| Huangshui is one of the main by-products produced in the solid state production of Baijiu in China.It has a huge output and contains a large amount of residual carbon and nitrogen and other nutrients.It has dual attributes of pollution and resources.At present,Huangshui is treated as wastewater by Baijiu enterprises,resulting in a huge waste of resources.According to reports,there are abundant polysaccharide nutrients in Huangshui,with a total sugar content of up to 53.2～54.0 mg/m L.At present,domestic and international research has found that foodborne polysaccharides have the function of protecting or repairing intestinal epithelial cell barrier damage,thus exploring the use of foodborne nutrients to alleviate or improve intestinal barrier damage has become a research hotspot in recent years.Therefore,in-depth analysis of polysaccharides from Huangshui,exploring their role and mechanism in improving intestinal barrier function damage,enhancing the nutritional and functional value of Huangshui polysaccharides,can provide new research ideas for the highvalue utilization of Huangshui.However,there is currently a lack of research data on the separation and characterization of polysaccharides from Huangshui,as well as their nutritional functions,which has become one of the “bottleneck” issues in the high-value utilization of Huangshui.Therefore,the purpose of this project is to develop the extraction and separation methods of HSPs,and to further explore the structure of HSPs and the mechanism of intestinal barrier protection activity.It provides a theoretical basis for the deep development and high value utilization of Huangshui.The main research contents and results are as follows:(1)A method to separate and extract polysaccharides from Huangshui was established,and 30 kinds of HSPs were successfully separated from Huangshui,the by-product of Baijiu brewing with different flavor types.Statistical analysis showed that the HSPs from different flavor types of Huangshui could not be distinguished by monosaccharide composition,but different eluting components of polysaccharides could be distinguished by monosaccharide composition;In the evaluation of antioxidant activity in vitro,it was found that the antioxidant capacity of HSPs eluted by 0.05 mol/L Na Cl and 0.2 mol/L Na Cl was stronger than that of water eluted fraction;The quantitative PCD-HPLC fingerprint activity relationship model showed that the monosaccharide composition was closely related to the antioxidant activity,while the glucose content was not significantly related to the antioxidant activity.In addition,the purity,molecular weight,and protein content of polysaccharides are also important factors affecting antioxidant activity.(2)The detailed structures of three representative HSPs(HSP-W: water eluted fraction,HSP-2: 0.05 mol/L Na Cl eluted fraction,and HSP-3: 0.2 mol/L Na Cl eluted fraction)were analyzed and characterized by HPGPC,FT-IR,GC-MS,NMR,AFM,and SEM.The results were as follows: The molecular weights of HSP-W,HSP-2,and HSP-3 were 166.00 k Da,39.80 k Da,and 26.40 k Da,respectively;The results of monosaccharide composition,FT-IR,methylation,and NMR analysis showed that HSP-W was a branched glucan consisted of a 1,4linked α-D-Glcp backbone with the substitution at O-6 with 1,6-linked α-D-Glcp residue;the backbone of HSP-2 was →2)-β-D-Manp-(1→2,6)-β-D-Manp-(1→6)-α-D-Glcp-(1→4)-α-LRhap-(1→3,4)-α-L-(Rhap-(1→;the skeleton of HSP-3 inferred as →2)-β-D-Manp-(1→2,6)-β-D-Manp-(1→6)-β-D-Galp-(1→3,6)-β-D-Galp-(1→4)-α-L-Rhap-(1→3,4)-α-L-Rhap-(1→;AFM,SEM,and particle size analysis results showed that HSP-W is composed of aggregated single spherical particles with holes,while HSP-2 and HSP-3 showed an irregular sheet shape,rough and uneven surface;particle size distribution of HSP-W,HSP-2,and HSP-3 ranges from nanometer to micrometer.(3)Based on the Caco-2 cell model,the intestinal barrier protective function of the selected HSPs with high activity was evaluated.HSPs obtained from the first part were screened by their ability to scavenge ROS in Caco-2 cells.It was found that JGJ-2,NLS-2,and WLY-0 had a higher ability to scavenge ROS.Subsequently,the antioxidant system of intracellular enzymes was studied.The results showed that these three polysaccharides could all play an antioxidant role by reducing the content of MDA and increasing the activities of SOD,CAT,and GSH-Px;However,in the anti-inflammatory activity,JGJ-2 can promote the release of inflammatory factor TNF-α,while NLS-2 and WLY-0 can inhibit the release of TNF-α,IL-6,and IL-1β and play an anti-inflammatory role.Therefore,the intestinal barrier function of NLS-2 and WLY-0 was further studied using Caco-2 model.It was found that NLS-2 and WLY-0 could reverse the decrease of TEER value and the increase of FD-4 flux induced by LPS,and could improve the expression of tight junction proteins Occludin,Claudin-1,ZO-1,and JAM-A to alleviate the barrier dysfunction of Caco-2 cell monolayer model caused by LPS.(4)The protective mechanism of two kinds of HSPs NLS-2 and WLY-0 on intestinal barrier function was investigated by RNA-seq and other biological techniques.The results showed that the differential genes between NLS-2 treated group and LPS group were enriched in MAPK signaling pathway,Toll like receptor signaling pathway,and NF-κB signal pathway,and Western Blot verification results showed that NLS-2 intervention can significantly inhibit the expression of TLR4 and MyD88 proteins,reduce the phosphorylation rate of p-IκB/IκB and p-NF-κB/NF-κB,and inhibit the occurrence of NF-κB nuclear translocation after LPS exposure.It was observed that the expression of downstream proinflammatory factors TNF-α,IL-6,and IL-8 was reduced.At the same time,it was also found that NLS-2 supplement inhibited the phosphorylation of p38 and JNK,but the phosphorylation level of ERK was promoted,indicating that NLS-2 could change inflammation and TJs related genes by regulating TLR4/MyD88/NF-κB and MAPK mediated anti-inflammatory pathway,thus resisting LPS damage to the intestinal barrier.The differential genes between WLY-0treatment group and LPS group were enriched in MAPK signal pathway.WLY-0 intervention had no significant effect on TLR4 protein expression.However,when WLY-0 was supplemented,it was observed that the phosphorylation of p38 and JNK was significantly inhibited,the phosphorylation level of ERK was significantly promoted,and the expression of downstream proinflammatory factors TNF-α,IL-6,and IL-8 was reduced,showing that WLY-0 changed inflammation related genes through regulating MAPK mediated anti-inflammatory pathway,thus preventing LPS damage to intestinal barrier. |
