Study On Cold-induced Sweetening Of Chinese Chestnut And Purification And Characterization Of Polyphenol Oxidase

Chestnut(Castanea mollisima BL.)is a plant of Castanea genus of beech family,which has a long planting history in China.Chestnut fruits are delicious in tastes and rich in nutrients.It is deeply loved by people,because it not only can be fried,but also as a dish ingredients.After harvest,chestnuts are usually stored in large quantities in cold storage.Due to the long storage time,the nutritional quality,microstructure,physiological status andmolecular level of chestnut have changed during storage.This study starts with the changes of chestnut before and after storage,we analyzed the quality changes of fresh chestnut just picked and aged chestnut stored in cold storage for one year,then explored the cold-induced sweetening process,finally purified and characterized the PPO of Chinese chestnut,the main research results are as follows:(1)Quality analysis of chestnut before and after storage: quality analysis showed that the content of reducing sugar and soluble sugar of aged Chinese chestnut increased significantly compared with the fresh chestnut,while the content of starch decreased,and the indexes of protein,flavonoids and polyphenols did not change significantly.The analysis of physiological indexes showed that the respiration rate and water content of aged chestnut decreased,and its color turns white.Besides,there was no significant difference in germination between fresh chestnut and aged chestnut.After sugar roasted chestnuts,the results of sensory evaluation showed that the aroma and sweetness of aged chestnut were better,the appearance was worse,and the color was brown obviously.Microstructural observation showed that chestnut cells became larger and starch granules decreased after storage.In addition,the transcriptome data of chestnut before and after storage were also analyzed,and it was found that themolecular level of chestnut before and after storage was significantly different,and there were significant differences in phenylpropanoid substance synthesis,phytohormone signal transduction,MAPK signal transduction and other pathways,especially in starch and sucrose metabolism pathways,and the number of differential expressed genes was significant.(2)Analysis of cold-induced sweetening of chinese chestnut: the samples were taken on October 1,2021 when the Chinese chestnut was started store,November 25,2021 when the Chinese chestnut cold-induced sweetening was in progress,and January3,2022 when the Chinese chestnut cold-induced sweetening was completed,respectively,for transcriptome sequencing and related index analysis.The results showed that the contents of amylopectin,sucrose,fructose and glucose increased first and then decreased slightly,while the contents of amylose and maltose increased all the time.The activity of α-amylase was decreased,the activity of β-amylase was first unchanged and then increased,the activity of sucrose synthase was first decreased and then increased,and the activity of acid invertase was first increased and then decreased.Analysis of transcriptome data revealed that starch synthase,starch phosphorylase,α-amylase,β-amylase,isoamylase,sucrose synthase,sucrose phosphatase,acid invertase,4-α-dextransferase and aquaporins play an important role in the cold-induced sweetening process.(3)Purification,identification and characterization of chestnut PPO: the crude PPO enzyme solution of chestnut was separated by ammonium sulfate,anion exchange chromatography on DEAE Sepharose FF and Sephacryl S-100 gel column chromatography,and we finally succeeded in obtaining 25 times purified target protein.Native-PAGE and SDS-PAGE showed that the apparentmolecular weight was 108 KDa and the monomermolecular weight was 27 KDa,indicating a tetraploid structure.It was identified as a novel protein A0A8J4VKD7 by mass spectrometry,and was confirmed to have PPO activity when expressed in E coli by constructing p ET28 a vector.Enzymatic characterization of chestnut PPO showed that the optimal temperature and p H value of chestnut PPO were 45℃ and 6.0,and it is stable in neutral environment and low temperature.Chestnut PPO showed substrate affinity to pyrogallic acid and catechol,and ascorbic acid and citric acid had the best inhibitory effect on it.The active center of chestnut PPO was in the helical bundle formed by α-helix and β-fold,in which the α-helix was 37% and β-fold was 37.3%,and the maximum fluorescence intensity was 1674 at the emission wavelength of 327 nm,which proved that its tryptophan residue was inside the protein.