Characterization Of Featured Trace Components In Feng-flavor Baijiu And Their Effects On Alcohol-induced Liver Injury

Baijiu is a traditional Chinese solid-state fermented alcoholic beverage,which has a brewing history of thousands of years in China and witnesses the development of Chinese history and culture.In addition,as a hobby drinking,Baijiu is popular among many consumers and bears important economic value.The existing researches show that Baijiu contains about 98%of water and ethanol,and 2%of trace components,and there are many kinds of trace components in Baijiu,of which more than 2,000 have been reported so far.At the same time,the studies also found that about 2%trace components in Baijiu not only determine the style and characteristics of Baijiu,but also contain many bio-active substances that are beneficial to human health.However,as far as the research objects are concerned,most of the existing researches focus on basic aroma types(such as strong-flavor,sauce-flavor,etc.),or distinctive minority aroma types(such as sesame-flavor,etc.),and few reports are made on Feng-flavor Baijiu of Shaanxi province.As far as the research contents are concerned,the existing researches mainly focus on the characterization of key flavor substances in volatile trace components of Baijiu,lacking of systematic research on overall trace components of Baijiu.Furthermore,the researches on bio-active substances in Baijiu are carried out by using the bio-active substance standard for in vitro or in vivo activity research,while the efficacy of all trace components in the whole Baijiu under the synergistic effect is still not clear.Therefore,in this research,Feng-flavor Baijiu was taken as the main research object,and was compared with Western distilled spirits(Whiskey,Vodka,Rum,Gin,Brandy)and China’s four basic flavor Baijiu(Fenjiu(FJ),Wuliangye(WLY),Maotai(MT),Guilinsanhua(GLSH)).Different pretreatment methods combined with Gas Chromatography-Mass Spectrometry(GC-MS)and Ultra-high Performance Liquid Chromatography-High Resolution Mass Spectrometry(UPLC-HRMS)were used to comprehensively detect the trace components in spirits samples.Afterwards multivariate statistical analysis was employed to find the differences of trace components between Feng-flavor Baijiu and other spirits samples,screening out the featured trace components of Feng-flavor Baijiu.In addition,the synergistic effects of all the trace components in Feng-flavor QZXF whole wine on alcohol liver injury were explored through cell experiment and animal model.The specific research contents include the following four parts:(1)Characteristic trace components in Feng-flavor Baijiu were characterized based on UHPLC-Q-Orbitrap-HRMS.Direct injection method combined with UHPLC-Q-Orbitrap-HRMS was used to detect the trace components of 10 spirit samples,and then multivariate statistical analysis combined with peak area ratio analysis were carried out to screen out the characteristic trace components of Feng-flavor Baijiu.The detection results showed that 946 trace components were identified in Feng-flavor QZXF Baijiu.The results of multivariate statistical analysis showed that Feng-flavor Baijiu group had a high degree of separation with other distilled spirit samples,and the aggregation degree within groups was good,which indicated that Feng-flavor Baijiu had potentially different trace components compared with other spirit samples.Further data analysis showed that the contents of betaine,choline,allopurinol,adenosine,adenine and D-(+)-proline in Feng-flavor Baijiu were significantly higher than other spirit samples,which were define as the characteristic trace components of Feng-flavor Baijiu based on UHPLC-Q-Orbitrap-HRMS.Finally,six characteristic trace components in Feng-flavor QZXF Baijiu were quantified as follows:allopurinol of 591.341μg/L,choline of 269.296 μg/L,D-(+)-proline of 103.726 μg/L,betaine of 78.026μg/L,and adenosine of 20 μg/L.(2)Characteristic trace components in Feng-flavor Baijiu were characterized based on GC-MS.Firstly,ortex-assisted liquid-liquid microextraction(VA-LLME)combined with GC-MS was used to detect volatile trace components in 10 distilled spirit samples,and MSTFA derivatization combined with GC-MS was used to detect non-volatile trace components in 10 distilled spirit samples.Then,multivariate statistical analysis combined with peak area ratio analysis were carried out to screen out the volatile and non-volatile characteristic trace components in Feng-flavor Baijiu.The qualitative results showed that there were 118 volatile trace components and 58 non-volatile trace components in Feng-flavor QZXF Baijiu.The multivariate statistical analysis results of volatile and non-volatile trace components showed that Feng-flavor QZXF Baijiu gathered well in the group and could be clearly separated from other distilled spirit samples,which indicated that there were potentially different trace components in Feng-flavor Baijiu.Further data analysis showed that there were 5 volatile characteristic trace components in Feng-flavor QZXF Baijiu,which were ethanone 1-(2-furanyl)-,acetoin,1-propanol,2-acetyl-5-methylfuran,and benzene 1,3-bis(1,1-dimethylethyl).There were 5 non-volatile trace components,including 2 non-volatile organic acids(D-malic acid,Glycolic acid)and 3 sugars(D-Fructose,D-Glucose,D-lyxose).(3)Cell experiment was carried out to study the effects of whole trace components in Feng-flavor Baijiu on alcoholic liver injury.Firstly,the alcohol-induced injury model of human normal liver cells LO2 was established by screening the optimal alcohol concentration,and the repair model of LO2 cells was established by adding different concentrations of betaine.The intervention effect of betaine on the injury of LO2 cells induced by alcohol was evaluated by detecting the contents of intracellular reactive oxygen species(ROS),malondialdehyde(MDA),reduced glutathione(GSH),alanine aminotransferase(ALT)and aspartate aminotransferase(AST).The results showed that the optimized alcohol concentration was 3%,which was applied to set up mold group.Furthermore,after the intervention of betaine with different concentrations,the intracellular content of ROS,ALT,AST and MDA decreased significantly(p<0.05),while the GSH content increased significantly(p<0.05),and such effects were dose-dependent.All these results indicated that betaine could alleviate the injury of LO2 cells induced by alcohol.Secondly,considering the "dose-effect relationship" of betaine and the "synergistic effect" among various trace components in QZXF,the whole Feng-flavor QZXF Baijiu was employed as the research object,and the industrial alcohol and edible alcohol were used as the control to study the differences of damage effects on cells LO2.MTT,ALT and AST results all showed that the damage degree of liver cell LO2 in QZXF Baijiu group was significantly lower than that in industrial alcohol and edible alcohol group,which indicated that there might be synergistic effects between different trace components such as betaine in QZXF Baijiu,resulting in a certain degree of protective effect on cell injury induced by alcohol.(4)The effect of Feng-flavor Baijiu whole wine intervention on liver injury was studied by animal experiment.Kunming mice were fed with low(0.93 mL/kg/d),middle(2.80 mL/kg/d)and high(4.66 mL/kg/d)doses of edible alcohol and Feng-flavor QZXF Baijiu for 12 weeks.The liver injury differences of edible alcohol and QZXF Baijiu on mice were compared by comparing the phenotypic indexes,biochemical indexes,and pathological sections of liver of mice.In addition,the structure of intestinal flora of mice was analyzed based on 16S rRNA high-throughput sequencing to explore the mechanism of QZXF whole wine affecting liver in mice.The results of phenotypic indexes,biochemical indexes and pathological sections of liver in mice all showed that Feng-flavor QZXF whole wine and alcohol both caused different degrees of liver damage in mice,but compared with the same dose of alcohol,QZXF whole wine had a lower degree of liver damage in mice,which indicated that whole trace components in QZXF could jointly antagonize the liver damage caused by alcohol.The analysis results of serum ethanol concentration and intestinal microflora structure in mice showed that the antagonistic effect of trace components in Feng-flavor QZXF Baijiu on alcoholic liver injury was not achieved by enhancing the ability of liver to decompose and metabolize ethanol,but possibly by regulating the intestinal microflora structure in mice.The results of phylum and genus level analysis of intestinal flora in mice showed that long-term alcohol intake could significantly increase the relative abundance of Bacteroidetes in the intestinal flora of mice,and significantly decrease the relative abundance of Firmicutes,Proteobacteria and Actinobacteria,but there was no significant difference in the structure of intestinal flora between QZXF group and alcohol group at phylum level.The difference is mainly reflected in the genus level of Polyangia,P-unidentified-Bacteria;C-Bacteroidia,etc.In this study,the characteristic trace components in Feng-flavor Baijiu were characterized and the influence of the trace components on alcohol-induced liver injury was explored.The research,on the one hand,can fill the research gap of Feng-flavor Baijiu to some degree,and on the other hand,can let more people know and understand Feng-flavor Baijiu and its health effects,which would provide scientific guidance on healthy drinking for people,lay a foundation for the quality control and standardized production of Feng-flavor Baijiu,and promote the high-quality development of Feng-flavor Baijiu.