Separation And Enrichment Of Cysteine-containing Peptides From Wheat Gluten And Their Binding Interaction With Pb²⁺

Wheat gluten is one of the products of wheat deep processing and contains 70%?75%protein.However,its high value application is less at present.Recently,with more attention to bioactive peptide products,wheat peptide products has gradually been interested by wheat deep processing enterprises and consumers.Wheat gluten is abundant in cysteine,and it is reported that cysteine-containing peptides have many bioactivities such as antioxidant activity,heavy metal-chelating property,and maintaining sulfhydryl group balance in vivo.In this paper,wheat gluten was selected as the raw material to produce cysteine-containing peptides.First,the effect of different hydrolysis on the cysteine content and recovery ratio of wheat gluten hydrolysates was investigated,then the method for separating and enriching cysteine-containing peptides was explored,finally the interaction of cysteine-containing peptides with Pb²⁺was studied,thus to provide a new direction for the development and utilization of wheat gluten hydrolysates.The main content and results are as follows:Firstly,the effect of hydrolysis on the content and recovery ratio of cysteine in wheat gluten hydrolysate was investigated.For acidic hydrolysis,it was found that with the increase of acid concentration and temperature,the contents of sulfhydryl and protein in the hydrolyzed supernatant increased gradually.After being treated with 4 mol/L hydrochloric acid at 85?for8 h,the recovery of protein in the supernatant reached 83.62±2.10%,and the content of total sulfhydryl reached 200.00±12.30?mol/g.Compared with acid hydrolysis,after enzymolysis by Alcalase,the protein recovery,total sulfhydryl content and total SH recovery of the hydrolysate were up to 84.96±4.14%,207.07±11.52?mol/g and 87.96±4.07%,respectively.On the contrary,pepsin had the worst enzymatic hydrolytic efficiency on wheat gluten,with protein recovery of only 35.56±2.35%and the total SH content of 179.75±8.12?mol/g.We compared the effects of hydrolysis before and after reduction on the preparation of cysteine-containing peptides.And the optimal conditions for preparing cysteine-containing peptides were as follows:wheat gluten were reduced and then hydrolyzed by pepsin and acidic protease.Under these conditions,the content of sulfhydryl in the supernatant was 260.45±10.34?mol/g protein,and the recovery rate of sulfhydryl was 80.45±4.43%.Secondly,cysteine-containing peptides were separated from wheat gluten hydrolysate.After the enrichment by Thiolpropyl-Sepharose 6B we obtained a wheat peptide that the content of SH was 1294.45±12.45?mol/g protein.At the same time,it was found that the gel had a higher adsorption capacity for cysteine-containing peptides with large molecular weight and strong hydrophobicity.Then the enriched peptides were further characterized with SEC-HPLC after mBBr labeling.It was found that cysteine-containing peptides had higher fluorescence/UV value and higher total SH content in low molecular weight fraction.It means the low molecular weight fraction were enriched more.We compared gel filtration chromatography,ultrafiltration and ion exchange chromatography to prepare cysteine-containing peptides.The results showed that Sephadex G-15 was chosen to be used for the further separation with SH content of484.05±16.26?mol/g and SH recovery of 67.83±1.86%.Finally,the properties of cysteine-containing peptides and their ability to chelate heavy metals were determined.Stability tests showed that the sulfhydryl group had good stability under the conditions of acidic(pH 3.0)and low temperature(4?)environment.We investigated the influence of the ratio of HCPs to Pb²⁺on the binding interaction of the two.The results showed that when the sulfhydryl group was constant,the amount of chelating metal increased and the chelating rate decreased with the increase of Pb²⁺content.When the ratio of SH/Pb²⁺was 1:1,the binding rate was more than 80%.We used pH potentiometric titration to investigate the interaction of HCPs and Pb²⁺.When Pb²⁺was added,the pH titration curve of HCPs shifted to the right,indicating that sulfhydryl group played a major role in the formation of HCPs and Pb²⁺.In addition,it showed that the two format of HCPs-Pb²⁺chelates(ML₁and ML₂)with the stability constant of 10^11.89 and 10^16.57were formed.HCPs had a better ability to chelating with Pb²⁺.The electrochemical experiments confirmed the existence of the HCPs-Pb²⁺chelates.Full-wavelength scanning experiments showed the strengthening of the absorption peak at 275 nm due to the formation of the HCPs-Pb²⁺chelates.In vitro simulated digestion experiments showed that even with the occurrence of high Pb²⁺concentration(HCPs:Pb²⁺=1:1),HCPs could still bind to Pb²⁺in the simulated intestinal environment at a high binding rate of 79.59%,thus to exert its detoxification effect.