature and the extension of treatment time,the relative molecular mass of pectin gradually decreased,with the number average molecular mass as low as 23.7 ku and the weight average molecular mass as low as 44.5 ku,and the degradation degree of pectin as high as 80.5% and 71.7%,respectively.Under the same condition,the degradation degree calculated by number average molecular mass is higher than that calculated by weight average molecular mass.The degradation of pectin by subcritical water conforms to the first-order kinetic equation.With the increase of subcritical water temperature and the extension of treatment time,the content of reducing sugar in pectin solution gradually increased,and the degree of hydrolysis of total sugar was between that calculated by number average molecular mass and that calculated by weight average molecular mass.(2)A rheometer was used to measure the rheological characteristics.The formula of power law equation of non-newtonian fluid was used for fitting.It was found that with the increase of subcritical water temperature and the extension of treatment time,the pectin solution gradually changed from a pseudo-plastic fluid to an expanded plastic fluid,and the rheological index gradually increased from less than 1 to more than 1.The rheological index of untreated pectin was 0.7919,and rose to 1.7864 after treatment at 130℃ for 180 min.Datas of apparent viscosity change over time were fitted by regression equation of first-order kinetics similar to that of polysaccharide degradation.Apparent viscosity of pectin decreased significantly after subcritical water treatment,which was more significant with the increase of treatment temperature and treatment time.(3)The untreated pectin(A)and the pectin treated at 120℃ for 40 min(B),130℃ for60 min(C)and 130℃ for 180 min(D)were selected for comparative study.It was found that with the increase of subcritical water temperature and treatment time,the relative molecular mass of pectin decreased,the polydispersity index(Mw/Mn)increased,the content of rhamnoose and galactose decreased first and then increased.It was found that the subcritical water treatment had no significant effect on the basic structure of pectin,but the content of hydroxyl and carboxyl groups in the molecule was increased,and the esterification reaction occurred.Under certain treatment conditions,the content of galacturonic acid carboxyl group was slightly increased.The ordered structure of pectin was reduced,and the disordered structure of relaxation and irregular aggregation was increased.Microscopic image analysis showed that after subcritical water treatment,the pectin molecules disappeared in bending,the aspect ratio decreased,the surface area increased,and the small molecule fragments increased,accompanied by the Browning reaction.Based on the above structural analysis,it was found that subcritical water treatment can hydrolyze pectin molecules,and it is possible to repolymerize pectin molecules through esterification and caramelization reactions.The subcritical water is more likely to degrade pectin by breaking the branched chains or glycoside bonds near the end of pectin molecules.After subcritical water treatment,pectin molecules degrade,the disordered structure increases,and more active groups are exposed,which may show better biological activity.(4)The DPPH free radical scavenging ability,ABTS free radical scavenging ability,reducing power and inhibitory activity on tumor cell growth of pectin obtained from four different subcritical water treatments were studied.It was found that the antioxidant activity of pectin increased significantly after the subcritical water treatment.Compared with untreated pectin,the DPPH free radical scavenging ability,ABTS free radical scavenging ability and reducing power of pectin were increased by 4.6-9.7 times,15.5-32.7 times and9.9-19.9 times respectively after treatment at 130℃ for 180 min.Compared with the common antioxidant Vc,the DPPH free radical scavenging ability,ABTS free radical scavenging ability and reducing power of pectin after being treated at 130℃ for 180 min were similar to those of Vc with concentrations of 0.9%-1.5%,9.9%-15.7% and 5.0%-17.5%,respectively.The inhibitory effect of four pectin samples on human colon cancer cells was higher than that on human liver cancer cells.With the increase of subcritical water temperature and treatment time,the inhibitory effect on cancer cells was gradually enhanced.After treatment at 130℃ for 180 min,half of the inhibitory concentrations of pectin on the proliferation of Hep G2 cells,ht-29 cells and SW620 cells(72 h)were 0.13 mg/m L,0.038mg/m L and 0.026 mg/m L,respectively.(5)The immunosuppressive mouse model was induced by cyclophosphamide to evaluate the regulation of intestinal flora and immune function of the low molecular pectin with subcritical water degradation.It was found that the intake of low molecular mass pectin help immunosuppressive mice to gain weight,reduce the atrophy of the spleen,increase the concentration of cytokines both in the serum and colon,thus enhancing immunity.Ingestion of low molecular mass pectin could restore the intestinal flora structure of mice altered by cyclophosphamide treatment,which was close to that of the normal control group.Cluster analysis showed that feeding low molecular mass pectin could normalize the abundance of bacteroidetes and firmicutes and inhibit the increase of proteobacteria.At the level of family and genus,the relative abundance of bacteria changed by cyclophosphamide treatment could be reversed to a level similar to that of normal control mice.Immunosuppressive mice supplemented with low molecular mass pectin resulted in differences in the KEGG metabolic pathways of functional genes of the bacterial community,which were close to the normal control group.It was found that the pectin treated with subcritical water showed better functional characteristics and biological activity in immune regulation than the untreated orange pectin.It is a potential immune regulator,which can regulate the intestinal flora structure and prevent and treat immunosuppressive diseases. |
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