Study On Formation,Structure Of Green Pigment And Mechanism Of Garlic Discoloration

Garlic(Allium sativum L.)is processed for various forms such as powder,puree and granules as a flavouring agent because of its unique flavor and taste.Garlic discoloration is a major concern in garlic processing,which limits commercial utilization,and decrease the economic value of garlic.Simultaneously,garlic greening is necessary for the procedure of‘Laba'garlic.Therefore,understanding the mechanism of garlic discoloration and the characteristic of green pigments is beneficial to control the quality of garlic during processing and improve the quality of‘Laba'garlic.In this thesis,the mechanism of garlic greening,characteristics of green pigments and mechanism of green pigment degradation were investigated.A greening model containing alliin,isoalliin,alliinase,and the amino acid was used to explore the effect of amino acid R groups in the formation of pigment precursor in garlic greening using UPLC-ESI-Q-TOF-MS analysis and density functional theory.The effects of amino acids on the formation and degradation of green pigments were studied using a model reaction containing thiosulfinate and amino acid by ATR-FTIR combined with chemometric methods.The structural changes of green pigment formed in garlic discoloration and the mechanism of green pigment degradation in different p H environments were also investigated.Multiple analysis methods were used in the degradation of pigment,including ATR-FTIR and FT-NIR.The main results are as follows:(1)Based on the accurate molecular weight and fragmentation ions provided by UPLC-ESI-Q-TOF-MS/MS analysis,the main compounds in greening models were identified.It was found that in the reaction model involving arginine or lysine,only one corresponding N-(3,4-dimethyl)pyrrole compound was produced,namely 2-(3,4-dimethyl-1H-pyrrol-1-yl)-5-guanidinopentanoic acid(PP-252)and 6-amino-2-(3,4-dimethyl-1H-pyrrol-1-yl)hexanoic acid(PP-224),respectively.Combined with the DFT results,it is shown that N-(3,4-dimethyl)pyrrole compounds are generated by the reaction of the?-NH₂ with 3,4-dimethylbutanediamine S-oxide.The primary amino group in the R groups of arginine and lysine did not participate in the reaction.The pigment precursor substances 2-(3,4-dimethyl-1H-pyrrolyl)propanoic acid(PP-167),2-(3,4-dimethyl-1H-pyrrol-1-yl)-3-(4-hydroxyphenyl)propanoic acid(PP-259),2-(3,4-dimethyl-1H-pyrrol-1-yl)acetic acid(PP-153)and 2-(3,4-dimethyl-1H-pyrrol-1-yl)pentanedioic acid(PP-225)were observed in the reaction model containing alanine,tyrosine,glycine and glutamate,respectively.When the amino acid R group has a hydroxyl group or a carboxyl group,the closer it is to the?-carbon,the more unstable the N-(3,4-dimethyl)pyrrole compound is produced.At the same time,there was a pigment-precursor-like substance 2-(3,4-dimethyl-1H-pyrrol-1-yl)acrylic acid founded in each reaction model.In the garlic greening model,for blue compounds with a?_max value between575 and 590 nm,after 24 h,the amount of pigment and the absorbance increased significantly compared with 4 h.The DFT results show that during the formation of blue compounds,the most active site of pigment precursor substances PP-167,PP-225,PP-252 and PP-224 is the C1site,the most active site in PP-153 and PP-259 is the C4 site.(2)The FTIR spectra of pigments produced in greening models containing amino acid and thiosulfinates are with some differences.There is a strong absorption peak at 1721 cm^-1observed in FTIR spectra of pigments formed in model containg Glu,Val,Ile,GABA and Leu respectively.The FTIR spectra of pigments produced by Arg,Asn and Gln models showed a unique absorption peak appears at 1675 cm^-1.Only in the FTIR spectrum of the pigment produced by Tyr,the absorption peak at 1515 cm^-1 appeared.There is a characteristic peak at1494 cm^-1 in the FTIR spectra of pigments produced by His or Phe.The Savitzky-Golay first derivative spectral range of 1750 to 900 cm^-1 for FTIR was chosen for PCA to distinguish different pigment types.It was demonstrated that PCA was capable of separating the pigments into several groups based on their IR spectral data.Furthermore,the FTIR spectrum of green pigments extracted from garlic mud was predicated in the PCA model.Changes in the absorptions at 1068 and 1024 cm^-1(O=C–O–H and C–N bonds vibrations)were observed during garlic greening at different reaction times.(3)The structural changes of green pigment formed in garlic discoloration were investigated in this study.Multiple analysis methods were used in the degradation of pigment,including UV-vis spectrophotometry,ATR-FTIR and FT-NIR.From the UV-vis spectra,it can be obtained that an acidic environment(p H 5.0)suppressed the decomposition of the blue pigments compared with p H 6.5.Alkaline condition accelerated decomposition of the blue pigment.It was found that the degradation degree of green pigments in a solution with p H 5.0 was the lowest.ATR-FTIR and FT-NIR spectroscopy revealed the spectral features changes of green pigment during the degradation process.Changes in the absorptions of C=O,C=O-O-,C=N,C-N,O=COCH,=C-O-C-,COOH and N–H bonds vibrations are attributed to the decomposition of the pigments.The absorption at 5170 cm^-1(N–H bond first overtone)and4871 cm^-1(O=C–H stretching)correlated to pigment degradation were confirmed by FT-NIR spectra.The pigments undergo decomposition in water,which leads to the breakage of C–N in the pyrrole ring and O-H in O=C–OH bonds.Together with the decomposition reaction,C=N,N–H,C=O,COO^-=C–O–C–and O=COCH chemical bonds formed gradually.Principal component analysis of the FTIR and FT-NIR spectra classified the sample into two groups according to the p H values.The first group of samples was formed by heat treatment at p H 5.0,the second group,formed by heat treatment at p H 6.5 and 8.0.In assessing the FTIR and FT-NIR spectral data,the pathway of the decomposition of blue pigments in different p H is parallel.