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BITTERNESS IN CITRUS FRUIT-THE BIOCHEMISTRY, ANALYSIS AND APPLICATIONS.

MANPREET KAUR SAINI 21th march 2012

CONTENTS OF PRESENTATION

BIOSYNTHESIS & BIOCHEMISTRY OF LIMONOIDS

EXTRACTION AND QUALITATIVE & QUANTITATIVE ANALYSIS OF CITRUS BITTER PRINCIPLE.
GENE EXPRESSION AND TRANSCRIPTOME STUDIES

CLONING AND CREATION OF TRANSGENIC VARIETIES
APPLICATION OF THE BITTER PRINCIPLE

PRIMARY AND SECONDARY METABOLITES
Metabolites- compounds synthesized by organisms using enzymemediated chemical reactions called metabolic pathways

PRIMARY METABOLITES Essential to growth and development. Present in all plants as are components or products of fundamental metabolic pathways or cycles.

SECONDARY METABOLITES  Not Essential to growth and development.  colored, fragrant, or flavorful compounds  typically mediate the interaction of plants with other organisms.

EXAMPLES OF PRIMARY METABOLITES Energy rich fuel molecules, such as sucrose and starch, Structural components such as cellulose, informational molecules such a DNA and RNA Pigments such as chlorophyll.

EXAMPLES OF SECONDARY METABOLITES Alkaloids such as caffeine, nicotine, etc. Terpenoids such as monoterpenes, diterpenes, triterpenes like Limonin and tetraterpenes.  Phenolics such as flavonoids like naringin and anthocyanins etc.

The main focus of this presentation will be triterpenoids mainly the Limonoids.

INTRODUCTION TO BITTER PRINCIPLES

Kinnow mandarin, a hybrid of Citrus nobilis and Citrus deliciosa is considered one of the major crops of Punjab.

But processing of Kinnow juice faced formidable problems in terms of “bitterness” and “delayed bitterness” thus affecting its consumer acceptability. Biochemical basis of bitterness in kinnow:
bitterness due to flavonoids e.g. naringin– species related to pumello. Threshold of bitterness is 50 ppm. delayed bitterness due to limonoids e.g. limonin. Threshold of bitterness is 6 ppm.
Critical reviews in biotechnology(199 6)


seeds. S. F. SM-segment membrane.Albedo. J.Flavedo.36 (2009) . Sci.) 43 : 28 .juice Kasetsart J.CITRUS BITTER PRINCIPLES IN DIFFERENT PART OF THE FRUIT Structure of citrus fruit showing concentration of limonoids and flavonoids in different parts. A. (Nat.

3 hydroxy 5 methyl glutaryl Co A. July (1995) . GPP-geranyl pyrophosphate.Decarboxylase Mevalonate kinase IPP Isomerase GPP synthase Prenyltransferase s FPP synthase squalene synthase GGPP synthase Terpene synthases MEVALONIC ACID PATHWAY AND TERPENOID SKELETON BIOSYNTHESIS ( HMG Co A. MVAP-Phosphomevalonate. Vol.BIOSYNTHESIS OF LIMONOIDS Acetyl Co A thiolase HMG Co A synthase HMG Co A reductase PhosphoMevalonate kinase MVAP. DMAPP-Dimethylallylpyrophosphate. IPP-Isopentyl Pyrophosphate. 1015-1026.geranyl geranyl pyrophosphate) The Plant Cell.5-pyro Phosphomevalonate.franesyl pyrophosphate. FPP. GGPP. MVAPP. 7.

BIOSYNTHESIS CONT…. Bull. SQUALENE NOMILIN ACEYL LYASE DEACETYLNOMILINIC ACID DEACETYLNOMILIN ICHANGENSIN(KETO) METHYL DEACETYLNOMILIC ACID CALAMIN CYCLOCALAMIN NOMILIN HYDROLASE OBACUNONE OBACUNOIC ACID ICHANGIN ICHANGENSIN(KETAL) LARL DEHYDROGENASE LIMONIN LIMONOL DEOXYLIMONIN UDP-D-GLUCOSE: LIMONOID GLUCOSYL TRANSFERASE DEOXYLIMONIC ACID 17 β-D GLUCOPURANOSIDE Biol. 29(2) 191—201 (2006) . Pharm.

>50 isolated from the Rutaceae (36 from Citrus & related genera) 2. 12(4). Two forms in citrus:  1. Limonoid glucosides (LG) -. LARL and NARL. LG: non-bitter. LA: bitter. insoluble in water 2.BIOCHEMISTRY OF LIMONOIDS IN CITRUS . water-soluble 3.the precursors for limonoid synthesis can also be considered  Relation between LA and LG 1.(1996).  A group of highly oxygenated tetracyclic triterpenoids in Rutaceae and Meliaceae plant families causes delayed bitterness in citrus and is a secondary metabolite. Food review. Limonoid aglycones (LA) -. .17 isolated 3. Int. LA glucosidated to LG-during fruit maturation and this is known as natural debittering.

Food Chem.LIMONOID AGLCONES J. No. Agric. 2007 Reviews . 55. 21. Vol..

. 2007 Reviews . No. Agric. 21. Food Chem.LIMONOID GLUCOSIDES AND SYNTHETIC LIMONIN CARBOXYMETHOXIME J. 55. Vol.

SITES OF BIOSYNTHESIS OF LIMONOIDS 3 forms of limonoids monolactone dilactone glucosides Mature peel and flesh tissues In leaves and seeds Limonin glucoside Nomilin glucoside .

seeds and leaves .ACCUMULATION OF LIMONOIDS acetate. mevalonate. peels. or farnesyl pyrophosphate Nomilin in stem phloem region other limonoids translocated to fruit tissues.

Limonin group 2. Ichangensin group 4. 7-acetate limonoid group Biosynthetic pathways of each group of these limonoids have been elucidated:  .MAJOR BIOSYNTHETIC GROUPS  Four groups of Limonoid Aglycones 1. Calamin group 3.

nomilin.THE LIMONIN BIOSYNTHETIC GROUP  in all citrus species. deacetylnomilin. . 12(4).(1996). Int. Ichangin and obacunone are the major limonoids BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE LIMONIN GROUP Food review. citrus hybrids and many non citrus members of family rutaceae.  Limonin.

retrocalamin. 6-keto-7bdeacetylnomilol and 6-keto-7b-nomilon. Int. methyl iso-obacunoate diosphenol. And major limonoids include calamin. 6-keto-7b-nomilon contains structural features of both calamin and limonin groupsrepresent a biosynthetic link between them.  BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE CALAMIN GROUP Food review.CALAMIN BIOSYNTHETIC GROUP  Found only in tissues of Fortunella and its hybrids like calamondin.(1996). 12(4). .

deacetylnomilin. . enzyme not found in any other citrus species. Nomilin converted to deacetylnomilin by nomilin deacetylase. Nomilin deacetlylase BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE ICHANGENSIN GROUP Food review.   present as ketal and keto group in chloroform solution but as ketal only in citrus.THE ICHANGENSIN BIOSYNTHETIC GROUP Found in tissues of Citrus ichangenesis and its hybrids Yuzu.(1996). 12(4). ichangenesin.Hanayu  and Ichang lemon. and deacetylnomilinic acid being the major compounds. Int. Kabosu. Sudachi.

. BIOSYNTHETIC PATHWAYS OF LIMONOIDS: THE 7-ACETATE lIMONOID GROUP Food review. limonyl acetate [20] and 7a-obacunyl acetate.(1996). Int. .THE 7-ACETATE LIMONOID BIOSYNTHETIC GROUP   Found only in tissues of Poncirus and its hybrids This limonoid group includes 7a-obacunol. 12(4).

The peel (rind) of the citrus fruit contain very high amount of flavonoids like naringin.   .Aring lactone). Hence the fresh citrus juice does not taste bitter but turns highly bitter on storage. But there is a difference between flavonoid and limonoid bitterness. The limonoids are present in the form of non-bitter compound (limnoate . The fruits containing high flavonoids are bitter even when consumed as fresh. neohesperidine etc.  The two classes of chemical compound namely flavonoids and limonoids were found responsible for bitterness in citrus juices. which is converted to bitter limonin and other bitter limonoids in the presence of enzyme limonoate-D-ring lactone hydrolase on storage.DELAYED BITTERNESS  Most citrus fruits do not taste bitter if eaten fresh or if freshly squeezed juice is consumed but within a few hours after juice extraction. the juice becomes bitter. making it highly bitter. This phenomenon is generally referred to as delayed bitterness.

Different temperature conditions affect the bitternesss content of same fruit.  Less the storage temperature.   With more time bitterness decreases. less the bitterness as enzyme activity reduced Promotes enzme activity    Initiates conversion of LARL to bitter limonoid aglycone .FACTORS RESPONSIBLE FOR DELAYED BITTERNESS  Cultivar Location Maturation time Storage temperature Acidic medium Field Freeze injure Mechanical damage Different varieties has different level of bitterness like in valencia orange and navel orange.

MECHANISM OF DELAYED BITTERNESS Food review. . 12(4).(1996). Int.

(1996). Int. isolated from citrus albedo tissues. 12(4).NATURAL LIMONOID DEBITTERING PROCESS   Limonin bitterness occurs in early to mid season harvested fruits only Natural debittering process occurs when in late stages of fruit growth and maturation limonoid aglycones were converted to their respective glucosides . found only in mature fruit tissues and seeds . . Enzyme involved is UDP-D_glucose transferase.glucosidic linkage such as limonin 17.   Food review.beta – D. Limonoid glucosides – contain one D.glucose molecule attached to the C-17 position of each corresponding aglycone via a beta.glucopyranoside.

etc. fruit tissues. fruit peels and seeds seeds biosynthesis of other limonoids from Nomilin.(1996) E-4 limonoid D. limonoid UDPglucopyranoside tranferase D- phloem region of citrus stem tissues biosynthesis of nomilin from acetate and mevalonate E-2 all citrus tissues including leaves. Enzymes like lyase.beta-Dglucopyranoside betaglucosidase dormant seeds and germinating seeds . dehydrogenase. esterase. reductase.ENZYMES INVOLVED IN BIOSYNTHESIS AND BIODEGRADATION OF LIMONOIDS IN CITRUS ENZYME GROUP NAME OF ENZYME SITE OF ACTION FUNCTIONS E-1 Various enzymes of terpenoid pathway like thiolase. isomerase. synthase. E-3 convert monolactones to glucosides during late stages of fruit growth and maturation lactonization of the Dring converts monolactones to dilactones limonoid biodegradation catalyzes the hydrolysis of limonoid glucosides and liberates limonoids and glucose Food review. hydrolase. Int.ring lactone hydrolase seeds E-5 limonoid 17. stems. etc. kinase. 12(4).

(1996) . 12(4).DEBITTRING METHODS DEBITTERING METHODS column and batch methods using adsorbent and ion exchange resins blend bitter juice with non bitter juice. Int. diluting out the bitter taste Creating new citrus varieties through Genetic engineering Transgenic citrus varieties free from limonin bitterness Limonoid UDP-D-glucose transferase Three specific target enzymes Limonoate Dehydrogenase Nomilin deacetylase Insertion of one of the three gene codings Yields nonbitter fruits in transgenic plants Food review.

(1996) . and ion-exchange highperformance liquid chromatography (HPLC) Isolated by ammonium sµlfate fractionation. affinity chromatography. and ion-exchange HPLC Enhancement of enzyme activity through genetic Engineering reduces aglycone concentration that reduces delayed bitterness In low level in citrus fruits so isolated from bacteria N terminal sequence determined Gene being from cDNA library prepared from bacterium diverts biosynthetic pathway of limonin  Nonbitter deacetylnomilin accumµlated instead of limonin Enzyme not isolated yet Food review. Int. 12(4). Blue dye-ligand affinity chromatography.Cont… Limonoid UDP-D-glucose transferase Limonoate Dehydrogenase Nomilin deacetylase Converts limonoid aglycones to glucosides Convert limonin to nonbitter limonoids Convert nomilin deacetylnomilin to Isolated from albedo tissues by a combination of ammonium sµlfate fractionation.

.EXTRACTION & QUALITATIVE & QUANTITATIVE ANALYSIS OF CITRUS BITTER PRINCIPLE.

for limonoid detection  Nuclear Magnetic Resonance (NMR) -.Analytical Methods of Citrus Limonoids Thin-layer chromatography (TLC) -.detection & quantification  Radioimmunoassay –detection & quantification  HPLC-MS – detection & quantification  .determination of limonoid structure  HPLC -.

Filtered liquid used to prepare three samples. JOURNAL OF CHROMATOGRAPHY A.45 um filter.5ml) . 1064(2005) 187-191 . Juice(150 µl)+CA internal standard solution(75 µl. LARL finally eluted with solution B (CH3CN-MeOH-Water) (1 ml).5 ml) and then CHCL3 (0. Supernatant collected and filtered through 0.EXTRACTION OF LARL & NARL FROM CITRUS JUICE Juice sample centrifuged at16000*g for 5min at 10˚ C. 100 ppm) Juice (150 µl)+internal calibrator solution(15 µl) Juice (150 µl)+ water (1. Thereafter column washed first with water(0.26 ml) Samples mixed and loaded(1 ml) onto strata X solid phase extraction columns that was washed with MeOH(1 ml) and preconditioned with water(1 ml).

3709-3714 . 51. It was evaporated to dryness and redissolved in MeOH or THF(300 µl) 3 µl diluted extract was injected into LC-MS J.5 ml) for 30 s CHCL3 extracts combined & 100 µl of it removed. FOOD CHEMISTRY 2005.EXTRACTION OF BITTER LIMONOIDS/ LIMONOID AGLYCONES FROM CITRUS JUICE Juice obtained by hand squeezing 2 ml juice transferred to test tube & 40 µl internal standard added (167 ng/µl) Juice gently liquid/liquid extracted with CHCl3 (2*2. AGRIC.

EXTRACTION OF LIMONOID GLUCOSIDES Solid samples (peel. VOL. AGRIC. 100 mg weighed into 10 * 50 mm cellulose extraction thimbles. Wet samples (juice. Diluted to 30 ml with MeOH Sample for injection prepared by combining 75 µl sample.45 um filter. seeds. NO 3.49. Supernatant collected and filtered through a 0. 925 µl water and 300 µl MeOH and 200 µl internal standard solution 300 µl of above extract + 700 µl water + 200 µl carminic acid solution(30 mg/l) added to autosampler vial Samples analyzed by ESI-LC-MS Sample analyzed by ESI-LC-MS J. Sample extracted overnight in soxhlet extracter with 25 ml MeOH. 2001 . etc) oven-dried but pulp samples dried overnight at 60 ˚C and ground to pass 2mm mesh screen. wash water) centrifuged at 16000 * g for 5 min at 10 ˚C. FOOD CHEMISTRY 2005. molasses.

GENE EXPRESSION AND TRANSCRIPTOME STUDIES .

19(4):333-362.. 2007 .STUDY OF TRANSCRIPTOME CHANGES DURING FRUIT DEVELOPMENT AND RIPENING FROM DIFFERENT PARTS OF THE FRUIT. J. STRUCTURE OF CITRUS FRUIT Braz. Plant Physiol.

CITRUS FRUIT STRUCTURES TARGETED FOR ENZYMES INDUCING NATURAL DEBITTERING IN CITRUS SEED FLESH ALBEDO FLAVEDO RNA ISOLATION AND CHARACTERIZATION AT DIFFERENT STAGES OF FRUIT DEVELOPMENT ANALYZING THE MECHANISM OF LIMONOID GLUCOSIDE ACCUMULATION IN CITRUS DEVELOPMENT OF TRANSGENIC PLANT WITH LOW LEVEL OF BITTER LIMONOIDS TROUGH ENHANCEMENT OF NATURAL DEBITTERING PROCESS .

Oases.HTS RNA ISOLATION AND cDNA LIBRARY PREPARATION BIOINFORMATICS USAGE OF SOFTWARES FOR ILLUMINA GENOME ANALYSER DATA ASSEMBLY(Velvet. 13:10 .CITRUS FRUIT TRANSCRIPTOME SEQUENCING. CLC GENOMICS) TRANSCRIPTOME COMPARISON OF CITRUS FRUIT UNIGENE WITH OTHER PLANT SPECIES SEQUENCE ANALYSIS AND FUNCTIONAL ANNOTATION THROUGH GENE ONTOLOGY(GO) CLASSIFICATION TRANSCRIPT PER MILLION(TPM)-STATISTICAL CALCULATION BETWEEN SAMPLES FOR DATA NORMALIZATION AND COMPARISON PURPOSE AND ITS EXPRESSION PROFILING DIFFERENTIAL EXPRESSION OF SELECTION OF GENES VALIDIATED BY APPLYING REAL TIME QUANTITATIVE RT-PCR(q RT-PCR) Yu et al. BMC Genomics 2012.

and to identify the compound(s) and/or protein(s) associated with them. such as antioxidants. THE CONSTRUCTION OF A GENE. to identify compounds of pharmaceutical.SCANNING AND DATA ANALYSES TRANSCRIPT PROFILING.first step in correlating gene expression with specific biological processes.specific oligonucleotide chip based on EST/genomic sequence data will expand microarray applications METABOLOMICS AND PROTEOMICS DONE TO 1. correlate between compounds and/or proteins and a given trait or process. industrial and commercial values. and unique aroma and flavor molecules Plant Molecular Biology (2005) 57:375–391 . 2. to compare between cultivars displaying different traits.CITRUS FRUIT TRANSCRIPTOME SEQUENCING USING MICROARRAY ANALYSIS RNA ISOLATION AND cDNA LIBRARY PREPARATION EST PROCESSING AND FUNCTIONAL ANNOTATION MICROARRAY HYBRIDIZATION. microarray being the high throughput method for it. 3.

CLONING AND CREATION OF TRANSGENIC VARIETIES .

Inoculation of plants with virus vectors a direct way to assay the function of specific genes without the time consuming process of plant transformation and regeneration.DEVELOPMENT OF CITRUS VIRUS VECTOR       sequencing plant genomes resulted in identification of large numbers of novel open reading frames (ORFs) using large-scale functional genomic. virus vectors for expression or silencing of plant genes.offering an interesting model system to study genes involved in growth and development of leaves and fruits. CLBV replicates in all citrus tissues. complete sequence of a new virus. useful in woody plants like citrus. the citrus leaf blotch virus (CLBV) obtained.mechanically transmissible to citrus & facilitate inoculation of engineered versions carrying the foreign genes .monopartite genome of 8747nt containing three ORFs & easy to manipulate 4. used as a vector for expression or silencing genes in citrus plants because: 1. with long juvenile periods (up to 6-8 years).viruses like CTV being phloem limited. 2. 3.accumulates mainly in meristematic tissues.

CREATION OF TRANSGENIC CITRUS FREE FROM LIMONIN BITTERNESS GENETIC TRANSFORMATION OF CITRUS EFFICIENT TRANSFORMATION IN CITRUS REQUIRED FOR TWO MAIN REASONS: TO IDENTIFY GENE FUNCTION DEVELOPMENT OF NEW AND IMPROVED CITRUS VARIETIES DEVELOPMENT OF TECHNOLOGIES FOR EFFICIENT TRANSFORMATION IN CITRUS GENE STACKING. AND WOULD TAKE A LONG TIME. THIS WOULD PROVIDE THE BENEFITS OF TRANSGENIC TRAITS BUT RESULT IN NONTRANSGENIC FRUIT. •GENE STACKING REQUIRES TRANSFORMATION WITH MULTIPLE GENES OR THE SEQUENTIAL TRANSFORMATION. MODIFYING MULTIPLE OR COMPLEX CHARACTERISTICS OF A TREE : •INSERTION OF MULTIPLE GENES. GENE REMOVAL REMOVING GENES THAT ARE NO LONGER USEFUL USEFUL WHEN THE PLANT IS SMALL AND JUVENILE. NOT REQUIRED IN THE MATURE TREE. ..

G. . ALLOW DISRUPTION OF GENE FUNCTION (KNOCKOUTS)..TECHNOLOGIES CONT……… TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS NON-ANTIBIOTIC SELECTION SYSTEMS SELECT TRANSFORMED CELLS & TISSUES WITHOUT USING ANTIBIOTICS. TARGETING GENE INSERTION BY HOMOLOGOUS RECOMBINATION INTO THE CITRUS GENOME. GENE TARGETING.. TRANSFORMATION BY LARGE DNA INSERTS CONTAINING MULTIPLE GENES IN LINKAGE GROUPS FACILITATING POSITIONAL CLONING & FUNCTIONAL ANALYSIS INTRODUCING MULTIPLE GENES FOR SECONDARY PRODUCT PATHWAYS INTO CITRUS . RESTORATION OF FUNCTION OF DEFECTIVE GENES (GENE THERAPY) REPLACEMENT OF A GENE WITH A NEW OR ALTERED VERSION.G. MANNOSE) SELECTION COMPOUNDS BE DEVELOPED LINKAGE GROUP TRANSFORMATION. PHOSPHINOTHRICIN) AND POSITIVE (E.  NEGATIVE (E.

TECHNOLOGIES CONT. DIRECT TESTING OF PUTATIVE GENE CANDIDATES. REQUIRE A NONPATHOGENIC VIRULENT VIRAL VECTOR & A METHOD TO DISPERSE THE VIRUS.. MATURE TISSUE TRANSFORMATION. . TECHNOLOGIES FOR GENETIC TRANSFORMATION OF CITRUS PROMOTER LIBRARY. VIRAL-MEDIATED TRANSFORMATION...  REPERTOIRE OF PROMOTER & CIS-ACTING ELEMENTS CONTROLING GENE EXPRESSION. AT SPECIFIC DEVELOPMENTAL STAGES. BYPASS THE LONG JUVENILITY PERIOD. COORDINATION OF APPROPRIATE EXPRESSION LEVELS IN SPECIFIC TISSUES OR CELL TYPES. PRESUMABLY AN INSECT VECTOR. PHENOTYPE OF THE TREE MODIFIED BY SIMPLY DEPLOYING A DIFFERENT ENGINEERED FORM OF THE VIRUS. AND UNDER VARIOUS ENVIRONMENTAL INDUCTION CONDITIONS.

APPLICATIONS OF THE BITTER PRINCIPLES .

8285-8294 .Obacuone. Information as such can be used to evaluate existing classification schemes or to modify those schemes J. 55. Iso-obacunoic acid and ichangin Limonoids acts as insecticides against Colorado beetle. Larvicidal effects.BIOLOGICAL ACTIVITY OF CITRUS LIMONOIDS Anticarcinogenesis Activity (Inducers for Glutathione STransferase Activity) Antifeedant Activity Taxonomic Markers Citrus limonoids posses furan moiety attached to the D-ring at the C-17 position that induces GST activity. Major limonoids with GST activity includes nomilin. spruce budworm and tobacco bud worm Can be used as taxonomic markers as certain biosynthetic pathways are unique to species and genus. Food chem 2007. fall armyworm. AdUlt repellent and oviposition deterrent effect of limonoids. Agric. corn earworm.

including flavonoids and limonoids. Agric. J. expanded the anecdotal evidence that secondary metabolites. 8285-8294 . 55. could function endogenously to lower LDL cholesterol. They inhibit viral replication. limonin and nomilin have also been evaluated for their capacity to act as antiretroviral agents.Cont…… HYPOCHOLESTEROLEMIC ACTIVITY ANTIVIRAL ACTIVITY The results of the rabbit study. Food chem 2007. themechanism of action being the inhibition of in Vitro HIV-1 protease activity.

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