| Carrageenans,a family of sulfated linear polysaccharides from red seaweeds,are commonly used in the food industry as gels,thickers and agents.Besides being the source of food additives,carrageenan polysaccharides are also the sources of bioactive carbohydrates that possess antioxidant,antiviral and immuregulatory activities.However,macromolecular carrageenans are high viscosity and difficult of being absorbed by organisms,which limit the functional utilization.Carrageenan oligosaccharides are absorbable and low toxicity.Therefore,efficient preparation of carrageenan oligosaccharides is an important way to promot deep processing technology of red seaweed oligosaccharides.In our study,a novel strain that could effectively hydrolyze K-carrageenan was isolated from Chondrus crispus and identified by 16S rDNA,the fementational kinetics models were as well built.The immobilized bacteria technology was carried out to prepare concentrated enrichment TF-332 microsphere,the Fe3O4-chitosan was chosen as the carrier,and the connection mechanism was studied.K-Carrageenan oligosaccharides were filtered by tangential flow ultrafiltration and purified through a system coupling with medium pressure liquid chromatography with an evaporative light scattering detector.Furthermore,ESI-MS,CID-MS/MS and NMR were used to characterize the structural sequences.To evalue the immune regulation activities of four types of K-carrageenan oligosaccharides,an inflammatory model was built using LPS induced RAW264.7 macrophages.Based on the analysis of systematic biology,we drawed the protein network of κ-carrageenan oligosaccharides treatment LPS-induced model,and studied the immunological regulation pathway and mechanism of K-carrageenan oligosaccharides reacton in proteomics level.Firstly,a novel strain that could effectively hydrolyze K-carrageenan was isolated and identified as Thalassospira sp.Fjfst-332(TF-332)by 16S rDNA sequencing.Based on Box-Behnken experimental design and response surface methodology,the optimal fermentation conditions were as follows:2.0 g/L K-carrageenan,1.0 g/L yeast extract,1.0 g/L FOS,20.0 g/L NaCl,2.0 g/L NaNO3,0.5 g/L MgS04 7H20,0.1 g/L K2HPO4,and 0.1 g/L CaCl2,at 25 ℃,pH 7.0,125 r/min,with 3 mL inoculum size and 30 mL culture volumn.The highest enzyme activity exhibited by Thalassospira sp.Fjfst-332 was 267 U/mL.In order to guide scaled-up production,two empirical models including the logistic equation and Luedeking-Piret equation were proposed to predict the strain growth and enzyme production,respectively.Then,immobilized bacteria(IMB)microsphere was prepared by embedding K-carrageenase-producing TF-332 onto a magnetic Fe3O4-chitosan carrier.Several physical methods including XRD,VSM,FTIR,SEM,CLSM and Raman spectra were used to characterize and verify that strain TF-332 combined with Fe3O4-Chitosan carriers through the amide Ⅱ(-NH2)groups.The optimal parameters of Fe3O4-chitosan carriers immobilized TF-332 were at 20 ℃ for 3 h,and the optimal fementation parameters of IMB were incubating with 700 mg IMB in 30 mL media at 30 ℃,pH 7.0.Additionally,compared with free TF-332,the efficiency of IMB producing K-carrageenase increased 41.7%,and the yield of K-carrageenase increased 66.7%.What’s more,the enzyme production was relatively stable in the first 7 cycles of incubation using the same IMB and the residual activity of IMB was about 66.18%.The results indicated that the IMB showed fementation stability and enhanced the yield and efficiency of preparation κ-carrageenase.Four types of purified K-carrageenan oligosaccharides were separated using MPLC-ELSD,and the total yield was approximately 5.02%.The optimal purified parameters were as follows:4 mL/min flow rate,1.0 g/mL sample concentration,1 mL loading amount and with 0.1M NH4HCO3 as eluent.The molecular weights of types of K-carrageenan oligosaccharides were 404 Da,807 Da,1210 Da and 1273 Da,respectively,which were detected by ESI-MS and CID-MS/MS.The structural sequences were inspected using NMR analyses and identified as follows:α-DA-1→3-G4Sra/p(κ-neocarrabiose),α-DA-1→3-β-G4S-1→4-α-DA-1→3-G4Srα/β(κ-neocarratetraose),α-DA-1→3-β-G4S-1→4-α-DA-1→3-β-G4S-1→4-α-DA-1→3-G4Srα/β(κ-neocarrahexaose),α-DA/DA2S-1→3-β-G4S-1→4-α-DA-1→3-β-G4S-1→4-α-DA-1←3-G 4Srα/β(heterozygous κ/t-neocarrahexaose).The in vitro immune defence effects of κ-carrageenan oligosaccharides were studied by LPS-induced RAW264.7 macrophages model.Results showed that K-carrageenan oligosaccharides have no evidence of cytotoxicity on RAW264.7 cells within a concentration of 75 μg/mL,while the inhibition rate of macrophages was increased at a concentration of 300 μg/mL.In addition,four types of κ-carrageenan oligosaccharides dose-dependently(p<0.01)decreased the levels of pro-inflammatory mediators including ROS,NO,TNF-α,IL-1β and IL-8,and the inhibitions also performded on transcription levels.Moreover,K-carrageenan oligosaccharides suppressed the expression of iNOS and COX-2 proteins,indicated that they exhibited well immune defence.The KCO-4 performed the best immune regulation effects,followed by KCO-3,KCO-2 and KCO-1,respectively,suggesting that the higher number of sulfate units and molecular weights ofκ-carrageenan oligosaccharides revealed the better immune defence.Based on the proteomic technology of Label-free quantification couple with LC-MS/MS,a total of 4,547 proteins were identified.Compared to LPS group,236,370,450 and 135 proteins showed significant differences(p<0.001)in KCO-1,KCO-2,KCO-3 and KCO-4 group,respectively.The differential regulated proteins in KCO-1 group were different from other three groups in the comprehensive functions,which most involved in cell cycle,cell death and survival,DNA replication.In other three groups,the differential proteins mainly associated with RNA post-transcriptional modification,RNA trafficking,organismal injury and abnormalities.Furthermore,the NF-κB pathway mediated by CD 14 coupled with Rel and p50(CD14/Rel@p50)was suppressed by KCOs,resulting in reduced expression levels of NO,TNF-α,IL-1β,IL-8,iNOS,COX-2 and other cytokines. |
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