| Citrus is the most cultivated fruit in the world.In addition to fresh fruit,it is also processed into juice,sweetmeats and other products.Orange juice accounts 2/3 of the fruit juice production in the world.In citrus juice processing industry,bitter taste is one important factor affecting the taste and consumption of citrus and juice,and limonoid is the main bitter substance accumulated in orange and juice,which has a negative effect on the flavor of orange juice.However,these substances have been proved to be a kind of biological active substances,which have obvious biological effects as anti-cancer,anti-oxidation,bacteriostatic,anti-aging et al.,and might play an important role in human health and medical health care as well as the resistance of plants themselves.So it is not only of great significance to citrus processing industry,but also of value to rational utilization of limonin biological activity to understand the changes of limonoid and its formation mechanism.In order to provide reference for the selection of processing materials,processing time and processing technology in citrus juice processing industry,as well as breeding of suitable cultivars for processing,we carried out this study.In the study,different varieties of sweet orange as the main test materials,the contents of limonin and nomilin in the fresh juice and pasteurized juice were analyzed by high performance liquid chromatography(HPLC),and the gene expression of limonin glucosyltransferase related to the metabolism of limonoid was analyzed by real-time fluorescence quantitative PCR.The main results are as follows:1?Optimizing and establishing of extraction and detection methods on limonin and nomilin in juiceUsing dichloromethane as extractant,limonin and nomilin were extracted from juice by ultrasonic method.A single factor experiment was designed to determine the effects of the volume ratio of juice to dichloromethane,ultrasonic time and extraction times on the extraction efficiency.The results showed that when the volume ratio of juice to dichloromethane was 30: 30,ultrasonic treatment for 40 minutes and extraction for 3 times,the extraction efficiency from orange juice was the best.The accuracy of the extracting method was evaluated by the recovery of adding standard sample method.The recoveries of limonin and nomilin were over 97% and 90%,and the relative standard deviation(RSDs)were 0.28% and 2.91%,which indicated that the extraction method was feasible and accurate.In addition,water and acetonitrile were used as fluidity in HPLC,the detection wavelength was 210 nm,the injection amount was 15 ?L,the column temperature was 30 ?,and the flow rate was 1 m L/min for gradient elution.The results showed that the peak area was significantly correlated with the concentration of limonin and nomilin,and the linear correlation coefficient of regression equation was 0.9998.The concentration of limonin and nomilin in the juice can be determined by the peak time of sample chromatographic icon and the peak area of chromatogram.2?The changes of limonin and nomilin contents in different fruit maturation stages The contents of limonin and nomilin in different citrus fresh juices and pasteurized juices were determined at different maturation stages in two consecutive years.Pasteurization is a routine and necessary process to ensure the safety of juice storage and transportation.The results showed that during the maturation of citrus fruits,the content of limonin decreased obviously as a whole.In 2015,the average content of limonin in all tested varieties was 6.60 mg/L at the beginning of the test and decreased to 1.94 mg/L at the end of the test.In 2016,the average content of limonin in all tested varieties was 13.23 mg/L at the beginning of the test and decreased to 2.45 mg/L at the end of the test.Around mid-November,in fresh and pasteurized juice of most citrus varieties,the content of limonin declined to the limonin bitterness threshold of humanity(6 mg/L).It can be used as a reference time point for starting orange juice processing.Similar changes of limonin content were observed in fresh juice and pasteurized juice of all tested varieties in different maturation stages.In the same test stage of tested varieties,the content of limonin in pasteurized fruit juice was higher than that of fresh juice,usually with an average about 2 times that of fresh juice.The changes of nomilin contents was very similar to limonin,but the content is much lower when compared with limonin,and is about 1/10 of that of limonin.At the same time,the content of nomilin changed little after pasteurization.So it may not be the main factor leading to juice bitterness.Moreover,contents differences of limonin and nomilin were found in citrus juice at similar time point in different years.It might be due to the maturity difference in different years since the annual climate difference.3?The content difference of limonin and nomilin in juice of different citrus cultivars The contents of limonin and nomilin in juice of different citrus cultivars were analyzed.The results showed that the contents of limonin and nomilin were significantly different among citrus cultivars.Take the test on October 10,2015 as an example,among the tested sweet orange cultivars with very similar genetic background,the highest content of limonin was found in low or acidless sweet orange cultivars with an average value of 13.56 mg/L,followed by navel orange cultivars with an average value of 6.42 mg/L,and common sweet orange cultivars were the lowest with an average value of 3.65 mg/L.But there were also significant differences among the same type of sweet orange varieties.Such as in low or no acid-sweet orange,the Bingtangcheng is 28.71 mg/L and the Taoyecheng is 12.73 mg/L in the first test.Among the varieties tested for two consecutive years,Bingtangcheng had the highest content of limonin,Wuhe Xuegan were significantly different in different years.The difference of nomilin content between different varieties was consistent with that of limonin during the test period.The measure of loose-skinned citrus varieties were carried out in the second year only,their contents of limonin in the juice were lower when compared with the sweet orange,and the average content of the initial and final tests is 2.31mg/L and 0.62 mg/L.4?The expression difference of limonin glucosyltransferase among varieties during different maturity stages Limonin glucosyltransferase is responsible for the converting of limonoid-A-ring lactone into limonoid glycoside.It is one of the key enzymes in the metabolism pathway of limonoid.The expression of limonin glucosyltransferase in juicy sac during fruit ripening was analyzed.The results showed that during the development of citrus fruits,the expression of limonin glucosyltransferase gene in juicy sac increased first and then decreased.The highest expression of the gene was observed on October 28 in all cultivars except Wuhe Xuegan.The contents of limonin and nomilin decreased significantly on October 28,with the mean of 3.28 mg/L and 0.58mg/L.In Wuhe Xuegan,the highest expression of limonin glucosyltransferase gene occurred in the middle of November and the contents of limonin and nomilin decreased obviously after this period.From the above analysis,it was suggested that limonin glucosyltransferase gene may play a negative role in the accumulation of limonin and nomilin but not a decisive factor for their content. |
PDF Full Text Request