The Role Of Lignin In Affecting Gastrointestinal Enzymatic Digestion And Gut Microbial Metabolism In Vitro

Dietary fiber,hailed as the seventh essential nutritional element for the human body,has gained significant attention from scholars due to its important role in preventing and treating chronic diseases.Lignin,a kind of foodborne dietary fiber,are ubiquitous in various plant-based foods and their by-products.Despite being the most plentiful polyphenolic macromolecule in plants,the role of lignin in gastrointestinal digestion is not well understood due to its complex and heterogeneous structure,which limits its application in the food industry and results in significant waste of resources.Therefore,based on the established in vitro digestion method,this study systematically investigated the inhibitory activity of modified lignin,obtained through a combination of alkali washing and thermal processing,on the apparent hydrolysis rate ofα-amylase（α-AA）during starch digestion,and revealed their interaction mechanism.Additionally,the role of lignin in inhibiting in vitro gastric pepsin and pancreatic proteases hydrolysis was investigated,and the key proteases-inhibitive components were identified,along with their interaction mechanism.Furthermore,the study investigated the function of lignin on colonic microbial metabolism through in vitro fecal fermentation.These investigations could provide theoretical guidance for further research and high-value utilization of foodborne lignin,and offer technical support for the personalized design and development of lignin functionalized products to meet the specific nutritional requirements of different groups.The main contents and results are as follows:Combined with first-order kinetic fitting and advanced techniques,the inhibitory mechanism of modified lignin onα-AA was elucidated.Alkali-washing successfully facilitates thermal-processed lignin to slow the hydrolysis of pancreaticα-amylase in starchy foods in food grade processing.The lignin sample treated with a combination of p H=10 alkali-washing and heating at 200°C（referred to as 10/200）achieved a～70%inhibition on the apparent hydrolysis rate of pancreaticα-amylase,leading to a significant drop in soluble lignin content from 279.3μg/m L to 7.8μg/m L.It was confirmed that inhibition ofα-amylase activity only occurred when the soluble lignin content fell below the threshold of～10μg/m L.Sample 10/200also exhibited a significant slowing（～74%and～43%reduction in starch digestion rate k_f value）in both pancreaticα-amylase hydrolysis and complex pancreatin hydrolysis during in vitro digestion of pasta,indicating its potential in controlling postprandial GI of starchy foods in the small intestine.The mechanism hypothesis is confirmed that a modification of insoluble-lignin surface morphology which might immobilizes theα-AA content in solution.Lignin was subjected to an in vitro stomach digestion assay to assess its inhibitory activity against pepsin,followed by further investigation of the inhibitory mechanism using enzymatic kinetics and analytical techniques.The addition of lignin resulted in over 90%inhibition on the apparent rate of pepsin hydrolysis.Furthermore,there was no significant difference observed in the particle size distribution in the presence of lignin.Soluble lignin was identified as the essential pepsin-inhibitive component（s）with an IC₅₀ value of approximately 0.17 mg/m L.The inhibitory activity of soluble lignin gradually decreased with thermal pre-treatment.The type of inhibition induced by soluble lignin was determined to be mixed-inhibition.Soluble lignin could dynamically interact with pepsin,quenching its intrinsic fluorescence.The interaction between lignin and pepsin was characterized as weak,with an affinity value（Ka）of（1.18×10⁵）,which is less than 1×10⁷.The intermolecular interaction force between lignin and pepsin was primarily hydrogen bond and van der Waals force.Semi-flexible molecular bonding analysis revealed that lignin could form 4 hydrogen bonds with the amino acid residues in the inactive center of pepsin,resulting in changes in the enzyme conformation and inhibition of its enzymatic activity.The effect of lignin on protein digestion in the small intestine was systematically investigated through in vitro protein digestion experiments,combined with acid precipitation fractionation technology and modern structure characterization technology,and the key components of enzyme inhibition were revealed and its mechanism was clarified.Lignin showed a similar～60-70%inhibition on the pancreatic hydrolysis of the gluten substrate.Soluble lignin was confirmed to play a crucial role in inhibiting pancreatic proteases with an IC₅₀ value of 0.3 mg/m L,and showed a～20%and 50%inhibition on pancreatic trypsin and chymotrypsin,respectively.To further study the components responsible for pancreatic protease inhibition,five different p H gradients were obtained through acid precipitation.All evidence indicated the presence of protease-inhibitory components in Lg_{p H5-4} and Lg_{p H<1},with Lg_{p H<1}being notably over 3.5 times more inhibitory per milligram than Lgp H_5-4.This was mainly due to the acid-differentiated soluble fraction(namely Lg_{p H<1},R_h 500,molecularly uncharged),which showed stronger molecular binding affinity with the two proteases due to its abundant polar groups（e.g.,-OH,-CHO）in aromatic rings that favor hydrogen bond formation.Soluble lignin interacted with trypsin and chymotrypsin,dynamically quenching their fluorescence,occupying the entrance to the active center of the enzyme,and interacting with catalytic key residues through hydrogen bonds and hydrophobic interactions,thereby inhibiting their hydrolytic activity.The effects of representative lignin samples（Lg,Lg200,Lg10-200,and SLg）on human colonic microbial metabolism were further investigated through in vitro fecal fermentation using 16s RNA sequencing.It was found that colonic microbial metabolism could partly use SLg for fermentation,as indicated by gas production and the p H of the fermentation broth.Moreover,the contents of acetic acid,propionic acid,N-butyric acid,and N-valeric acid in SLg were higher than in the blank.However,the fermentation degree of the other lignin samples was similar to that of the blank,indicating that colonic microorganisms could hardly use them for fermentation activities.Results of the Alpha diversity index and Venn plot analysis showed that SLg had the highest microbial species diversity.Additionally,the Firmicutes/Bacteroidetes（F/B）ratio in SLg was lower than in the blank,indicating that intake of SLg may reduce the risk of obesity and diabetes in the host.Results of the community Heatmap showed that SLg could significantly promote the relative abundance of beneficial bacteria Ruminococcus-torques-group and Megamonas,and significantly inhibit that of pro-inflammatory bacteria Collinsella.