Investigation Into The Accumulation Of γ-aminobutyric Acid In Coffee Leaves And The Associated Mechanism In Response To Ultrasound Stress

Gamma-aminobutyric acid(GABA),a non-protein amino acid ubiquitously found in animals and plants,possesses various physiological functions such as anti-hypertension,anti-anxiety,and anti-Alzheimer,et al.However,it normally does not meet the daily requirement of the middle-aged and elderly people due to the low content of GABA in animals and plants.Ultrasound is a green processing technology that has been used to increase the GABA content in germinated seeds.Coffee leaves are rich in a variety of bioactive components that are beneficial to human health,thus,they have been processed into a novel healthy beverage,coffee leaf tea.Therefore,the in-depth investigation into the GABA accumulation conditions and mechanisms in coffee leaves under ultrasound stress will provide a theoretical foundation for the preparation of GABA-enriched coffee leaf tea,which has important academic and practical significances.In the present study,the effects of ultrasonic treatment on the GABA content,total phenol content(TPC),phytochemical compositions,the activities of key enzymes,antioxidant activity,and the surface microstructure of coffee leaves were investigated.The mechanism of the accumulation of GABA in coffee leaves under ultrasound stress was preliminarily studied using metabolomics analysis.The results are as follows:(1)Ultrasonication together with the pH control activated glutamate decarboxylase(GAD)and inhibit GABA transaminase(GABA-T),thereby promoting the accumulation of GABA in coffee leaves.Under optimized conditions(UT1): pH =5.9,incubation time = 6.6 h,ultrasonic power = 150 W,ultrasonic frequency = 20/40 k Hz,ultrasonic temperature = 28℃,ultrasonic time = 5 min,and MSG solution concentration = 2%,GABA content was increased to 2.6 times compared with that of fresh coffee leaf(CTL).The angiotensin-converting enzyme(ACE)inhibitory activity of the coffee leaves treated under the optimal condition was 79.00%,which was25.30% higher than that of untreated coffee leaves.However,it was significantly lower than the 91.17% ACE inhibitory activity of coffee leaves treated under the ultrasonic condition(UT2)that includes ultrasonic frequency of 20/40 k Hz,power intensity of 60 W,and incubation of 2 h.(2)The effects of ultrasonic conditions on the phytochemical composition,antioxidant activity,and the activities of polyphenol oxidase(PPO)and peroxidase(POD)in coffee leaves are different from that of GABA.Different ultrasound conditions can either inhibit or increase the activities of PPO and POD as well as the polyphenol contents and antioxidant activity.Triple-frequency(20/35/50 k Hz)ultrasound significantly reduced the total phenolic content(TPC),trigonelline,caffeine,chlorogenic acid,mangiferin,and rutin,as well as antioxidant activity,and PPO activity.After single-frequency(35 k Hz)ultrasonic treatment,the antioxidant activity of coffee leaves reached the highest level while the POD activity was significantly reduced.With the extension of the incubation time,the content of polyphenols and the antioxidant activity were gradually decreased,whereas the activities of PPO and POD were increased first and then decreased.Under UT1 conditions,the TPC and antioxidant activity were reduced by 45.51% and 15.45%,respectively compared with the fresh coffee leaves,however,they were only decreased by 22.07% and 7.14%,respectively when coffee leaves were treated under UT2 conditions.Therefore,ultrasound can simultaneously affect the polyphenolic compounds and GABA content of coffee leaves.The decrease of ACE inhibitory activity in the coffee leaves treated with UT1 conditions is related to the decrease of polyphenolic compounds.(3)Ultrasound stress changed the permeability,surface microstructure,and metabolites of coffee leaves.The electrical conductivity of coffee leaves was significantly higher than that of fresh leaves under ultrasound stress,indicating that the permeability of the cell membrane was increased.The images of the surface microstructure showed that the number of pores on the surface of coffee leaves was increased,which accelerated the migration of substrate into the coffee leaf cells,thus increased the accumulation of GABA.The results of metabolomics research showed that the significant difference metabolites(VIP > 1,P < 0.05)of UT1 vs CTL,UT2 vs CTL,and UT2 vs UT1 were 177,153,and 188 kinds,respectively,and the main categories were lipids and lipid-like molecules,organic acids and derivatives.The results of the KEGG pathway analysis showed that the metabolic pathways related to GABA synthesis in the three comparison groups were the amino acid metabolism pathway and carbohydrate metabolism pathway,and the changes in the metabolites were mainly GABA,α-ketoglutarate,lysine,and other amino acids.