Investigation On Quality Evaluation Of Garlic Based On Metabonomics

Garlic is an important condiment and vegetable in daily life of people all over the world.And garlic has become a research hotspot for its enormous nutritional function and medicinal value.China is by far the largest garlic producer not only owing to the planting area but also the yield.However,there are still some problems for the development of garlic in China.For example,the production and management of garlic are not standardized,leading to the varieties confusion during the process of long-term cultivation in various regions.Besides,the garlic products in our country are simple,which are only limited to raw garlic and primary garlic products.Thus,applying scientific methods to evaluate the quality of garlic and garlic products comprehensively and objectively is of great significance in many areas,including the determination of quality characteristics of different garlic cultivars,developing deep processing products,optimizing the industrial structure of garlic in China and promoting the development of garlic industry in our country.Furthermore,it is also imperative to establish new methods for the determination of constitutes in garlic.In this dissertation,garlic and garlic products("Laba" garlic)were selected as the research objects.Firstly,the volatile metabolites and primary metabolites of different varieties of garlic were preliminarily studied by gas chromatography and mass spectrometry(GC/MS)based on method of metabonomics,and the characteristic components of different varieties of garlic were analyzed.Secondly,a method for evaluating the sensory quality of fresh garlic and garlic products was established by integrating quality indicators based on metabonomics method,which offers a new sight for the evaluation of garlic quality.Thirdly,a systemic metabolite profile of garlic product("Laba" garlic)during traditional processing was performed by using GC-MS and high performance liquid chromatography and tandem mass spectrometry(HPLC-MS/MS),which provides a new theoretical basis and experimental data for evaluating the quality of garlic products.In the end,a rapid,simple and environmentally friendly supercritical fluid extraction and supercritical fluid chromatography coupled to mass spectrometry(SFE-SFC-MS/MS)method has been developed for the analysis of phenolic compounds in garlic.The main results of this research were as follows:1.The volatile metabolites and primary metabolites of 25 varieties of garlic were preliminarily studied by GC/MS based on method of metabonomics,and the results showed that the metabonomics method is a powerful tool to study constituents of garlic.The compositions of 25 varieties of garlic were investigated and analyzed in detail,including a total of 47 volatile metabolites and 32 primary metabolites.Among the metabolites,diallyl disulfide,diallyl tetrasulfide,diallyl trisulfide,3-vinyl-1,2-dithiocyclohexene-4-ene,allyl methyl disulfide and propylene were the main volatile metabolites,while the major primary metabolites included L-tyrosine,L-proline,malic acid,isocitric acid,D-fructose,D-galactose and D-glucose.By using hierarchical cluster analysis(HCA)models and partial least squares discriminant analysis(PLS-DA)models,25 varieties of garlic were successfully classified into different groups with identifying characteristic metabolites of garlic in different groups,which supplies a new idea for estimate the quality of different varieties of garlic.2.The quality indicators of 28 varieties of fresh garlic from four main garlic producing areas in China were systematically determined by method of metabonomics.Then,89 quality indicators of fresh garlic were integrated,the obtained data of which were subsequently studied by PLS-DA,variance analysis,analysis of variation coefficient,principal component analysis and correlation analysis.Finally,the results showed 4 core quality indicators of garlic,including a*value,L-alanine,L-proline and gluconic acid.A BP neural network model for predicting garlic quality was also established based on these 4 core quality indicators,and regression analysis was made between sensory score and BP neural network model prediction score with R2=0.9603,indicating that this BP neural network model can evaluate the quality of fresh garlic reasonably.3.A systemic metabolite profile of "Laba" garlic during traditional processing was investigated by using GC-MS and HPLC-MS/MS with multivariate analysis,companying with the investigation of the antioxidant activity changes of "Laba"garlic.The results showed that the colour of garlic samples during traditional processing changed from white to green and then to yellow,and the strongest green colour of garlic sample was observed at day 15.In addition,a total of 20 volatile metabolites,16 primary metabolites and 15 secondary metabolites were identified as notable changed compounds along with the traditional processing(P<0.05).The characteristic flavor of "Laba" garlic was formed mainly by the decreased content of organosulfur compounds and the increased content of non-organosulfur compounds.In addition,this study also proposed the metabolic pathway of primary metabolites and phenolic compounds in garlic samples during processing.Most primary metabolites including lactic acid,isocitric acid,L-leucine,L-proline,D-fructose,D-glucose and erythritol increased from day 3 to day 12,which were thought to be foundation for the sweet and sour taste of "Laba" garlic.The concentration of the 15 phenolic compounds from day 3 to day 12 was increasing,and reached the maximum level at day 12,indicating the potential nutritional value of "Laba" garlic.Multiple metabolites of Laba garlic changed significantly at day 12 during processing.Hence,it can be concluded that day 12 is the important processing time of "Laba" garlic.In addition,notable positive associations between total organosulfur compounds,total organic acids,total amino acids and antioxidant activities were identified,and the 15 secondary metabolites contributed little to the antioxidant activity in "Laba" garlic.4.A total of 28 varieties of fresh garlic from four main garlic producing areas in China were processed into "Laba" garlic.Then,quality indicators of these garlics were detected using metabonomics method.And 106 quality indicators of "Laba" garlic were integrated and subsequently investigated by PLS-DA,variance analysis,analysis of variation coefficient,principal component analysis and correlation analysis.At last,5 core quality indicators of"Laba" garlic were choosed,including a*value,b*value,ferulic acid,vanillic acid and L-tyrosine.A BP neural network model for predicting garlic quality was also established based on these 5 core quality indicators,and regression analysis was made between sensory score and BP neural network model prediction score with R2=0.9641,suggesting that this BP neural network model can assess the quality of "Laba" garlic reasonably.5.A rapid,simple and environmentally friendly supercritical fluid extraction and supercritical fluid chromatography coupled to mass spectrometry(SFE-SFC-MS/MS)method has been developed for the analysis of phenolic compounds in garlic.The effects of different chromatographic columns,modifiers,column temperature,backpressure and flow rate on supercritical fluid chromatography were investigated,while the best conditions of SFC-MS/MS was as follows:Shim-pack UC-X Diol column,methanol as modifier,0.1 mM oxalic acid and 1 mM ammonium formate as mobile phase additives,column temperature was 40?,back pressure was 10 MPa,and flow rate was 2 mL/min.In this condition,15 phenolic compounds in the garlic showed good separation performance in less than 12 min.Moreover,through the optimization of the SFE parameters using response surface methodology(RSM),15 phenolic compounds were successfully extracted at 50? in 9 min with the addition of 30%methanol.Furthermore,only 9 phenolic compounds(including ferulic acid,p-coumaric acid,naringenin,apigenin,protocatechuic acid,isorhamnetin,luteolin,phthalic acid,quercetin)showed acceptable selectivity,linearity,sensitivity,recovery and precision by the validation data.And the above constructed method achieved its practical application through the analysis of phenolic compounds in garlic from different origins in China.