Study On The Formation Of Bitterness In The Pericarp Of Green Prickly Ash(Zanthoxylum Schinifolium Sieb.et Zucc) At Different Developmental Stages

Prickly ash(Zanthoxylum bungeanum)is an aromatic plant of the Rutaceae family,native to China and is one of the traditional spices of China,beloved by consumers for its unique numbing taste and rich aroma.However,on the other hand,prickly ash produces a certain bitterness during the process of consumption.With the increasing demand for prickly ash consumption and processing,the scale and scope of prickly ash cultivation in China continue to rise.As a result,problems such as variety confusion,adulteration,outdated technology,and incomplete industrial chain have emerged.These issues have led to uneven quality of prickly ash,with an increasingly prominent phenomenon of bitterness,particularly in the bitter taste of the fruit pericarp of the green prickly ash(Zanthoxylum schinifolium Sieb.et Zucc).Consumers typically have an aversion to bitterness in food,which has gradually become one of the factors hindering the consumption and development of Z.schinifolium.Thus,it has become an urgent task to elucidate the source of bitterness in the Z.schinifolium pericarp and the mechanism underlying the formation of bitter compounds.Considering this,the present study employed “Jiuyeqing” Z.schinifolium from Chongqing Jiangjin,which exhibits particularly prominent bitterness,as the experimental material.Firstly,the composition and flavor characteristics of Z.schinifolium pericarp at different developmental stages were studied.Then,combined with bitterness sensory evaluation and metabolomics technology,the bitterness changes of Z.schinifolium pericarp during growth were monitored,and key bitterness metabolites were screened.Subsequently,highperformance liquid chromatography(HPLC)was used to determine the content changes of key bitterness metabolites in Z.schinifolium pericarp.Finally,through the combination analysis of metabolomics and transcriptomics,the key metabolic pathways and key regulatory genes of the key bitterness metabolites in Z.schinifolium pericarp were constructed and elucidated.The main research results are as follows:1.The components and flavor characteristics of Z.schinifolium pericarp at different developmental stages.The Z.schinifolium pericarp harvested in mid-July presents the most vibrant and bright green appearance,and is rich in alcohol soluble extract,nonvolatile ether extract,total flavonoids(123.38 mg·RE/g)and total phenols(53.66 mg·GAE/g).At the same time,sanshool and volatile oil in the pericarp of Z.schinifolium rapidly accumulate in the mid to late July,and their major contributing components to the numbing and fragrant flavor,hydroxy-α-sanshool and linalool,respectively,reach their peak content of 115.89 mg/g and256.02 mg/m L,endowing the Z.schinifolium pericarp with its most prominent numbing and fragrant flavor quality.Overall,mid-July is the most suitable harvesting period for “Jiuyeqing”Z.schinifolium.However,through bitter taste sensory evaluation,it was also found that the bitterness of the Z.schinifolium pericarp gradually increases during the growth and maturation process,and the bitterness intensity is highest in mid-July,suggesting that bitter substances accumulate in the Z.schinifolium pericarp as their growth metabolism progresses.2.Metabolomics analysis of Z.schinifolium pericarp at different developmental stages and investigation of the dynamic accumulation of bitter metabolites.Based on metabolomics analysis of Z.schinifolium pericarp at different developmental stages,a total of 93 bitter metabolites potentially contributing to the bitterness of Z.schinifolium pericarp were screened.Bitter metabolites that accumulated differently at different developmental stages were mainly enriched in biosynthesis of secondary metabolites,phenylalanine metabolism,phenylpropanoid biosynthesis,flavonoid biosynthesis,flavone and flavonol biosynthesis metabolic pathways.The content and taste activity value(TAV)of 18 key bitter metabolites were determined dynamically.Eleven bitter metabolites(arbutin,epicatechin,quercetin-3-galactoside,isoquercitin,umbelliferone,quercetin-3-arabinoside,quercetin,quercitrin,kaempferol,isorhamnetin,and procyanidin B2)had TAV values greater than 1 at all developmental stages and significantly contributed to the bitterness.Most bitter metabolites,except epicatechin and procyanidin B2,showed low TAV values during the fruit setting stage and high TAV values during the mature stage,positively correlated with the sensory evaluation of bitterness.Quercetin-3-galactoside,isoquercitin,quercitrin,kaempferol,and isorhamnetin had TAV values >10 and reached their maximum values in mid-July,and were the main sources of bitterness in the pericarp of mature Z.schinifolium.3.Study of transcriptome analysis and bitter metabolites biosynthesis pathways in the pericarp of Z.schinifolium at different developmental stages.The differential expression genes(DEGs)in Z.schinifolium pericarp at different developmental stages were mainly enriched in metabolic pathways,biosynthesis of secondary metabolites,phenylpropanoid biosynthesis,flavonoid biosynthesis,plant hormone signal transduction,starch and sucrose metabolism,galactose metabolism,photosynthesis,phenylalanine,tyrosine and tryptophan biosynthesis,and phenylalanine metabolism.Weighted gene co-expression network analysis(WGCNA)revealed that the expression of 13 structural gene families including PAL,C4 H,4CL,COMT,F5 H,F6H,F3 H,F3’5’H,F3’H,DFR,ANS,ANR,and FLS were strongly correlated with the dynamic accumulation of bitter metabolites in Z.schinifolium pericarp,indicating that these genes may potentially regulate bitter metabolites formation.Based on these findings,a key biosynthesis pathway of bitter metabolites in Z.schinifolium pericarp was constructed,which is mainly composed of phenylpropanoid biosynthesis,flavonoid biosynthesis,and flavone and flavonol biosynthesis pathways.4.Study on the correlation between transcription factors and bitterness formation related genes in different developmental stages of Z.schinifolium pericarp.b HLHs and MYBs are the most differentially expressed transcription factors in different developmental stages of Z.schinifolium pericarp,and the differentially expressed b HLHs and MYBs show high coexpression correlation with 29 DEGs(including 8 COMTs,2 ANSs,1 ANR,7 4CLs,6 PALs,1F3 H,1 DFR,1 F3’H,1 F3’5’H,and 1 C4H)with Pearson correlation coefficient > 0.90.These MYBs and b HLHs are key transcription factors that influence the expression of bitterness-related genes in Z.schinifolium pericarp and are closely related to the biosynthesis pathway of Z.schinifolium pericarp bitter metabolites.In conclusion,this study has determined the optimal harvest time for “Jiuyeqing” Z.schinifolium,enriched the metabolic and transcriptomic information of Z.schinifolium pericarp at different devleopmental stages,clarified the formation process of bitter metabolites in Z.schinifolium,with quercetin-3-galactoside,isoquercitin,quercitrin,kaempferol,and isorhamnetin being the main sources of bitterness in mature Z.schinifolium pericarp.Additionally,a key bitter metabolite biosynthesis pathway in Z.schinifolium pericarp was constructed,consisting primarily of the phenylpropanoid biosynthesis,flavonoid biosynthesis,and flavone and flavonol biosynthesis pathways.