Study On The Enzymatic Mechanism Of Lactiplantibacillus Plantarum Dy-1 Fermentation Promoting The Release Of Bound Phenolics In Barley

Phenolics in grains mostly occur in bound forms.Most of the bound phenolics are released and metabolized in the colon,resulting in low bioavailability which due to the lack of digestive enzymes in stomach and intestine to promote the release of bound phenolics.Our previous studies have found that fermentation with Lactiplantibacillus plantarum dy-1 could effectively promote the release of bound phenolics in barley.However,the key enzymes involved in the release of bound phenolics during fermentation and its enzymatic mechanism were still unclear.In this study,the key enzyme differently-expressed during the L.plantarum dy-1fermentation was selected by multi-omics method,and further explored its ability of releasing barley bound phenolics and its enzymatic mechanism.The main research contents and results are as follows:1.Combination screens of the key enzyme that releasing barley bound phenolics during fermentation with L.plantarum dy-1.The effects of fermentation on phenolics composition in raw barley dietary fiber（RBDF）were analyzed by ultra performance liquid chromatogram-high resolution mass spectrometry.In order to screen the key enzyme that may promote the release of bound phenolics,we compared the differentially-expressed proteins during fermentation,and analyzed the results of whole-genome sequencing and functional gene annotation of L.plantarum dy-1.The results showed that after fermentation with L.plantarum dy-1,the total content of free and bound phenolics in RBDF increased by 1074.58g/g and decreased by 2408.69g/g,respectively.The whole genome sequencing and functional gene annotation results showed that L.plantarum dy-1 encoded 142 glycoside hydrolases,53 esterases and 158 glycosyltransferases.It was found that the expression yield of 50 proteins were significantly up-regulated during fermentation with L.plantarum dy-1,among which the expression yield of aβ-glucosidase was found to increase more than 4times,which ID was KN812-14630.Therefore,we speculated that it might be the key enzyme promoting the release of bound phenolics during fermentation with L.plantarum dy-1 and named it LPBG.2.Heterologous expression and characterization ofβ-glucosidase LPBG.The LPBG was heterologous expressed in E.coli BL21（DE3）and was purified by nickel column affinity chromatography,followed by characterizing its enzymatic properties.The results showed that the expression of recombinant LPBG induced at 15℃was higher than that at 37℃,and was about 40 mg/L.The enzyme activity of purified LPBG was increased by 74%with a recovery of 16.43%,and its optimum temperature was 45℃and its optimum p H was 7.0.The Mg²⁺,Fe³⁺and Ca²⁺could promote the enzyme activity of purified LPBG,while the Mn²⁺,Zn²⁺and Cu²⁺can significantly inhibit its enzyme activity.3.The ability of LPBG to promote the release of barley bound phenolics and its enzymatic mechanism were analyzed.The enzymatic-hydrolyzed barley dietary fiber（EBDF）was obtained by enzymatic hydrolysis the RBDF with purified LPBG,and the ability of LPBG to promote the release of barley bound phenolics was analyzed by determining the composition of phenolics in enzymatic hydrolysates.Moreover,the enzymatic mechanism of LPBG to release barley bound phenolics was explored by analyzing the change of the molecular structure in EBDF.The results showed that the total free phenolics content of EBDF increased by 1074.58g/g,in which the free syringic acid,gallic acid and benzoic acid content increased significantly.The proportion of high molecular weight polysaccharide in EBDF decreased significantly,while its low molecular weight polysaccharide content increased and the monosaccharide composition changed.The structure integrity of EBDF was damaged,and the fluorescence of bound phenolics was faded obviously under the confocal laser scanning microscope.The X-ray diffraction analysis showed that the crystallinity of EBDF decreased by about 16%,while the infrared spectroscopy and the nuclear magnetic resonance spectroscopy showed that the composition of functional groups and chemical bonds of EBDF were changed.These results indicate that LPBG may reduce the covalent and non-covalent binding intensity between dietary fiber and bound phenolics by destroying the structure of cellulose and hemicellulose,thus promoting the release of barley bound phenolics,it may also break the glycosidic bond between dietary fiber and bound phenolics,and promote further conversion of phenolics from the bound form to the free form.