Extraction And Purification Of γ-GTP From Toona Sinensis And Its Effect On The Formation Of Characteristic Flavor

Toona sinensis(TS)is known as a traditional woody vegetable unique to China,and its young leaves have a unique aroma and are loved by Chinese consumers.The characteristic flavor of TS is one of the important influencing factors to determine the edible value and commercial value,therefore,preserving the characteristic flavor of TS during processing has become one of the research hotspots.However,the characteristic flavors of TS,especially sulfur-containing characteristic flavors,are very unstable and are easily dissipated during thermal processing.Combined with the similar synthesis pathway of sulfur-containing volatile flavor substances in aromatic vegetables such as Welsh onion,garlic and onions after tissue damage,it is assumed that the flavor precursors in TS form propene thiol under the action of enzymes.The unstable propene thiol undergoes polymerization or pyrolysis to produce a series of volatile sulfur-containing compounds,resulting in the characteristic flavor of TS.It has been found that the flavor precursor substance of TS isγ-glutamyl-propeny-thioglycine,which is not easily volatile and destructive,and γ-glutamyl transpeptidase(γ-GTP)can specifically catalyze the transfer reaction of γ-glutamyl,which is presumed to be the key enzyme in the formation pathway of the characteristic flavor of TS.So far,there are few studies on which sulfur-containing characteristic flavors are associated with,how to affect and the role of γ-GTP of TS.In this study,we isolated and purifiedγ-GTP from TS,and then added the purified γ-GTP into different systems(flavor precursor solution and TS inactivator solution)to explore the response pattern of the sulfur-containing characteristic aroma components of TS to γ-GTP,so as to verify the key enzyme status ofγ-GTP in the characteristic flavor formation pathway of TS.This will provide a technical basis for the in-depth analysis of flavor formation mechanism and directional regulation.The main findings are as follows:(1)Drying method screening: Firstly,γ-GTP was extracted from different growth stages(seeds,young leaves and old leaves)of TS using ice bath ultrasonication.The results of enzyme activity assay showed that the γ-GTP activities in different growth stages of TS samples differed greatly,among which young leaves had strong aroma and the highestγ-GTP activity,which was the best material for the isolation and purification of γ-GTP.Secondly,different drying methods were used to dry the young leaves of TS in order to improve the extraction rate of γ-GTP.The volatile components of fresh,dried and rehydrated TS were analyzed and identified by gas chromatography-ion mobility spectrometry(GC-IMS),headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS),combined with internal standard method and relative odor activity value(ROAV)method,and the results showed that after rehydration of dried TS,the total content of sulfur compounds in vacuum freeze-dried TS increased from 1.08 μg/g to 189.61μg/g,about 175 times higher,and the total content of sulfur compounds in heat pump-dried TS increased from 24.53 μg/g to 176.12 μg/g,about 7 times higher.The vacuum freeze-dried and heat pump-dried samples were able to recover more sulfur-containing flavor compounds,and the overall flavor was closer to that of fresh TS.Comparing fresh TS with different drying methods of TS,the highest γ-GTP activity(6.06 U/g)was found in the vacuum freeze-dried TS samples.By comparing the effects of different drying methods on the volatile components and γ-GTP activity of the TS,it was determined that the vacuum freeze-drying treatment could better retain the volatile aroma components and γ-GTP activity of the TS and was the best pretreatment method.(2)Separation and purification of γ-GTP: The γ-GTP was obtained by vacuum freeze-dried TS(young leaves),prepared by ultrasound-assisted extraction,ammonium sulfate graded precipitation,dialysis desalination,DEAE-Sepharose FF ion exchange chromatography column,Sephadex G100 gel chromatography column,etc.The purification multiple was 15.10 times,and the yield was 2.16%.The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that γ-GTP contained two subunits with molecular weights of 35 KDa and 50 KDa,respectively,and reached the electrophoretic pure grade.(3)In vitro enzymatic reaction verification: Firstly,GC-MS was used to analyze and identify the volatile components of the inactivated solution of TS.The results showed that no sulfur-containing compounds were identified in the inactivated system compared with fresh TS,reflecting that the formation of sulfur-containing characteristic flavors was closely related to the enzymatic reaction.Secondly,the purified γ-GTP and the precursor substances were subjected to enzymatic reaction in the aqueous phase,and the characteristic flavor substances in the reaction system were determined by GC-MS.It was found that the enzyme solution alone and the reaction precursor were almost devoid of sulfur-containing compounds,while 19 sulfur-containing characteristic aroma compounds were detected in the reaction system,including the characteristic aroma components of TS Z/E-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene,(E,Z)-di-1-propenyl disulfide,di(1-propenyl)sulfide,etc.Thus,the key position of γ-GTP in the formation pathway of the characteristic sulfur-containing flavors of TS was verified.(4)Study of reaction conditions: Based on the constructed in vitro enzymatic reaction system,single-factor tests and orthogonal tests were conducted with reaction time,temperature and p H as influencing factors and the total content of characteristic sulfur-containing aroma components as indicators.The results showed that the factors influencing the content of sulfur-containing characteristic aroma components of TS were reaction p H,reaction time and reaction temperature in order.The optimal enzymatic reaction conditions were: 40℃ water bath reaction for 30 min and reaction p H of 7.0.At this time,the content of sulfur-containing characteristic aroma components was 1815.49±31.22 μg/g.When different concentrations of γ-GTP were added to the TS inactivating enzyme substrate,the content of sulfur-containing characteristic volatile components of TS increased with the increase of enzyme solution concentration(1 mg/m L～30 mg/m L)at a low concentration.However,when the enzyme solution concentration increased to a certain value(30 mg/m L),the substrate concentration was insufficient and the content of the characteristic sulfur-containing volatile components of TS stabilized.The effect of γ-GTP addition on the characteristic flavor of TS was systematically analyzed to further verify the status of γ-GTP as a key enzyme in the flavor metabolic pathway of TS.It provides technical support for maintaining and enhancing the characteristic flavor of TS in production practice in the future.In summary,the results of this study verified that γ-GTP is a key enzyme in the formation pathway of the sulfur-containing characteristic flavor of TS,which provides a technical basis for the later in-depth analysis of the formation mechanism and targeted regulation of the characteristic flavor of TS.