| Due to the call for building a healthy China,more functional oligosaccharide products need to be developed.In view of the current waste treatment problems in the citrus processing industry,this study tried to prepare pectin oligosaccharides from citrus pectin by enzymatic hydrolysis and investigate their functional properties.However,there is no uniform method for the quantitative analysis of pectin oligosaccharides in current research,and the complexity of the pectinases makes it challenging to obtain specific enzyme suitable for pectin oligosaccharide production.To solve the above problems,this paper used the citrus peel residues as raw material and then carried out research on the extraction of pectin polysaccharides,the separation of oligosaccharides from enzymatic products,the splitting of pectinase component,and the effect of glycosyl composition on the intestinal probiotic activity and immune activity of pectin oligosaccharides.The main results are as follows:Effects of different extraction methods on the structural characteristics of pectin polysaccharides were firstly investigated.Three pectin samples were extracted from citrus peel residues by acidic,alkaline and hydrothermal methods,and their physicochemical properties,glycosyl composition,surface morphology and characteristic functional groups were all analyzed.Results showed that the extraction techniques significantly affected the structural properties of pectin polysaccharides,particularly in terms of glycosyl composition.The proportion of homogalacturonan(HG)in acidic-extracted pectin was as high as 64.42%,which was 2.70 times higher than that of rhamnogalactan-I(RG-I),suggesting that HG was its main structural unit.The contents of RG-I in alkaline-and hydrothermal-extracted pectin were 48.17% and 37.56%,respectively,both significantly higher than that of acidic-extracted pectin(23.83%),confirming that the alkaline and hydrothermal methods could significantly retain the neutral sugar side chain structure of pectin.The feasibility of separating oligosaccharides from pectin enzymatic hydrolysates by organic reagents was investigated.The enzymatic products of polygalacturonic acid(PGA)were separated by ethanol with dfferent concentrations,and the molecular weights of each precipitate were analyzed.Results showed that ethanol fractional precipitation technology could achieve effective separation of PGA degradation products with different molecular weights.The supernatant fraction after the 60% ethanol precipitation was strictly oligosaccharide with heavy average molecular weight,number average molecular weight and degree of polymerization were detected as 2.58 k Da,2.34 k Da and 15,respectively.In addition,ethanol precipitation treatment and chromatographic analysis were also suitable for the quantitative characterisation of commercial pectin oligosaccharides.In order to improve the yield of oligosaccharides in pectin hydrolysis products,the pectinase from Aspergillus niger was split using a 30 k Da ultrafiltration membrane,and the activities of endo-and exo-pectinase were measured.Results showed that the ratio of endo-and exo-pectinase activity in the treated enzyme was 3.16,which was 4.94 times more than that of the original pectinase,indicating that the enzyme with low exo-pectinase activity was suitable for the production of pectin oligosaccharides.When the pectinase was used to hydrolyse PGA,the highest yield of oligosaccharides was up to 85.9% under the optimal enzymatic conditions.And two pectin oligosaccharide samples with significant differences in monosaccharide composition were obtained by the enzymatic hydrolysis of PGA and hydrothermal extracted-pectin.Among them,oligo-GA only contained galacturonic acid(97.83%),while oligo-HEP contained both galacturonic acid(46.37%)and neutral sugars(49.80%).Effects of glycosyl composition on the intestinal probiotic property of pectin oligosaccharides were studied.Oligo-GA and oligo-HEP were used as the sole carbon sources in in vitro fecal fermentation experiments.And the short-chain fatty acids(SCFAs)production and microbial distribution of each oligosaccharide group were analyzed.Results revealed that the total SCFAs yields of oligo-GA and oligo-HEP were 59.19 m M and 56.62 m M,which were 2.88 and 2.75 times higher than those of the blank control group,respectively.It was also found that the difference in glycosyl composition significantly influenced the structure of the intestinal flora in both oligosaccharide groups.However,both oligo-GA and oligo-HEP appeared to significantly inhibit the growth of intestinal pathogenic bacteria(Escherichia-Shigella,Lachnospiraceae＿UCG-004 and Lachnospiraceae＿UCG-010),while oligo-HEP sample could significantly increase the relative abundance of the probiotic Bifidobacterium and oligo-GA sample could significantly increase the relative abundance of the probiotic Lachnospira.In general,all pectin oligosaccharide samples have beneficial effects on regulating the balance of intestinal flora and promoting the health of the organism.Effects of molecular weight and glycosyl composition on the immunomodulatory activity of pectin and its oligosaccharides were evaluated.The results showed that all samples had no toxic effect on macrophages and were able to promote cell proliferation to some extent.Compared to the polysaccharide samples,oligo-GA and oligo-HEP had a more significant ability to stimulate cell activation and induce cell morphological changes.Furthermore,oligo-HEP sample showed much higher cytokine secretion than oligo-GA sample,including nitric oxide,tumor necrosis factor-α,interleukin-6 and toll-like receptor 4.It was further confirmed that molecular weight and neutral sugar side chain structure were key factors influencing the immunological activity of pectin.According to the TAK-242 addition assay,both oligo-HEP and oligo-GA were able to activate macrophage and promote cytokine secretion by binding to TLR4 receptors on the cell membrane.In summary,this study established a scientific and accurate quantitative analysis method for pectin oligosaccharides based on ethanol fractionation precipitation and obtained the enzyme with low exo-pectinase activity using membrane technology.It was also discovered that the oligo-HEP sample containing large amounts of neutral sugars had more significant intestinal modulating activity and immune enhancing ability.Taken together,these findings lay the foundation for the subsequent industrial production and application of pectin oligosaccharides and also provide a theoretical basis and technical support for the further processing of agricultural and forestry wastes. |
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