Antioxidant Activity Of Peptides From Sesame Flavor-type Baijiu And Their Interactions With Aroma Compounds

Baijiu is a traditional sprit drink in China and its chemical composition is very complex.There are numberous volatile and nonvolatile compounds reported in Baijiu besides alcohols and water.The volatiles are foundations of the unique aroma characteristics of Baijiu,whereas the nonvolatiles influence the whole aroma profiles in Baijiu and they are gerenally bioactive ingredents.Clarifying the nonvolatile compounds and their concentrations is a prerequisite to regulate the whole aroma of Baijiu and determine the relationships with human helath.It is of great significants for the improvement of quality and international competitiveness of Baijiu.However,previous researches on the nonvolatiles in Baijiu were not systematic and in-depth.Therefore,the thesis was concerned with the peptides in Sesame flavor-type Baijiu,which was a new flavor-type Baijiu from the early 1990s.Optimized research approaches were employed to detect the peptides qualitatively and quantitatively.The antioxidant activities of the peptides were evaluated by the chemical and cell models,and the mechanisms underlying the intracellular protective effects were further studied.Meanwhile,the interactions between the peptides and the volatile aroma compunds in Baijiu and the mechanisms were explored.The main research contents and results are as follows:?1?Methods based on direct concentration,gel column chromatography and liquid-liquid extraction concentration coupled with high-performance liquid chromatography and quadrupole-time-of-flight-mass spectrometry?HPLC-Q-TOF-MS?were developed for the analysis of peptides in Guojing Sesame flavor-type Baijiu.Compared with the qualitative results of three methods,liquid-liquid extraction concentration was the best way for peptides??1000 Da?extraction because of the high recovery?82.2¹20.2%?.The gel column chromatography method was more suitable for the nonvolatile with molecular weight less than 200 Da.The direct concentration was less efficien due to the extremely low contens of peptides in Baijiu.?2?Selecting Jingzhi and Guojing Baijiu samples as the subjects,peptides in the two kinds of Baijiu were explored by liquid-liquid extraction concentration coupled with HPLC-Q-TOF-MS.Results showed that four peptides were isolated and identified from the samples,and the sequence of the peptides were respectively Ala-Lys-Arg-Ala?AKRA?,Asp-Arg-Ala-Arg?DRAR?,Pro-His-Pro?PHP?and Pro-Pro-Asp-Gly?PPDG?.The peptides were quantified by HPLC-Q-TOF-MS using an internal standard method,and their contents are range from 8.497 to 66.524?g/L.?3?The antioxidant activities of the 4 synthetic peptides were evaluated by in vitro chemical experiments,including DPPH,ABTS,ORAC,reducing power and Fe²⁺chelating ability assays.Results showed all peptides displayed very weak radicals scavenging abilities with Trolox equivalent?TE?values ranging from 0.006⁰.04?mol TE/?mol,and none of them exhibited Fe²⁺chelating ability.However,DRAR and AKRA showed very strong reducing power with TE values 12.58 and 12.91?mol TE/?mol,respectively,followed by PPDG?0.31?mol TE/?mol?and PHP?0.22?mol TE/?mol?.Effects of amino acids on the reducing power of the peptides were further discussed,and results showed that the sequence was Lys>Ala>Arg>Asp=Gly>Pro with TE values 16.98,13.51,12.38,11.58,11.58 and6.45?mol TE/?mol,respectively.These findings indicated that the presence of such antioxidant amino acid residues in peptides was the determining factor for their antioxidant activities.?4?Protective effects of AKRA,DRAR,PHP and PPDG on 2,2'-azobis?2-methylpropanimidamidine?dihydrochloride?AAPH?-induced oxidative stress in HepG2cells were investigated.The cellular antioxidant activity assay results showed that all the peptides protected AAPH-inducedoxidative stress inHepG2 cells in a concentration-dependent manner.Pretreatment of the cells for 2 h with peptides in a certain concentration ranges caused strong intracellular reactive oxygen species?ROS?scavenging activities and prevented a decrease in reduced glutathione?GSH?and increases in oxidized glutathione?GSSG?and malondialdehyde?MDA?.In addition,peptides treatment prevented significant decreases in glutathione peroxidase?GSH-Px?,superoxide dismutase?SOD?,and catalase?CAT?induced by AAPH.Thus,peptides treatment ameliorated AAPH-induced oxidative stress in HepG2 cells.?5?The involved molecular mechanisms of the protective effects of the peptides in AAPH-induced oxidative stress were further explored.Results showed that pretreatment with peptides in a certain concentration range stimulated the mRNA and protein expression levels of antioxidant enzymes and nuclear factor E2 related factor 2?Nrf2?.Meanwhile,AKRA,DRAR,PHP and PPDG markedly reduced the level of Kelch-like ECH-associated protein 1?Keap1?,suggesting that the peptides effectively activated Nrf2/antioxidant response element?ARE?-mediated activity to up-regulated the activities of cellular antioxidant enzymes to eliminate free-radicals in AAPH-indecued oxidative stress in HepG2 cells.?6?Headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry and gas chromatography-olfactometry?HS-SPME-GC-MS/GC-MS-O?was used to evaluate the influnces of different concentrations of peptides ranged from 10²000?g/L on the whole aroma profiles of Guojing Baijiu.Results showed the peptides selectively suppressed the volatility of aroma compounds in a concentration-dependent manner,especially to esters,alcohols,acids,aldehydes and phenols.Selected AKRA as the object to study the interactions with p-Cresol,which was an off-odor in Baijiu,by ultraviolet absorption spectroscopy and nuclear magnetic resonance spectrum.Thermodynamic analysis indicated hydrogen bonds played a major role in stabilizing AKRA-p-Cresol complexes.Furthermore,the mixture of AKRA and p-Cresol revealed intermolecular cross-peaks between the carboxyl hydrogen of AKRA and the phenolic hydroxyl of p-Cresol,by using nuclear overhauser effect spectroscopy?¹H-¹H NOESY?.