Identification Of Key Bitter Compounds In The Formation Of Bitter Taste Of Red Huajiao(Zahthoxylum Simulans Hance) Peel

Zanthoxylum bungeanum,belonging to Rutaceae family and Zanthoxylum L.genus.The fruits of Z.bungeanum are one of the traditional “eight major condiments”,which is an important ingredient in many kinds of traditional Chinese herbal medicine.Z.bungeanum is widely distributed and planted in most provinces of China.Recently,with the adjustment of the agricultural industrial structure and the implementation of the policy of returning farmland to forests,Z.bungeanum has been planted in large areas as an economically tree species,and its planting area increases by20%-30%annually.However,some varieties or origins of Z.bungeanum have a bitter taste which will have negative effects on the overall taste of food when cooking,resulting in a large adverse reaction in the hot pot industry in China.Food choices are mainly driven by acceptability,bitter taste attributes associated with foods has been recognized as one of the most influential factors limiting consumption,especially in hot pot industry.Undoubtedly,the bitter compounds have greatly restricted the development of Z.bungeanum industry.Nevertheless,the current researches on Z.bungeanum are mainly focused on its volatile oils,alkylamide compounds,and their biological activities,while the studies on bitter compounds in Z.bungeanum is very few.Thus,the wild Zanthoxylum simulans Hance with intense bitter taste found by the research team in Hanyuan County,Sichuan Province,China,was used as the test material,and the Z.bungeanum with weak bitterness obtained from the same place was used as the control.Firstly,the non-targeted metabolism technology was used to screen out the differential metabolites between Z.simulans and Z.bungeanum,and the possible bitter compounds were screened by comparing to the public bitter compounds database and references related to bitter compounds.Then,Based on sensory-guided fractionation techniques and subsequent identification by UPLC-MS/MS analysis,the primary bitter compounds were identified.The results of this experiment can provide some theoretical reference for the quantitative analysis and debittering of the bitter compounds in Z.simulans.The main findings are as follows:1.The potential bitter metabolites in Z.simulans were analyzed and identified based on non-targeted metabolomics technology.Combined with taste profile analysis(TPA)with a scale from 0 to 5,the suitable solvent for extracting bitter compounds in Z.simulans was determined,and then the differential metabolites between Z.simulans and Z.bungeanum were analyzed based on ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Finally,the possible bitter compounds in differential metabolites were screened through the comparison of the public bitter compounds database and relevent references.The results showed that the bitter compounds in Z.simulans were mostly soluble in ethanol and water,and the75% aqueous ethanol could fully extract the bitter compounds in Z.simulans.Through principal component analysis and orthogonal partial least squares discriminant analysis,there were 2457 and 1825 different metabolites in positive ion mode and negative ion mode between Z.simulans and Z.bungeanum,respectively.According to the comparison of the public bitter compounds database and relevant references,a total of 50 bitter compounds were screened in the two ion modes,including 5 L-amino acids,12 alkaloids,11 flavonoids,2 phenols,2 coumarin(and its derivatives),3(terpenoids)terpenes,4 Glycosides and 11 other metabolites.Compared with Z.bungeanum,36 bitter compounds were up-regulated in Z.simulans,and many of which with high intensity of bitter taste were significantly up-regulated.The remaining 14 bitter compounds were down-regulated in Z.simulans,but the fold changes of them were small.2.The key bitter compounds in Z.simulans were isolated and identified.After extracting the bitter compounds in Z.simulans with 75% aqueous ethanol,the compounds with intense bitter taste under the guidance of taste dilution analysis(TDA)were purified and identified by preparative HPLC and UPLC-MS/MS.The results showed that a total of 148 components were isolated from Z.simulans,16 of which had bitter taste.In this paper,only 10 components with higher bitterness intensity were further analyzed.15 key bitter compounds were finally identified,including phenylalanine,valine,isoleucine,sinapic acid,salicylic acid,arbutin,quercetin,quercetin,isoquercetin,quercetin-3-O-α-L-arabinoside,isorhamnetin,(2E,4E,9E,11E)-N-(2-hydroxy-2-methypropyl)-8-hydroxy-13-oxo-2,4,9,-11-Tetradecatetraenamide(Zanthoamide A),magnoliaine,berberine,adenosine.In summary,there were many types of bitter compounds in Z.simulans,including amino acids,alkaloids,flavones,polyphenols,coumarin(and its derivatives),glycosides and terpenes and so on.In this paper,15 key bitter compounds including 3 amino acids,3 alkaloids,5 flavonoids,2 phenolic acids,1hydroquinone glycoside and 1 nucleoside,were identified.