Determination Of Seed Vigor And Analysis Of Phenolic Compounds In Soybean With Different Aging Times

Soybean(Glycine max(L.)Merr)is a kind of legume rich in oil and protein,is one of the most important food crops in the world.However,soybean seeds are short-lived seeds,with the increase of storage time,soybean seeds have deteriorated,seed vigor decreased,germination rate and germination potential decreased,resulting in the use value and species value reduction.Therefore,it has great practical significance to find an effective method for the determination of soybean seed vigor.In this study,we used extractive electrospray ionization mass spectrometry(EESI-MS)to obtain chemical fingerprints directly from the water extracts of soybean seeds with different vigor.The raw mass spectra data was analyzed using multivariate analysis,including principal component analysis(PCA),cluster analysis(CA)and discriminant analysis(DA)to establish a method to identify different soybean seed vigor.At the same time,the dynamic changes of phenolic compounds in soybean seeds were analyzed by high performance liquid chromatography(HPLC)and electrospray ionization mass spectrometry(EESI-MS).The main contents and results of research are as follows:1.In this study,we used artificial accelerated aging method to obtain soybean seeds with different vigor,which were measured by standard germination test,with unaged seeds as controls,to obtain the changes of germination index and seed vigor of soybean seeds during aging.The results showed that there was a significant difference between the vigor indexes of the soybean seeds with different aging treatments(P < 0.01).For different aging times,the seed germination index and vigor index showed a downward trend,and for the seeds with a certain aging time,the germination index and vigor index were dropped to 0,indicating that the seed with the aging of the processing time,seed vigor decreased until completely lost.The correlation analysis of seed vigor index and aging time showed that vigor index and aging time were significant negative correlation.2.In order to distinguish the soybean seeds with different vigor quickly,this paper used EESI-MS to obtain chemical fingerprints directly from the water extracts of soybean seeds with different vigor.The cumulative contribution rate of the first 3principal components of PCA was 81.50%.The correct rates of CA and DA were100%.In DA,the cross validation analysis success rate was 90%.Gammaaminobutyric acid,glutamine and acetyl genistin were identificated by mass spectrometric.The experimental results showed that the contents of Gamma aminobutyric acid increased and the contents of glutamine and acetyl genistin decreased with increasing of soybean seed aging time.EESI-MS combined with multivariate analysis can distinguish soybean seeds with different vigor effectively.3.We used HPLC to carry out qualitative and quantitative analysis of 19 kinds of phenolic compounds in soybean seeds during aging treatments.The results showed that the contents of 19 kinds of phenolic compounds in soybean seeds had obvious change during aging process.In the soybean seed aging process,protocatechuic acid,rutin and Morin decreased with the increase of aging time decreased respectively from 183.60 μg/g,14.03 μg/g and 55.03 μg/g to 138.10 μg/g,12.37 μg/g and 38.60μg/g,the contents of daidzein,glycitein and genistein and baicalin emodin increased with the increase of aging time increased,from 12.57 μg/g,trace(Trace,Tr),9.10μg/g and 0.83 μg/g increased to 91.97 μg/g,6.60 μg/g,76.10 μg/g and 3.53 μg/g,respectively.The contents of caffeic acid,epicatechin ferulic acid,daidzin,genistin and resveratrol were first increased and then decreased along with the increase of aging time,ferulic acid content reached the maximum at 2 d after aging,and the remaining 5 phenolic compounds reached the maximum at 4 d.The contents of chlorogenic acid,myricetin,cinnamic acid,kaempferol,p-nitrobenzoic acid and biochanin A showed irregular changes with the increase of aging time.The variation tendency of contents of chlorogenic acid,myricetin and kaempferol was same,and the change trends of cinnamic acid,p-nitrobenzoic acid and biochanin A have the same trend.The total content of the 19 phenolic compounds increased firstly and then decreased with the aging treatment time,and the maximum value was 6830.88 μg/g at 4 d.4.EESI-MS was used to carry out the direct mass spectrometric analysis of the ethanol extracts of soybean seeds at different aging time,the fingerprint patterns were obtained in positive ion mode,and qualitative and quantitative analysis of the four typical isoflavones were carried out,and the contents of daidzein,genistein,daidzin and genistin were significantly different at different aging treatments.The contents of daidzein and genistein increased with aging time,and the contents of daidzin and genistin increased firstly and then decreased with the aging treatment time,and the contents reached maximum at 4 d.At the same time,the fingerprint of soybean seed alcohol extract with different aging time was analyzed by PCA,the results showedthat the differences of soybean seed alcohol extract materials with different vigors were obviously,soybean seed with different vigors can distinguish in the two-dimensional space of PCA.In this study,we used EESI-MS and HPLC to rapid identifysoybean seeds with different vigor,and systematically studied the dynamic changes of phenolics in the aging process of soybean seeds,and established a new method for the rapid,sensitive and effective soybean seed vigor detection.The study provides a theoretical basis for the seed storage and the research and utilization of germplasm resources.