| The comprehensive utilization of apple pomace remains a problem hindering the sustainable development of apple deep processing.At present,only a small part of apple pomace is used as animal feed after rough processing,and most of it is discarded as waste,which seriously wastes resources and pollutes the environment.Therefore,the high additional utilization of apple pomace is an urgent problem to be solved.Lipid metabolism disorder can lead to obesity,and it is one of the main risk factors of coronary heart disease,arteriosclerosis,fatty liver,diabetes,hypertension and other diseases.Studies have shown that plant polysaccharides have the function of regulating lipid metabolism.In this paper,apple pomace was modified by enzymatic hydrolysis,and the enzymatic hydrolysis process of apple pomace was optimized by synthesizing the changes of in vitro physical properties related to the yield of polysaccharide in apple pomace and lipid-lowering performance.Compared with polysaccharides from apple pomace(PAP),the structural changes,in vitro antioxidant activity and structure-activity relationship of polysaccharides from enzymatic apple pomace(PEAP)were studied.Based on the obese mouse model induced by high-fat diet,the lipid-lowering activity in vivo and the regulation mechanism of lipid metabolism of PEAP were studied.The main research contents and results are as follows:(1)Taking the yield of PEAP as the index,the preparation process parameters of enzymatic apple pomace(EAP)were optimized by single factor and response surface experiments,and the lipid-lowering related physical properties of EAP were discussed.The experimental results show that the microstructure of EPA has changed significantly,EPA forms more large cavities and obvious cracks,has larger specific surface area,exposes more hydrophilic and lipophilic groups,and the water holding capacity,oil holding capacity,expansion capacity and adsorption energy of cholesterol and cholate of EPA have been improved.Pearson correlation analysis showed that the yield of PEAP was closely related to the physical properties of EPA.The optimum technology of apple pomace enzymatic hydrolysis is as follows:the ratio of material to liquid is 1:20,the compound enzyme is composed of cellulase and pectinase(cellulase 20 U/g;pectinase 40 U/g),the time of enzymatic hydrolysis is 5.5 h,the pH of enzymatic hydrolysis is 5.1,the temperature of enzymatic hydrolysis is 44℃,and the yield of PEAP can reach 16.84%.(2)Compared with PAP,the structure and antioxidant activity of PEAP in vitro were studied.Structure:both of them are acidic polysaccharides with the same monosaccharide type,and their main chains contain the same content αType and β Type glycosidic bond,but the content of monosaccide was different.The galacturonic acid content of PEAP was 20.01%higher than that of PAP;the main Mw of PAP was greater than 50 kDa,in which 200≥Mw≥50 kDa accounted for 93.9%of PAP,while the main Mw of PEAP was in the range of 50≥Mw≥4kDa,accounting for 92.1%of PEAP.The scavenging activities of PAP and PEAP to ·OH,O2-·,DPPH· and ABTS+·were evaluated.The results showed that the antioxidant activities of PAP and PEAP were concentration dependent.PEAP had stronger antioxidant activity than PAP,which was attributed to the higher galacturonic acid content in PEAP and the lower molecular weight of PEAP.(3)Structural characterization and in vitro antioxidant activity of PEAPw,PEAP1 and PEAP2,were studied.PEAPw was neutral sugar,and its main chains contained α-glycosidic and β-glycosidic bond.PEAPw is composed of six monosaccharides:Fuc,Ara,Rha,Gal,Glc and Xyl.The main glycosidic bond is 1,4-α-D-linked Glcp,1,4-β-D-linked Glcp,1,4-β-D-linked Galp and 1,4,6-β-Dlinked Glcp,Mw=6.461kda,and its molecular configuration was irregular coil configuration;PEAP1 is an acidic sugar,and its main chains contained αglycosidic and β-glycosidic bond.PEAPi is composed of nine monosaccharides:Fuc,Ara,Rha,Gal,Glc,Xyl,Man,GalAc and GlcAc.The content of uronic acid is 14.67%.The main glycosidic bond is 1,4-α-D-linked GalAcp,1,4-β-Dlinked GalAcp,T-a-L-linked Araf and T-β-D-linked Xylp,Mw=45.164 kDa,the morphology is irregular flake aggregation state,and many spherical protrusions are formed on the surface of the lamella,which should be formed by the mutual winding and bonding force of polysubstituted polysaccharide structure;PEAP2 is an acidic sugar,and its main chains contained a-glycosidic and β-glycosidic bond.PEAP2 is composed of eight monosaccharides:Fuc,Ara,Rha,Gal,Glc,Xyl,GalAc and GlcAc.The content of uronic acid is 18.17%.The main glycosidic bond is 1,4-a-D-linked GalAcp,1,4-β-D-linked GalAcp,1,2-α-L-linked Rhap and T-β-D-linked Xylp,Mw=41.698kda,and the molecular configuration is a highly branched structure.The scavenging activities of PEAP,PEAPw,PEAP1 and PEAP2 on ·OH,O2-·,DPPH·and ABTS+· were concentration dependent and positively correlated with the change of concentration.The free radical scavenging ability of PEAP was higher than that of purified sugar;The free radical scavenging ability of the three purified sugars was comprehensively evaluated,and the best one was PEAP2.(4)The lipid-lowering effect of PEAP in vivo was studied based on the obese mouse model induced by high-fat diet.The phenotypic data,biochemical indexes and histomorphological section structure of mice showed that PEAP had lipid-lowering effect.The study showed that PEAP could reduce the contents of TC,TG,LDL-C,AST,ALT and MAD in obese mice,increase the contents of SOD and CAT,It shows that PEAP can improve blood lipid and repair the disorder of lipid metabolism.The experimental results of histomorphology showed that the volume and number of lipid droplets in the liver of mice in PEAP group decreased,and the diameter of adipocytes also decreased,indicating that PEAP can reduce adipogenesis in mice and improve the damaged liver of mice to a certain extent.Spearman correlation analysis showed that there was a close correlation between biochemical indexes and oxidation indexes in the lipid-lowering process of PEAP.(5)Study on the mechanism of PEAP regulating lipid metabolism in obese mice induced by high-fat diet:On the basis of the metabonomics method of UPLC-Q-TOF/MS,51 potential biomarkers related to lipid metabolism and 5 metabolic pathways involved were screened out.The results showed that there were significant changes in serum metabolism spectrum of mice induced by high-fat diet.PEAP could regulate lipid metabolism and reduce fat production by increasing the contents of L-carnitine and phosphatidylcholine(PCs)in serum of mice;at the same time,they could reduce the contents of LysoPCs,improve the level of oxidative stress in mice and regulate the metabolism of glycerophosphingolipid;PEAP might regulate pentose.(6)Western blotting was used to further confirm that PEAP could improve the lipid metabolism disorder of obese mice through the following ways:First,it inhibited PPAR-γ by enhancing the activation of AMPK to reduce preadipocyte differentiation and triglyceride formation.Second,by increasing the phosphorylation level of PI3K/Akt pathway,the activation of pathway can be increased,islet resistance can be improved,lipid decomposition can be promoted,lipid disorder can be improved,and the balance of glucose and lipid metabolism can be realized.Through this study,it is discovered that PEAP had better antioxidant activity in vitro and lipid-lowering activity in vivo,and it is explained the structural reasons for the improvement of PEAP antioxidant activity,Structural characterization and microstructure of PEAPw,PEAP1 and PEAP2 were characterized and confirmed.On the basis of the metabonomics method and western,it was revealed that PEAP improved the disorder of lipid metabolism in obese mice through multiple ways.The completion of this paper lays a theoretical foundation for extending the apple processing industry chain and realizing the high added value utilization of apple pomace. |
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