The Interaction Mechanism Of Highland Barley Proteins-polyphenols And The Application Of Its Complexes

As a special type of barley,highland barley（HB）has higher nutritional value than rice,wheat and corn.It is a crop with edible and medicinal value.It is rich in different phenolic substances（phenolic acids,flavonoids,condensed tannins,etc.）.At present,the researches on highland barley phenolic substances mainly focus on the components and antioxidant activities.There are few reports on the effects of phenolic substances on the structural and functional characteristics of highland barley protein and the interaction between highland barley protein and phenolic substances.The purpose of this paper is to study the interaction between highland barley protein and phenolic substances,clarify the mechanism of the influence of phenolic substances on protein performance.This study will propose a new idea and method for scientific and rational use of highland barley.Distribution of highland barley protein and phenolic substances in highland barley cortex.With the increase of milling rate,the content of total protein,albumin,globulin,glutenin,bound polyphenol,free polyphenol,bound flavone,free flavone and condensed tannin in the cortex of highland barley increased first and then decreased,while the content of hordein increased.The content of bound polyphenol,free polyphenol,bound flavone,free flavone and condensed tannin reached the highest value when the milling rate was 8.30%,the content were 1174.57±44.78 mg GA/100 g,540.00±6.99 mg GA/100 g,229.15±8.75 mg TE/100 g,240.17±1.79 mg TE/100 g and 2215.58±33.85 mg PC/100 g,respectively.Ferulic acid,hesperidin and naringin were the main components in the highland barley cortex,and the highest content found in the outer layer of seed coat and aleurone layer,the contents were0.909±0.061,0.362±0.021 and 0.385±0.054 mg/g,respectively.The trends of antioxidant activity of bound phenol and free phenol were consistent with that of their content,and were significantly positively correlated with the content of phenolic substances（p<0.01）.The results of light microscope showed that the highland barley bran with milling rate 0%～3.79%was the HB pericarp layer;the highland barley bran with milling rate 3.80%～8.30%was the HB cortex;the highland barley bran with milling rate 8.31%～18.40%was the positions of HB aleurone layer;the highland barley bran with milling rate 18.41%～20.91%belonged to HB endosperm.Phenolic substances in highland barley cortex mainly existing form,and the effects of decolorization on the composition,structural and functional characteristics of highland barley protein were investigated.Phenolic substances in highland barley cortex were mainly in binding form.In different types of highland barley protein,the content of bound phenols was higher than that of free phenols.The content of bound phenols was the highest in the highland barley bran with milling rate 5.78%～8.30%,among which the content of covalently bound polyphenols in gluten was as high as 3.87±0.23 mg/g.The senior structure change caused by hydrogen peroxide（H₂O₂）oxidation treatment was the reason for the improvement of functional properties of highland barley protein.The molecular weight distribution of highland barley proteins（HBPs,including total highland barley protein,highland barley glutenin and highland barley hordein）was not changed by H₂O₂oxidative decolorization treatment.However,the content of sulfhydryl,disulfide,ionic and hydrogen bonds of HBPs increased after discoloring by H₂O₂.Compared with HBPs,the content ofβ-sheet（reduced from 43.51%,44.56%and 39.32%to 42.63%,42.69%and 37.60%,respectively）andα-helix（from 17.78%,14.24%and 12.33%to 15.60%,12.00%and 11.69%,respectively）of the decolorized highland barley protein（DHBP）,decolorized highland barley gluten（DHBG）and decolorized highland barley hordin（DHBH）decreased,while the content ofβ-turn and irregular curl increased significantly（p<0.05）.This result indicated that the structure of HBPs changed from ordered state to disordered state after decolorization.The change of protein spatial structure and microstructure significantly improved the solubility,water absorption,foaming,emulsifying and water holding capacity of HBPs.The effect of phenolic substances on the structure and functional properties of DHBP.The non covalent interaction between phenolic acids and DHBP significantly increased the surface hydrophobicity and decreased the Zeta-potential value of DHBP（p<0.05）,resulting in aggregation（when the concentration of phenolic acids was>0.1 mg/mg protein,the particle size of the complex was>1000 nm,and the polymer dispersion index of the complex was close to 1.0）.The results of endogenous fluorescence spectra showed that proanthocyanidin（PC）,tea polyphenol,hesperidin（HE）,naringin and three phenolic acids could significantly quench the endogenous fluorescence of DHBP except for salidroside.Phenolic substances combined with DHBP can significantly reduce the content of free sulfhydryl and amino groups in DHBP.The results of infrared spectrum and circular dichroism spectrum showed that eight phenolic substances can change the secondary structure of DHBP.PC,tea polyphenols,ferulic acid and HE made theα-helix content of reduce.In addition,the combination of eight phenolic substances and DHBP significantly improved the DPPH free radical scavenging ability,ABTS free radical scavenging ability and FRAP value of DHBP（p<0.05）.The antioxidant activities of DHBP-PC complexes and DHBP-HE complexes were significantly higher than that of PC and HE（p<0.05）,showing a synergistic effect,especially DHBP-PC complexes.Interaction mechanism between PC and DHBG.Under the conditions of p H 7 and p H9,the interaction between PC and DHBG significantly reduced the surface hydrophobicity（p<0.05）and increased the Zeta-potential value of DHBG.PC had obvious quenching effect on the endogenous fluorescence of DHBG,which was static quenching.At p H 7,hydrogen bond and van der Waals force were the main driving forces for the formation DHBG-PC complexes,and at p H 9,hydrophobic interaction was the main driving force.The combination of PC and DHBG resulted in a significant decrease in the content of free amino and free sulfhydryl groups of DHBG（p<0.05）.The secondary structure results showed that the interaction between PC and DHBG at p H 7 increased significantly the content ofα-helix and irregular curl（p<0.05）reduce theβ-sheet content.Simultaneously,the combination of PC and DHBG at p H 9 significantly reduced the content ofα-helix content（p<0.05）,increasedβ-turn and irregular curl content.In addition,the interaction of PC and DHBG can significantly increase the antioxidant activity of DHBG（DPPH free radical scavenging ability,ABTS free radical scavenging ability and FRAP value）（p<0.05）,and the antioxidant activity of DHBG-PC complexes formed at p H 7 was better than that of DHBG-PC complexes formed at p H 9.Proteomic results showed that there were 4 on cysteine,14 on histidine,47 on lysine and 52 on arginine during the interaction between PC and DHBG.Antimicrobial and antioxidant effects of DHBG-PC complex.DHBG-PC complexes did not affect the cooking characteristics of highland barley fresh wet noodles（p>0.05）.However,the complexes could significantly increase the hardness,viscosity,chewability,adhesiveness,resilience and cohesion of highland barley fresh wet noodles（p<0.05）,of which200 mg/100 g was the most significant.When the addition of DHBG-PC complexes was0～200 mg/100 g,it can significantly improve the water holding capacity of highland barley fresh wet noodles and promote the close combination of protein and starch.The total bacterial count test results showed that DHBG-PC complexes could significantly reduce the total bacterial count of highland barley fresh wet noodles（reduced from 5.81×10⁴to 2.2×10³）（p<0.05）.When the addition amount reached 200 mg/100 g,there was no obvious mold growth on the surface of highland barley fresh wet noodles（the total number of bacterial colonies was 5.12×10³）,and also can effectively prevent the browning of fresh wet highland barley noodles.