Studies On The Degradation Characteristic Of Cell Wall Polysaccharides And Regulation During Fruit Softening Of Annona Squamosa

Annona squamosa L.was a tropical fruit with sweet flesh and rich nutrients,but the fruit softening and cracking rapidly within a few days of harvest,leading to a shortened shelf life,which severely limited its commercial and economic value.In this thesis,the correlation between fruit softening and cell wall polysaccharide degradation was used as a starting point,which the structural properties of cell wall polysaccharide components were initially investigated.Combined with the results of transcriptomic analysis,the key enzymes of cell wall glycan metabolism affecting fruit softening were screened.Then,the effects of non-enzymatic factors on cell wall polysaccharide degradation were investigated.Finally,the correlation between post-harvest treatment on the softening quality of Annona squamosa and key enzymes of cell wall glycan metabolism was investigated to provide a theoretical basis for subsequent studies on the regulation of key enzymes of cell wall glycan metabolism in the softening process of Annona squamosa,which was important for promoting the application of Annona squamosa in the food industry.Firstly,the correlation between softening and degradation of cell wall polysaccharides was investigated in Annona squamosa.Through physicochemical methods to determine the hardness,color,moisture content,vitamin C（Vc）content,soluble solids（TSS）content,titratable acid（TA）content,water distribution,low-field nuclear magnetic resonance imaging and other quality indicators.The results showed that the fruit softening process,the appearance of the color from green to yellow-green and finally brown.The hardness,water content,Vc content of the fruit were gradually decreased.TSS content remained basically unchanged.The fruit in the combined water and free water were a lot of loss.Water-soluble pectin（WSP）,chelator-soluble pectin（CSP）,sodium carbonate-soluble pectin（NSP）,and potassium hydroxide soluble fraction（HC1 and HC4）were obtained by different solvent extractions.The molecular weight and monosaccharide composition of the cell wall polysaccharides of Annona squamosa at different storage times were determined by high performance liquid chromatography（HPLC）.The results showed that WSP,CSP and NSP appeared as small molecular weight polymers in the late storage period,and the content of high molecular weight polymers had a decreasing trend.HC1 and HC4 changed from one characteristic absorption peak to four characteristic absorption peaks during storage.The main monosaccharide components in WSP and CSP were glucose and galacturonic acid.In HC1 and HC4,the relative content of glucose was extremely high,but the relative contents of mannose and rhamnose among the five polysaccharide components were extremely low and fluctuated weakly throughout the storage period.Correlation analysis using SPSS 20.0software confirmed that the correlation between softening of Annona squamosa and degradation of cell wall polysaccharides was relevant.Secondly,the structural characterization of the five fractions of Annona squamosa cell wall polysaccharides was determined by HPLC,high performance anion exchange chromatography（HPAEC）,Fourier infrared spectroscopy（FT-IR）,dynamic rheometry and thermogravimetric analyzer.The results showed that the fractions of CSP,NSP,HC1 and HC4 mainly showed two peaks.The NSP fraction had the highest yield.The five polysaccharide fractions contained bothα-type andβ-type glycosidic bonds.The pyrolysis process of the five polysaccharide fractions mainly went through three stages,and their rheological properties all exhibited pseudoplastic fluid behavior.The cell wall polysaccharide fraction WSP,with a molecular weight of 1902.589 k Da,was significantly associated with the softening of Annona squamosa,which consisted mainly of galacturonic acid and dominated in the RG-I pectin region.The other HC4 fraction,which was significantly associated with the softening of Annona squamosa,consisted mainly of glucose and had a low degree of linearity or branching of hemicellulose.Thirdly,the key enzymes of cell wall glycan metabolism affecting the softening of Annona squamosa were screened in combination with transcriptome sequencing.The activities of polygalacturonase（PG）,pectin methyl esterase（PME）,cellulase（Cx）,xylanase（NEX）,β-galactosidase（β-Gal）,β-glucosidase（β-GC）andα-arabinofuranosidase（α-L-Afa）were determined by chemical methods.The results showed that during fruit storage,PG activity increased continuously.Cx,PME,NEX andβ-Gal activities showed an increasing and then decreasing trend.α-L-Afa activity was the opposite.β-GC activity remained basically unchanged.The expression of PG,PME and Cx genes was measured by real-time fluorescence quantitative PCR（RT-q PCR）.The results showed that the expression of Cx gene with higher expression increased and then decreased.As shown by transcriptional analysis of the reference-free genome,compared with the control group,a total of22,363,262 and 208,18626 filtered reads were obtained in the softening model group（6 d and 12 d Annona squamosa）,6.71G and 6.25G filtered bases,47.95%and 48.48%GC content,and 3,168 and 2897,respectively.DEGs were enriched to 107 and 86 pathways,respectively,which the most important pathway was starch and sucrose metabolic pathway（ko00500）.Combining the results of correlation analysis between the softening of Annona squamosa and cell wall material,cell wall polysaccharide fractions,and cell wall glycan metabolizing enzymes,preliminary screening determined that the key cell wall glycan metabolizing enzymes in the softening process of Annona squamosa were cellulases.Fourthly,non-enzymatic factors（p H,free radicals）and enzymatic activities of related antioxidants affecting the softening of Annona squamosa were studied.The p H of the fruit was determined by p H-meter.The results showed that the p H of Annona squamosa was maintained at around 4.2 after ripening showing acidity.The results showed that with the fruit softening,the H₂O₂content of Annona squamosa gradually increased.The·OH content increased and then decreased.The SOD and APX activities gradually decreased,while the POD and CAT content gradually increased.The results of correlation analysis showed that the fruit hardness was extremely significant negative correlated with H₂O₂content（p<0.01）and significant negative correlated with MDA content（p<0.05）,indicating that the free radical H₂O₂was involved in fruit softening.The H₂O₂content was extremely significant negative correlated with CWM content（p<0.01）and the correlation coefficient was the largest.The correlation coefficient between H₂O₂content and cell wall polysaccharide WSP and HC4 fractions was larger,indicating that free radicals were correlated with cell wall polysaccharide degradation properties but not significantly.Finally,1-MCP,chitosan and calcium chloride treatments were applied to the fruits of Annona squamosa to clarify the correlation between the effective preservation mode and the key enzymes of cell wall sugar metabolism that regulate the post-harvest softening of Annona squamosa in combination with changes in quality indicators,fruit nutrition,key enzyme activities and gene expression.The results showed that the skin color was brighter and the fruit hardness decreased slowly during storage in the 1-MCP treatment group.The activity of key enzymes and gene expression were determined by chemical and RT-q PCR methods.It was found that the Cx enzyme activity in the 1-MCP treatment group was always at a lower level compared to the other treatment groups.Fructose,sucrose and glucose contents and volatile flavor substances were analyzed by HPLC and gas chromatography-mass spectrometry（GC-MS）,respectively.The results showed that the fructose and glucose contents of Annona squamosa showed an increasing trend during storage,and（+）-limonene was one of the highest and most important chemical components in relative terms.Using SPSS 20.0 software for correlation analysis,the results showed that the correction between the hardness of 1-MCP treatment of Annona squamosa and glucose and fructose content was highly significant negative correlation（p<0.01）with large correlation coefficient.The fruit hardness and cellulase enzyme activity and gene expression was negatively correlated with large correlation coefficient.In summary,1-MCP treatment showed better results as a potential way to extend the shelf life of post-harvest Annona squamosa.Besides,there was a large correlation between 1-MCP treatment and the enzymatic activity and gene expression of key enzymes of cell wall sugar metabolism.