| The lemon(Citrus limon(L.)Burman f.),a perennial evergreen undershrub,belonged to the family of Rutaceae,can bloom and fruit each year.In 2012,the worlds' yards of lemon reached a total of 8.38 million hectares and the total production was 15.1 million,which corresponded to 26.208 billion US dollars,however,the above data in China was only 111.6 thousand hectares,2.32 million tons and 798 million dollars,respectively(FAO data,2013).Anyue,a county in Sichuan province,had 40 thousand yards of lemon,and the annual production was approximately 0.7 million tones,which corresponded to 1.041 billion US dollars in 2015.Therefore,Anyue was the top eighth major production area in the world and known as 'the hometown of lemon in China'.Lemon is rich in organic acids,sugars,amino acids,pectins,mineral elements and a multiple of bioactive substances such as flavonoids,limonoids,coumarin,carotenoids and vitamins.Among of which,the flavonoids,limonoids analogues,vitamins,essential oils,coumarin and carotenoids have medicinal and health functions.In addition,lemon is processed into lemonade,lemon oil,lemon tea,lemon wine and so on.The lemon tree can also play important roles in preventing soil erosion,soil desertification and windbreak.Thus,the lemon which has become the leading industry to increase farmers' income and has significant economic,social and ecological benefits.With the continuous improvement of modern living standards,the growing demands of natural medicinal health products and deep processing of products,lemon has become one of the favorite fruits.Currently,studies about the organic components in different lemon cultivars,especially the processing technical system,is relatively poor,and this has become an obstacle to the related product development.It urgent needed to study the main components in different lemon cultivars to guide the consumption of fresh fruit and the produce research on medicine and health,to provide theoretical basis and technical support for its further processing,to provide reference for selection of processing lemon cultivars.In this paper,the 8-year-old tree of EUR,ALL,FEM,FIN,KWT.MON,VER and VIL.which at high yield and high quality demonstration resources nursery in Anyue were used as materials.The main cultivar EUR was set as the control group.The fruit appearance qualityand the intrinsic quality were determined.A High performance liquid chromatograph(HPLC)technique was developed to determine the 11 flavonoids,6 water-soluble vitamins,limonin and nomilin in eight lemon cultivars during the period of fruit development.To study the accumulation and dynamic changes,the data of organic components were analyzed using multivariate statistical methods.We determined the aroma components,carbohydrate and acid components in fruits of eight lemon cultivars using gas chromatography-mass spectrometry coupled with similarity analysis,which provides theoretical basis and technical support for health care and medicinal products of lemon and a comprehensive utilization of by-products.The main results were as follows:1.Under the same management pattern in the same nursery,the color and fineness of the fruit did not change significantly,while the size of the mastoid changed significantly.The mean fruit weights of FIN(216.88±24.24 g)and EUR(207.79±20.59 g)group had no statistically difference(P>0.05).Similar results were also observed in the KWT(200.76±31.92 g)and EUR(P>0.05)group.The MON,VER and FEM were dramatically lower than that of EUR(P<0.01)group.The ALL group was significantly lower than that of the EUR group(P<0.05).The maximum fruit weight of FIN(277.95 g)and KWT(256.45 g)were significantly higher than that of the EUR group(241.67 g).In contrast,the maximum fruit weight of the VER(211.40 g),FEM(210.31 g),VIL(209.86 g),ALL(190.79 g)and MON(182.65 g)group was significantly lower than that of the EUR group.The fruit shape index of the VER(1.60±0.09)group was significantly higher than that of the EUR group(1.32±0.07)(P<0.O1).The fruit shape index in VIL(1.36±0.09),KWT(1.36±0.09)and MON(1.33±0.10)group had no statistically difference when compared to the EUR group(P>0.05).All fruits of eight lemon cultivars have reached the standard of superior fruit.Except the FIN(7.80±0.07%),the TSS content of EUR(7.71 ±0.05%)was significantly higher than that of the other six cultivars.The total acid content of FIN was significantly higher than EUR(6.67±0.06%)(P<0.01)while the contents of other six cultivars were significantly lower than EUR(P<0.01).The solid acid ratio of ALL(1.02±0.01)was significantly lower than that of the EUR group(1.15±0.01)(P<0.01),and FEM was significantly lower than EUR(1.10±0.04)(P<0.05),while the solid acid ratio in VER,FIN,KWT,EUR and VIL group were few of change(P>0.05).The edible rates of eight lemon cultivars were extremely significant(P<0.01).FIN,EUR,VER and VIL were screened for high soluble solids and high acidity.BSTFA derivative coupled with gas chromatography-mass spectrometry(GC-MS)was used to detect glycometabolism and acid metabolism components in mature fruit of the eight lemon cultivars.From the results,we have found 34 types of components including 23 sugars or sugar derivatives,11 organic acids,amino acids or their derivatives.13,14,14,13,10,10,9 and 12 kinds of main metabolic compounds were identified in the fruits of ALL,EUR,FEM,FIN,KWT,MON,VER and VIL lemon,respectively,which amounting to 85.61%,88.29%,81.10%,93.62%,91.40%,88.46%,79.02%and 87.63%of total peak areas.The difference between each group was from 0.17%to 14.60%.The similarity evaluation results showed that there were no significant negative correlations between EUR and VIL,and there were no significant positive correlations with ALL and other six cultivars.The results indicated that there were differences in the types and content of the compounds in the fruits components of eight lemon cultivars.2.A high performance liquid chromatograph assay was developed to determine the 11 flavonoids in lemon,including the hesperidin,neohesperidin,naringin narirutin,eriocitrin,hesperitin,naringenin,luteolin,nobiletin,eriodictyol and rutin.An ultraviolet detector was employed,the chromatographic separation was performed on an Agilent Eclipse XDB-C18 column(250 mm × 4.6 mm I.D.,5 ?m)kept at 30 ? using 0.1%formic acid aqueous solution-acetonitrile as mobile phase at a flow rate of 1.0 mL min-1 and detection wavelength was 287 nm,the gradient elution as follow:0-30 min,15-30%B,30-45 min,30-80%B,45-50 min,80%B,50-55 min,80-15%B,55-60 min,15%B?The same high performance liquid chromatograph method was also employed to determine the 6 water-soluble components as well as the limonin and nomilin in lemon except that the flow rate of mobile phase used to seperate Vc,Vbl,Vb3,Vb6,Vb9 and Vb12 was 0.9 mL·min-1 versus 1.0 mL·min-1 to isolate limonin and nomilin.The detection wavelength to separate Vc,Vbl,Vb3,Vb6,Vb9 and Vbl2 was set at 267 nm versus 210nm to separate limonin and nomilin.In addition,the elution procedure to separate Vc,Vbl.Vb3,Vb6,Vb9 and Vb12 was set as 0-5 min,1%B,5-10 min,1-20%B,10-20 min,20%B,20-22.5 min,20-70%B,22.5-25 min,70-1%B,which versus 0-30 min,15-30%B,30-45 min,30-80%B,45-50 min,80%B,50-55 min,80-15%B,55-60 min.15%B to separate the limonin and nomilin.The results of the methodological studies suggested that the RSD of precision,stability and reproducibility of HPLC methods was less than 3.0%,the recovery rates of the eleven flavonoids were 95.49-103.10%,the recovery rates of six water-soluble vitamins were 99.06-101.72%.For limonin and nomilin,the recovery rates were 100.36%and 99.79%,respectively,which indicated that the HPLC methods that we used were of great reliability and had highly accuracy.3.The dynamic changes of the three important secondary metabolites in fruits of eight lemon cultivars at 5 different periods were investigated using the methods described previously(2).The results demonstrated that the contents of 11 types of flavonoids in different lemon cultivars varied during the period of fruit development.The hesperidin,eriocitrin,narirutin and naringenin showed a rising-decreasing trend,the contents of these compounds in the three periods of second physiological fruit drop period,fruit maturity period and fruit color-changed period was the highest(19.59?26.68 mg·g-1,7.92?11.01 mg.g-1,1.08?2.79 mg·g-1,0.26?1.02 mg.g-1).With the fruit developing,the other 7 flavonoids declined all the way to fruit maturity,the contents of them at the first physiological fruit drop period was the highest(Neohesperidin:0.84?4.73 mg·g-1,Naringin:0.17?0.58 mg.g-1,Rutin:0.74?1.84 mg.g-1,Nobiletin:0.39?1.60 mg.g-1 Eriodictyol:0.35?1.33 mg·g-1,Luteolin:0.26?1.08 mg.g-1,Hesperitin:0.28?0.76 mg.g-1).The content of vitamins C at fruit maturity period was the highest(12.90?33.73 mg·100g 1),while that The contents of the five B-Vitamins showed a trend of stabilization after second physiological fruit drop period,fruitenlargement period or fruit color-changed period decreased to a certain level(Vb9:1.22?5.17 mg ·100g-1,Vb6:0.53?1.93 mg·100g-1,Vbl:0.30?1.66 mg·100g-1,Vb3:1.34?2.10 mg·100g-1,Vb12:2.35?3.43 mg·100g-1).The contents of limonin and nomilin showed a rising-decreasing trend,the contents of them in the four periods of first physiological fruit drop period,second physiological fruit drop period,fruit maturity period and fruit color-changed period was the highest(0.41?0.86 mg·g-1,0.31?0.57 mg.g-1).The results of multivariate analysis indicated that the quality of eight lemon cultivars was close to maturity.The EUR lemon was the cultivar with high content of hesperidin,neohesperidin,naringin and rutin,the FIN lemon was the cultivar with high content of eriocitrin and narirutin,the VER lemon was the cultivar with high content of nobiletin,hesperitin vitamin c,vitamin b6 and vitamin b9,the KWT lemon was the cultivar with high content of eriodictyol,luteolin,limonin and nomilin,while the KWT lemon was the cultivar with high content of vitamin b1,vitamin b3 and vitamin b12.4.Steam distillation extraction coupled with gas chromatography-mass spectrometry(GC-MS)was used to detect aroma components in mature fruit of eight lemon cultivars.The results suggested that 44 types of aroma compounds were identified,in which 31,31,33,34,33,32,32 and 32 kinds of major compounds were identified in the peels of ALL,EUR,FEM,FIN,KWT,MON,VER and VIL lemon,which amounting to 90.94%,93.42%,91.61%,91.02%,88.11%,89.58%,92.57%and 92.09%of total peak areas,respectively.Eight samples contained twenty-two common components which were D-Limonene,a-Terpineol,y-Terpinene,?-Pinene and Terpinen-4-ol etc.There were some differences in component types and percentages contents of eight lemon cultivars,this can be used as the important features for distinguished cultivars.This study provided scientific theory basis for integrated utilization of lemon peels and selection of lemon cultivars. |
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