Study on the composition of free amino acid and the effects on fruit flavor quality in 15 hybrid citrus varieties

Abstract: 【Objective】Hybrid citrus was derived from natural or artificial cross between different citrus species, such as from mandarin and orange or pomelo. Many hybrid citrus varieties have good character- istics such as wide adaptability, easy peeling, long storage period and rich flavor. The previous research mainly focused on the performance, botany traits, biological characteristics and cultivation techniques of introduced hybrid citrus. However, few studies on the content characteristics of free amino acids (FAA) and its influence on flavor quality were reported. The content and types of FAA could be used as an important index to evaluate the nutrition, taste and flavor of fruits. Hence, the present study identi- fied the composition of different FAA and their influence on the flavor quality of 15 hybrid varieties in- troduced into Zhejiang production area.【Methods】High-performance liquid chromatography (HPLC) system with a C18-H (250 mm×4.6 mm, 5 μm) was used to determine the composition and content of FAA in the citrus fruits. Phenyl isothiocyanate acetonitrile solution was used as pre column derivatiza  tion reagent. The column temperature was 40 ℃, the flow rate was 0.5 mL·min^-1, the detection wave-length was 254 nm and the mobile phase was sodium acetate acetonitrile solution(A) and 80% acetoni-trile aqueous solution(B), mobile phase gradient elution. N-leucine was used as internal standard and quantified by external standard method. The statistics and analysis of test data were processed by Micro- soft SPSS 19.0 and the Excel 2007, and Visio 2016 software were used for drawing. The effect of con- tents of 17 free amino acids in different hybrid citrus on fruit flavor was judged by taste intensity value (TAV), and the FAA quality was evaluated by principal component analysis (PCA) and cluster analysis.【Results】All 15 hybrid citrus varieties contained 17 FAAs, among which Arginine (Arg) content was the highest in 4 varieties, and Proline (Pro) content was the highest in the other 11 varieties. The total free amino acids (TFAA) of the varieties ranged from 2 605.99 to 6 572.68 mg·L^-1. The content of non-essential amino acids (NEAA) was the highest, followed by essential amino acid (CEAA) for children,and content of essential amino acids (EAA) was the lowest. EAA/TFAA was only 0.84%-4.90%. Medic-inal amino acids (MAA) showed a great variability from 13.39% to 67.80%, it was higher than 50.00% in 5 cultivars. The characteristics of amino acid components of different varieties showed significant dif-ference. For example, the contents of TFAA, taste amino acids and sweet amino acids (SAA) of Beni-bae were the highest followed by Tsunokagayaki, while delicious amino acid (DAA), bitter amino acid (BAA) and aromatic amino acid (AAA) of Himeruby, Hongmeiren and Harumi were dominant. Addi-tionally, the EAA and MAA contents of Mihaya and Kanpei were the highest . According to the flavor characteristics of each free amino acid, they were divided into four categories, including SAA, BAA, DAA and AAA. The order of the contents of 4 different flavor amino acids in the 15 hybrid citrus culti- vars was SAA>BAA>DAA>AAA, but BAA>SAA>DAA>AAA in Haruka. It was found that Arg was the main factor affecting the fruit flavor with the TAV of 0.92-3.37 and a mean value of 2.01. There were 13 kinds of hybrid citrus varieties with the TAV of Arg>1, and the one of Sweet Alanine(Ala), Pro-line(Pro) and Delicious glutamic(Glu) in a few varieties was also >1. The total variance contribution of the four principal components was 85.44% and it was identified by PCA analysis, which could reflect the comprehensive information of FAA in hybrid citrus. The evaluation model was established by inte- grating the four factors: F=0.471F1 + 0.221F2 + 0.169F3 + 0.136F4. The order of the comprehensive scores of 17 kinds of FAA in the 15 hybrid citrus varieties was Mihaya > Himekoharu > Hongmeiren >Tsunokagayaki > Benibae > Harumi > Haruka > Himeruby > Ehime No.31 > Kanpei > Setoka > Asu-mi >Tsunonozomi > Orri mandarin > Huangmeire. The 15 hybrid citrus varieties were divided into 4 groups by cluster analysis, the results showed that Mihaya, Tsunokagayaki and Benibae were clustered into the first group, Himekoharu and Hongmeiren in the second group, Harumi in the third group, and the other varieties were clustered into the fourth group separately.【Conclusion】The amino acid compo-sitions in fruits of some varieties were significantly different. Pro and Arg were preponderant in17 free amino acids of different hybrid citrus, followed by Asp and Ala. Bitter Arg was the main factor affect-ing flavor of hybrid citrus fruits. The results of cluster analysis and principal component analysis were basically the same, both could comprehensively reflect and evaluate the differences of the amino acids in various varieties. The research would provide important references to the selection of hybrid citrus varieties.