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Chlorophylls:ChemistryandBiologicalFunctions

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269

Chlorophylls: Chemistry and Biological FunctionsSunil Pareek,1∗ Narashans Alok Sagar,1 Sunil Sharma,1 Vinay Kumar,1 Tripti Agarwal,1

Gustavo A. González-Aguilar,2 and Elhadi M. Yahia3

1Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Ministry of FoodProcessing Industries, Kundli, Sonepat, Haryana, India2Technology of Food of Vegetable Origin, Research Center for Food and Development, Hermosillo, Sonora, Mexico3Faculty of Natural Sciences, Autonomous University of Querétaro, Avenida de las Ciencias s/n, Juriquilla, Querétaro, Mexico

14.1 Introduction

Chlorophylls are unique pigmentswith green color and arefound in diverse plants, algae, and cyanobacteria (Inanc,2011). The term chlorophyll is derived from the Greekchloros meaning “green” and phyllon meaning “leaf.” Iso­lation and naming of the chlorophyll was first carried outby Joseph Bienaimé Caventou (French pharmacist) andPierre-Joseph Pelletier (French chemist) in 1817 (Gopiet al., 2014).Chlorophyll ismadeupof carbon andnitrogenatoms along with a magnesium ion in central position.Chlorophyll is found in almost every green part of plants,i.e. leaves and stem, within the chloroplast, the mainorganellewhich contains thehighest amount.Chloroplastsare found in the mesophyll layer, in the middle of plantleaves. Chloroplasts possess thylakoid membranes whichcontain green chlorophyll pigment. Chloroplast can bereferred to as the “food factory” of the plant cell because itproduces energy and glucose for the whole plant in asso­ciation with CO2, water, and sunlight.The name “chlorophyll” was first given to the chloro­

plast of higher plants only, but later it was extended to allphotosynthetic porphyrin pigments (Vernon and Seely,1966). It comes under the special class of compoundscalled tetrapyrroles because it contains four pyrrole ringsjoined together with a covalent bond, as are vitamin B12and the heme molecule (Willows, 2004).

14.1.1 Importance

The main source of life on earth is the solar energy that iscaptured by green plants, algae, and various photosynthetic

∗Corresponding author.

bacteria. Although there are different photosynthetic pig­ments such as carotenoids and phycobilins which entrapsolar radiation, chlorophyll is the most important of thesemolecules. It converts solar energy into chemical energythat is used to build essential carbohydrate molecules(glucose) which are used as food source for the whole plant(Hynninen and Leppakases, 2002). The process can bedescribed by the equation shown in Figure 14.1.Broadly, the conversion process of solar energy into

chemical energy is called photosynthesis. Chlorophyllpigment absorbs blue light and red light of solar radiationat 430 nm and 660 nm, respectively, and it reflects thegreen spectrum (Inanc, 2011) (Figure 14.2).

14.1.2 Types and Distribution of Chlorophylls

The numbers of naturally occurring chlorophylls may notyet be fully known. Chlorophyll a and chlorophyll b arethe main components of photosystems in photosyntheticorganisms. The types of different chlorophyll pigmentspresent in nature in decreasing order of importance aregiven in Table 14.1.Initially, chlorophyll was classified into four– chlorophyll

a, chlorophyll b, chlorophyll c, and chlorophyll d (Vernonand Seely, 1966) – but later a new type of chlorophyll wasdiscovered within stromatolite (a hard rock structure madeby cyanobacteria) in western Australia, which was namedchlorophyll f. Thus eventually chlorophyll was divided intofive classes, a, b, c, d, and f.

14.1.2.1 Chlorophyll aThis type of chlorophyll is found in almost all photo­synthetic organisms, i.e. plants, algae, cyanobacteria, and

Fruit and Vegetable Phytochemicals: Chemistry and Human Health, Volume I, Second Edition. Edited by Elhadi M. Yahia.© 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

270 Fruit and Vegetable Phytochemicals

Figure 14.1 Photosynthetic reaction.

aquatic species (Jordan et al., 2001; Nakamura et al., 2003).Previously it had been called chlorophyll α. It is found in alllight harvesting complexes (LHCs) and both reactioncenters (RCs) in organisms, photosystem I (PS I) andphotosystem II (PS II). It absorbs mainly red light fromthe solar spectrum; the absorption peak is captured at420 nm and 660nm in organic solvents and at 453 nm and670–480nm in photosynthetic cells (in vivo). It works asprimary donor in the RCs of PS I and PS II (Scheer, 1991).Subsequently, two types of chlorophyll a, Ca 670 andCa

680, were identified which are responsible for absorptionof different wavelengths from the light spectrum (Frenchet al., 1972). This observation was confirmed by curveanalysis of the absorption spectra of many plants andalgae. Various experiments were conducted, and theresults reported were that Ca 670 possessed greaterhalf-width than Ca 680 in the fraction in PS I, and Ca680 possessed greater half-width than Ca 670 in PS II(French et al., 1969a, 1969b; French 1970).

14.1.2.2 Chlorophyll bPreviously, this was called chlorophyll β. Chlorophyll bhas been confirmed to be found in green algae and also inhigher plants. It assists chlorophyll a in the photo­synthesis process. This pigment has yellow color in itsnatural state but absorbs blue light from the whole solarspectrum. For chlorophyll b the characteristic absorptionpeak was observed at 453 nm and 625 nm in vitro and at480 nm and 650 nm in vivo (Strain et al., 1963).

14.1.2.3 Chlorophyll cChlorophyll c is a brownish-golden colored pigment thataccompanies chlorophyll a in the photosynthesis process

Table 14.1 Types of different chlorophylls in nature, in order ofabundance from most (1) to least (7).

Abundance Chlorophyll types

1 Chlorophyll a

2 Chlorophyll b

3 Chlorophyll c

4 Chlorophyll d

5 Chlorophyll f

6 Protochlorophyll

7 Bacteriochlorophyll

8 Chlorobium chlorophyll

as an accessory pigment. It has three subclasses, namedchlorophyll c1, c2, and c3, which have been found invarious algae (Beale, 1999). This pigment is widely assim­ilated in different marine organisms like diatoms, brownalgae, and other marine algae (Smith and Benitez, 1955;Strain et al., 1963). The absorption peak of chlorophyll cfor the photosynthetic spectrum was obtained at 445 nmand 625 nm in organic solvent and 645 nm in vivo.

14.1.2.4 Chlorophyll dChlorophyll d is the minor chlorophyll that was identifiedin red algae (Rhodophyta) by Strain (1958). It captures theextreme red end of the spectrum of sunlight. The absorp­tion spectra were obtained for chlorophyll d at 450 nmand 690 nm in vitro conditions and up to 740 nm on redband in vivo. It can be prepared in the laboratory with thehelp of permanganate by the oxidation of chlorophyll a(Holt and Morley, 1959; Holt, 1961).

14.1.2.5 Chlorophyll fChlorophyll f was the last main chlorophyll to be dis­covered. It was revealed in cyanobacteria from the deepstromatolites in the western region of Australia by Min

Figure 14.2 Light absorption of different photosyntheticpigments.

27114 Chlorophylls: Chemistry and Biological Functions

Chen and his colleagues. Among all known types ofchlorophyll, it is chlorophyll f that can utilize the lowestsolar light from the extreme end of the infrared spec­trum for photosynthesis. The in vitro absorption andfluorescence of chlorophyll f was obtained at 706 nmand 722 nm, respectively (Chen et al., 2010). Min Chenhighlighted the need for a revised view of the dynamicrole of chlorophyll, as “discovering this new chlorophyllhas completely overturned the traditional notion thatphotosynthesis needs high energy light.” However, allpigments are often considered accessory pigmentsexcept chlorophyll a.In addition to the chlorophyll types described above,

some other chlorophylls are found in nature, as discussedin the following sections.

14.1.2.6 Chlorophyll eChlorophyll e is a rare type, which was reported in golden-yellow algae named Vaucheria hamata and Tribonemabombycinum (Eugene and Govindjee, 1969). Its properworking mechanism has not been revealed clearly.

14.1.2.7 ProtochlorophyllProtochlorophyll occurs in very small amounts alongwithpheoporphyrin a and protochlorophyllide. It is found inpumpkin seeds at the inner coat and in the dark-grownyellow leaves of seedlings (Madsen, 1963).

14.1.2.8 BacteriochlorophyllBacteriochlorophyll is the main chlorophyll of variousphotosynthetic bacteria (French, 1969b; Sistrom andClayton, 1964; Jensen et al., 1964). Several forms ofbacteriochlorophylls have been reported, namely a, b, c,d, e, and g (Eimhjellen et al., 1963; Scheer, 1991).

14.1.2.9 Chlorobium ChlorophyllsThese are abundantly found in Chlorobacteriaceae(green-sulfur bacteria). Chlorobium chlorophylls some­times work in association with bacteriochlorophylls(Kaplan and Silberman, 1959; Moshentseva and Kondra­teva, 1962; Mathewson et al., 1963).

14.1.3 Distribution of Chlorophylls amongPhotosynthetic Organisms

The distribution of chlorophylls in their natural state issummarized in Table 14.2 (Scheer, 1991; Chen et al.,2010).

Table 14.2 Distribution of different chlorophylls in nature.

Types of chlorophylls Presence and occurrence

Chlorophyll a All photosynthetic plants (excludingbacteria)

Chlorophyll b Higher plants and various green algae

Chlorophyll c Brown algae and diatoms

Chlorophyll d Various red algae, reported in Rhodophyta

Chlorophyll e Golden-yellow algae (Vaucheria hamata)

Chlorophyll f Cyanobacteria

Protochlorophyll Found in yellow leaves of seedlings(grown in dark) and seed coat of pumpkin

Bacteriochlorophyll a Purple and green bacteria

Bacteriochlorophyll b Purple bacterium (Rhodopseudomonas)

Bacteriochlorophyll c Reported in Chloroflexaceae,Chlorobiaceae

Bacteriochlorophyll d Reported in Chloroflexaceae,Chlorobiaceae

Bacteriochlorophyll e Reported in Chloroflexaceae,Chlorobiaceae

Bacteriochlorophyll g Discovered in Heliobacterium chlorum

Chlorobium Green bacteriachlorophylls

14.2 Chemistry of Chlorophylls

The porphyrin unit has a very crucial role in naturebecause it participates in the fundamental skeleton ofchlorophyll (Eugene and Govindjee, 1969). Manyresearchers have performed various experiments, andboth analyzed the chemical properties and elucidatedthe structure of chlorophyll molecules (Aronoff, 1966;Seely, 1966). Research has revealed that the chlorophyllsare tetrapyrroles, a cyclic form of porphyrin and chlorin(the parent molecule of all chlorophylls). This cyclic formcreates an isocyclic ring with the help of ��CH bridges.Chemically, chlorophylls possess a magnesium ion in thecentral position which is found connected with the tetra­pyrrole ring (Scheer, 1991). Moreover, chlorophylls arehydrophobic molecules because they contain phytol, anesterified isoprenoid C20 alcohol. The phytol (C20H30OH)possesses a double bond in the trans configuration (Gross,1991).

14.2.1 Structures of Different Chlorophylls

Chemically, the basic skeleton of chlorophyll is composedof a cyclic tetrapyrrole ring, which is a large planarstructure of a symmetric arrangement in which the

272 Fruit and Vegetable Phytochemicals

four pyrrole rings are joined together bymethine (��CH=)bridges, and four nitrogen atoms are coordinated with acentral metal atom. In addition, they have a phytol groupthat confers a hydrophobic characteristic; the metalbound to the chlorophylls is magnesium. Therefore,this structure contains a chromophore of several conju­gated double bonds responsible for absorbing light in thevisible region, i.e. red (peak at 670–680 nm) and blue(peak at 435–455 nm). The reflection and/or transmissionof the non-absorbed green light (intermediate wave­length) give the characteristic green color to plants andchlorophyll solutions. Higher plants, ferns, mosses, greenalgae, and the prokaryotic organism prochloron only havetwo chlorophylls (a and b); the remaining chlorophylls are

present in algae and bacteria. The structures of differentchlorophylls are described below:

14.2.1.1 Chlorophyll aThe molecular formula of chlorophyll a isC55H72MgN4O5. It contains a chlorin ring in which amagnesium ion is surrounded centrally by four nitrogenatoms (Taiz et al., 2006) (the structures of the differentchlorophylls are shown in Figure 14.3). The side chains ofvarious chlorophyll molecules determine the charactersof other chlorophyll types and create changes in theabsorption spectrum of solar light (Niedzwiedzki andBlankenship, 2010). Chlorophyll a possesses a long

Figure 14.3 Structures of different chlorophylls.

27314 Chlorophylls: Chemistry and Biological Functions

hydrophobic tail that anchors the molecule to alternativehydrophobic proteins within the thylakoid membrane ofthe plastid (Raven et al., 2005).

14.2.1.2 Chlorophyll bThe chemical formula of chlorophyll b is C55H70MgN4O6.

There is only a minor difference between the structures ofchlorophyll a and chlorophyll b: in the latter, a ��CHOgroup is found in place of ��CH3 at the C-7 position(Figure 14.3).

14.2.1.3 Chlorophyll cChlorophyll c was confirmed as a mixture of magnesiumtetradehydro- and hexadehydropheoporphyrin a3 mono-methyl ester (Ia, Ib) (Dougherty et al., 1970). It has threesub classes, c1, c2, and c3. Their respective formulas areC35H30MgN4O5, C35H28MgN4O5, and C36H28MgN4O7.The c1 is considered the most common form of chloro­phyll c; its structure differs from c2 in that it contains anethyl group in place of a vinyl group at the C-8 position(Figure 14.3).

14.2.1.4 Chlorophyll dThemolecular formula of chlorophyll d is C54H70MgN4O6.The structure of chlorophyll d was elucidated by Manningand Strain (1943). They revealed that it differs from chlo­rophyll a in that it has a formyl group in place of divinylgroup in ring A of porphyrin (Larkum and Kuhl, 2005)(Figure 14.3).

14.2.1.5 Chlorophyll fThe molecular structure of chlorophyll f is similar to thatof chlorophyll b, namely C55H70MgN4O6. However, afteranalysis using nuclear magnetic resonance (NMR), massspectroscopy (MS), and high-performance liquid chro­matography (HPLC), amajor difference was revealed: thatis, the existence of ��CHO at the C-2 position of theporphyrin ring (Figure 14.3) (Willows et al., 2013).

14.2.2 Synthesis of Chlorophyll

Chlorophyll synthesis is a very crucial mechanism for theexistence of photosynthetic organisms as well as of othercreatures that are dependent on them. The chlorophyllbiosynthesis process is highly complicated because of thecomplex combination of different enzymes and the manyresulting compounds.The C-5 pathway, a complex biosynthetic pathway, is

responsible for naturally synthesizing the chlorophyll and

other tetrapyrrole pigments because this pathway pro­duces key precursors and the intermediate C-5 com­pound δ-aminolevulinic acid (ALA) (Wettstein et al.,1995; Tanaka and Tanaka, 2007). Chlorophyll bio­synthesis is depicted in Figure 14.4.The whole biosynthetic pathway of chlorophyll can be

elaborated in the steps described in the following sections.

14.2.2.1 Forming of 5-aminolevulinic acid (ALA)The first step in the C-5 pathway is glutamate activation byt-RNAGlu. A similar step is found in protein synthesis forglutamate incorporation (Hori and Kumar, 1996; Kumaret al., 1996). The second step is the formation of glutamyl-1­semialdehyde by the reduction of glutamyl t-RNAGlu, cata­lyzed by glutamyl t-RNAGlu reductase. In this step, t-RNA-Glu is released. It has been revealed that illuminationincreases the activity of glutamyl t-RNAGlu reductase inseedling cotyledons of green cucumber (Masuda et al.,1996). The final step is the conversion of glutamate-1­semialdehyde into ALA by the transamination process.The C-5 pathway occurs in the plastids and is the onlyprocess that has been reported in plants. Apart from plants,ALA synthesis also takes place in animals and fungi, but thepathway is different and is known as the C-4 pathway. Thispathway is found in mitochondria (Drechsler-Thielmannet al., 1993; Weinstein et al., 1993).

14.2.2.2 Formation of Protoporphyrin from ALAIn this process, two ALAmolecules create themonopyrroleporphobilinogen via the enzyme ALA dehydratase. Thenext step consists of the formation of hydroxymethylbilane,a tetrapyrrole from porphobilinogen. Four molecules ofporphobilinogen are converted by the enzyme porphobili­nogen deaminase (hydroxymethylbilane synthase). Thecondensation reaction takes place in this conversion(Jordan, 1991). The structure of the hydroxymethylbilanesynthase enzymewas elucidatedfirst among all the enzymesof tetrapyrrole biosynthesis (Louie et al., 1992).In the next step, uroporphyrinogen III is formed from

hydroxymethylbilane; the reaction is catalyzed by uropor­phyrinogen III synthase. Uroporphyrinogen III is thefirst cyclic compound of the chlorophyll biosynthesis path­way (Leeper, 1994). The subsequent steps involve thedecarboxylation of acetate chains of uroporphyrinogen IIIwhich gives coproporphyrinogen III, catalyzed by uropor­phyrinogen decarboxylase. Protoporphyrinogen IX forma­tion takesplace fromcoproporphyrinogen III by anoxidativedecarboxylation reaction. Coproporphyrinogen oxidase isthe catalytic enzyme for this decarboxylation reaction.The last step, proporphyrinogen formation, is catalyzed

by protoporphyrinogen oxidase, which has two features.First, this is the only enzyme of plant porphyrin

274 Fruit and Vegetable Phytochemicals

Figure 14.4 Chlorophyll biosynthesis.

biosynthesis that is found in two organelles, mitochondriaand plastid (Jacobs et al., 1982; Jacobs and Jacobs, 1984).Second, protoporphyrinogen is a colorless compound, asare previously formed intermediate compounds; proto­porphyrinogen oxidase produces the first pigment, pro­toporphyrin, of the chlorophyll biosynthesis process.

14.2.2.3 Addition of Magnesium IonThe identification and analysis of photosynthetic genecluster, a 46 kbp gene sequence, was the real discovery inthe field of chlorophyll and bacteriophyll biosyntheticpathways. This gene cluster was identified in Rhodobacter

capsulatus which possessed inter alia the genes respon­sible for the production of all the enzymes related to Mgbranch formation in the biosynthetic pathway.The insertion of the magnesium ion is catalyzed by the

Mg chelatase enzymewhich needs ATP for activation.Mgchelatase is a significant bottleneck enzyme that specifi­cally binds to Mg ions only (Castelfranco et al., 1979).

14.2.2.4 Conversion of Magnesium Protoporphyrin intoProtochlorophyllideThis step involves esterification at position 6 of thepropionic acid side chain to produce Mg protoporphyrin

27514 Chlorophylls: Chemistry and Biological Functions

IX monoethyl ester; the reaction is catalyzed by S-adeno- Table 14.3 Different conditions of chlorophyll degradation in

syl-L-methionine:Mg-protoporphyrin-O-methyltransfer­ plants along with the parts affected.

ase. The catalytic mechanism of this enzyme wasconfirmed as a pingpong mechanism in wheat (Richardset al., 1987) and as an ordered mechanism in Rhodobacter

Condition and factors Part affected (chlorophyllbreakdown)

sphaeroides (Bollivar et al., 1994). Various investigationshave demonstrated thatMg protoporphyrin IX works as asubstrate for the methylation step. After that, an isocyclic

At the point of life cycle initiation

1 Germination of seed Cotyledons loss

ring is formed that constitutes divinyl protochlorophyl­ 2 Initiation of first leaves

lide. The divinyl protochlorophyllide (Mg-2,4-divinyl­ 3 Maturation of inflorescence Bracts loss

pheoporphyrin a5) (MgDVP) is created by β-oxidation 4 Fruit ripening Green color loss of fruitand cyclization at position 6 in ring C. This reaction iscatalyzed by Mg protoporphyrin IX oxidative cyclase orMPE oxidative cyclase.

During the life cycle

Turnover (continuous synthesis) Degradation of chloroplastparts

Premature death

14.2.2.5 Reduction of Protochlorophyllide into Biotic (disease, harvesting, Whole plant

Chlorophyllide grazing, etc.)

The photoreduction process occurs at this step, which Climatic (UV rays, temperature, Whole plant

converts protochlorophyllide into chlorophyllide a, and darkness)

the macrocycle (the dihydroporphyrin or chlorin) is Edaphic (deficiency of water and Whole plantdeveloped. NADPH protochlorophyllide oxidoreductase minerals)

is the enzyme responsible for photoreduction. A chro­mophore is found in the macrocycle that provides the Source: Hendry et al., 1987.

green color to chlorophyll a. Prothylakoids of etioplastscontain this enzyme.

biological phenomena that degrade chlorophylls such asflowering, fruit ripening, germination, bleaching, etc.

14.2.2.6 Esterification of Chlorophyllide a These mechanisms are considered to be part of the agingThis is the last step in the process of chlorophyll a processes. However, the chlorophyll degradation processbiosynthesis. Here, phytol, a C-20 isoprenoid alcohol, is not fixed or the same in all cases (Gossauer and Engel,reacts with 7-propionic acid residue by an esterification 1996). Table 14.3 demonstrates some natural and biolog­process. Chlorophyll dihydrogeranylgeranyl ester and ical mechanisms in which chlorophyll breakdown occurstetrahydrogeranylgeranyl ester are formed as intermedi­ (Hendry et al., 1987).ates when the geranyl geraniol is esterified by chlorophyll Two main steps are involved in the chlorophyll degra­a, which is transformed into phytol by three gradual steps. dation process. These are phytol removal by chlorophyl­

lase, and removal of Mg catalyzed by magnesiumdechelatase, but these two steps may be different in plants

14.2.2.7 Biosynthesis of Chlorophyll b via Chlorophyll a (Amir-Shapira et al., 1987). Chlorophyllase is believed toAs discussed previously, the only difference between be present in thylakoid membrane of plastids but it haschlorophyll a and chlorophyll b is the presence of an also been detected near the light harvesting complexaldehyde (formyl) group at ring B in place of a 3-methyl (Schellenberg and Matile, 1995; Brandis et al., 1996).group. In the conversion process of chlorophyll b, trans- The phytol residue is esterified with acetic acid, since itformation occurs (Espineda et al., 1999). Earlier experi­ is not stored as free alcohol (Bortlik et al., 1990).ments suggested that chlorophyll a was the precursor of The opening of the chlorophyll macrocycle by an oxida­chlorophyll b (Rudiger, 1997). tion process is the key step in chlorophyll degradation,

which produces a different resulting structure (Matile andKräutler, 1995; Gossauer and Engel, 1996; Mühlecker and

14.2.3 Degradation or Catabolism of Chlorophyll Kräutler, 1996; Engel et al., 1996). The enzyme which hasthe key role here is called pheophorbide oxygenase (Curty

The global degradation of chlorophyll is found to be et al., 1995), which is localized in gerontoplast (senescentalways equal to the global synthesis of chlorophyll (Hen­ plastids) membrane (Matile and Schellenberg, 1996).dry et al., 1987). Senescence of plant leaves involves a Many structures of chlorophyll catabolites have beenchlorophyll degradation process, and there are various reported in algae and higher plants (Mühlecker and

276 Fruit and Vegetable Phytochemicals

Kräutler, 1996), algae (Engel et al., 1991, 1993, 1996;Iturraspe et al., 1994). This results in oxygenase catalysisand formation of a structure having 19-formyl-1(21H,22H)bilinone derivate.Catabolic products derived from chlorophyll a and

chlorophyll b have been reported among algae. Theresponsible modification behind this is the esterificationhydrolysis of the methyl ester group at ring E. In higherplants, catabolites are hydrogenated in a few metabolicsteps, and after ring opening a reductase reaction isindicated for higher plants (Gossauer and Engel, 1996).Subsequently, modification includes hydroxylationreactions, found only at ethyl group of ring B in Brassicanapus and at side chains of ring A and B in Hordeumvulgare. This change facilitates transport into storage andvacuole for further formation of malonic acid ester andβ-glucoside in Brassica napus.

14.3 Presence and Distribution in Fruitsand Vegetables

Chlorophyll is mostly composed of a lipophilic derivativewhich includes chlorophyll a and b and is found in freshfruits and green vegetables (Kimura and Rodriguez-Amaya, 2002). It is frequently found in the range 1000to 2000 ppm in some species (Khachik et al., 1986). Chlo­rophyll is awidely distributed plant pigment (Table 14.4) inall green fruits and vegetables, and it is the most abundantpigment on earth. Chlorophylls are registered as foodadditives used to give color and other properties to avariety of foods and green beverages.Generally, chlorophyll comprises 0.6% to 1.6% of plants

on a dry weight basis; however, wide variation has beennoted. In nature, six classes of chlorophyll exist in plantsand photosynthetic organisms: a, b, c, d, e, and f. However,only a and b are predominant in angiospermic plants,while c, d, e, and f are found in different photosyntheticalgal and diatomic species. Chlorophyll a is predominantover chlorophyll b by a 3:1 margin. Lichtenthaler (1968)surveyed the molar ratio of chlorophyll a to b in higherplants in 24 species, and the range was from 2.56 to 3.45.The a:b ratio was approximately 3 depending upon spe­cies. But the ratio can vary due to different environmental

Table 14.4 Distribution of chlorophylls in living organisms.

Chlorophylltype

Occurrence Chlorophylltype

Occurrence

Chlorophyll a

Chlorophyll b

Chlorophyll c

Universal

Mostly plants

Various algae

Chlorophyll d

Chlorophyll e

Chlorophyll f

Various algae

Cyanobacteria

Cyanobacteria

factors and growth conditions, and particularly with highexposure to sunlight.The concentration of chlorophyll correlates with the

photosynthetic capacity of plants, which gives some indi­cation of the physiological status of the plant system(Gamon and Surfus, 1999).

14.4 Biological Functions ofChlorophyll

Chlorophyll is an extremely important biomolecule foundin green plants. Plants utilize chlorophyll and sunlight tomake foodmaterials. Chlorophyll is important not only asa color pigment and for its physiological role in plants, butalso for its health benefits (Liu et al., 2007).Chlorophylls have been the subject of extensive

research efforts because of their eminent role in plantphysiology and their role as derivatives in the food sector.They have a major role in interrupting diverse diseasessuch as cancer, cardiovascular, and other chronic diseases(Sangeetha and Baskaran, 2010). Chlorophyll is used forbad breath, and it reduces colostomy odor. Chlorophyllshave been used for a long time as a traditional medicinefor therapeutic functions.Humans have no potential to synthesize chlorophyll

pigment, but they are able to deposit dietary chlorophyllas absorbed or with minor alteration to its structure(Larsen and Christensen, 2005). Healthcare providersuse chlorophyll intravenously for the pain and swelling(inflammation) with pancreas problems (pancreatitis).Common sources of chlorophyll used for medicine

are alfalfa (Medicago sativa) and silkworm droppings.Early research suggests that chlorophyll not only isresponsible for green color but also can prevent lungand skin cancer by intravenous injection along with thedrug talaporfin, followed by laser therapy, in early stagesof cancer.Both natural and artificial commercial grade deriva­

tives such as copper chlorophyll have been extensivelyused for their beneficial biological activities, whichinclude wound healing, control of calcium oxalate crys­tal (Tawashi et al., 1980), and anti-inflammatory prop­erties (Bowers, 1947; Larato and Pfau, 1970). Somereports do provide sufficient evidence for the beneficialeffects of chlorophyll. However, many researchers havemade claims regarding the healing properties ofchlorophyll, but most have been disproved by the com­panies that are marketing them. Quackwatch (www.quackwatch.org) claims that no deodorant effect canpossibly occur from chlorophyll added to products suchas gum, foot powder, and cough drops (Kephart, 1955).More evidence is essential to rate the effectiveness ofchlorophyll for different biological uses.

27714 Chlorophylls: Chemistry and Biological Functions

14.5 Changes in Chlorophyll duringProcessing of Fruits and Vegetables

Chlorophyll degradation is a unique phenomenon thathappens during the processing of fruits and vegetables.Color is one of the important sensory characteristics andplays a vital role in the acceptability of food items in thefood industry. Chlorophyll a imparts blue-green colorwhereas chlorophyll b imparts yellow-green color. Theyalso possess different thermal stabilities (Steet and Tong,1996). Chlorophyll b was reported to be thermally morestable, whereas chlorophyll a was found to be thermallyunstable (Schwartz and Elbe, 1983; Canjura et al., 1991;Schwartz and Lorenzo, 1991). In the temperature range70–100 °C, green peas showed degradation of chlorophylla and b following a first-order kinetics model. The deg­radation of chlorophyll a was reported to be 12–18 timesfaster than that of chlorophyll b, and was found to beprimarily dependent upon the temperature regime duringthe processing; at the same time, this indicated the highersusceptibility of chlorophyll a in a thermal environment(Erge et al., 2008). The higher thermal stability of chloro­phyll b has been attributed to the electron-withdrawingeffect of its C-3 formyl group (Belitz and Grosch, 1987).Chlorophylls are highly susceptible to degradation dur­

ing processing conditions, resulting in food color changes(Schwartz and Elbe, 1983). The simple reaction mecha­nism that occurs with the chlorophyll molecule duringdegradation is shown in Figure 14.5. The chlorophylldegradation in foods may occur via chemical and bio­chemical reactions. Chemical reactions involve the forma­tion of pheophytin from the chlorophyll by thereplacement of magnesium ions from the porphyrinring. The magnesium replacement can occur by acidicsubstitution, by heat treatment, or after the action of Mgchelatase.Decarbomethoxylationmay occurduring strongheat treatment, leading to the conversion of pheophytin topyropheophytin (Schwartz et al., 1981; Diop et al., 2011).Kräutler and Matile (1999) and Matile et al. (1999)

described the role of chlorophyllase, Mg dechelatase,andpheophorbideaoxygenase in chlorophyll degradation.

Figure 14.5 Reactions of chlorophylls.

Among these enzymes, chlorophyllase catalyzes the firststep in chlorophyll catabolism and removes the phytolchain from the porphyrin ring to form chlorophyllide.Lipoxygenase is widely distributed in vegetables and isinvolved in off-flavor development and color loss. Thelatter is due to hydroperoxide and radical formation byoxidation of lipids, which can destroy chlorophyll andcarotenoids during frozen storage (Vamos-Vigyazo andHaard, 1981; Adams, 1991). Lopez-Ayerra et al. (1998)concluded that lipid oxidation was related to chlorophylldegradation in spinach. Peroxidases are assumed to play animportant role in chlorophyll degradation, a processaccompanying ripening and senescence in most fruitsand vegetables. Peroxidase is responsible for off-flavordevelopment during storage of canned products, especiallyof non-acidic vegetables which often exhibit high levels ofactivity. They impair not only the sensory properties and,hence, the marketability of the product, but also its nutri­tive value (Vamos-Vigyazo and Haard, 1981). Martinezet al. (2001) reported that chlorophyll degrading peroxi­dase activity in strawberry and canola seeds decreasedwithchlorophyll content in their ripening stage, while thereverse occurred in mustard.Kato and Shimizu (1985) reported that a phenolic

peroxidaseH2O2 systemwas involved in in vitro bleachingof chlorophyll. The phenolics active in the system werederivatives of monophenols having an electron-attractinggroup in the para position. Yamauchi et al. (2004) con­cluded that in peroxidase-mediated chlorophyll degrada­tion, peroxidase oxidizes the phenolic compounds withhydrogen peroxide and forms the phenoxy radical. Thephenoxy radical then oxidizes chlorophyll to colorless lowmolecular weight compounds through the formation ofC-13-hydroxychlorophyll-a and a bilirubin-like com­pound as an intermediate.It is very important to consider the factors affecting

chlorophyll degradation during the processing of fruitsand vegetables. Diverse factors are responsible for degra­dation, including exposure to dilute acids, heat, andoxygen (Tonucci and Von Elbe, 1992). Under thermalprocessing conditions, chemical and structural variations

278 Fruit and Vegetable Phytochemicals

take place in the chlorophyll which in turn lead to changesin color (Canjura et al., 1991; Heaton et al., 1996). In greencellular tissues subjected to various processing condi­tions, chlorophyll undergoes distinct types of degradationreactions. The changes in the chlorophyll molecule dur­ing heating have been discussed by Gauthier-Jaques et al.(2001). Demetalation and epimerization were observedduring heat treatment, and prolonged heating leads toadditional demethoxycarbonylation of the molecule.Demethoxycarbonylation also occurs during the canningof vegetables. Dephytylation of chlorophyll is achievedenzymatically during fermentation and storage and isoften observed together with demetalation.In food products, chlorophyll degradation studies

revealed that chlorophyll degrades to pheophorbide viapheophytins or chlorophyllide. Shwartz and Lorenzo(1990) demonstrated that chlorophyll degradation termi­nates at pheophorbide. However, studies by Matile et al.(1992) and Ginsburg et al. (1994) have shown that chlo­rophyll degradation continues beyond pheophorbide tocolorless compounds. Heaton et al. (1996) observed thatchlorophyll degradation of cabbage heads in cold storagedid not lead to pheophorbide accumulation, leaving thedegradation of pheophorbide to colorless byproducts asthe only explanation.Chlorophyll degradation has been shown to follow

different pathways depending on the commodity (Heatonet al., 1996). However, the mechanisms and kinetics ofthose reactions have been only partially characterized.Heaton and Marangoni (1996) summarized the acceptedmechanism of chlorophyll degradation in fruits and veg­etables and extended it to include the degradation ofpheophorbides into colorless compounds. Takamiyaet al. (2000), in a study on the mechanism of chlorophylldegradation using gene cloning, determined that aftersuccessful removal of phytol and Mg2+ from the chloro­phyll molecule by the use of chlorophyllase and Mgdechelatase, pheophorbide is cleaved and reduced to yielda colorless, open tetrapyrrole intermediate.Variation in pH is one of the important factors that

influence the variations in the color of chlorophylls by theconversion of chlorophylls to pheophytins (Minguez-Mosquera et al., 1989). During the processing of peasfor puree formation, three chlorophyll-linked degradedproducts were reported at high pH under high-tempera­ture, short-time conditions after the storage of puree atroom temperature (Buckle and Edwards, 1969). Schwartzand Lorenzo (1990), in a study on continuous asepticprocessing and storage, concluded that oxidation duringstorage was not a dominant factor in chlorophyll degra­dation and color loss. A major problem in thermal proc­essing of green vegetables is the continuous decrease inpH values due to acid formation. To avoid this problem,Ryan-Stoneham and Tong (2000) conducted degradation

studies in a specially designed reactor with an online pHcontrol capability. The authors concluded that the acti­vation energy was independent of pH, with a magnitudevarying between 17 and 17.5 kcal/mol for both chloro­phyll a and b. The degradation rate was found to decreaselinearly as the pH increased.It was demonstrated that chlorophylls have higher sta­

bility at alkaline pH conditions (Clydesda and Francis,1968). Gupte and Francis (1964) reported that the use ofmagnesium carbonate in combination with high-tempera­ture, short-time processing (at 150 °C) initially improvedchlorophyll retention in pureed spinach. However, theeffect was not stable during storage. Magnesium com­pounds resulted in the formation of hard white crystalsof magnesium-ammonium-phosphate (Eheart and Odubi,1973). Ahmed et al. (2002) studied the color degradationkinetics of coriander leaf puree; however, they worked onpuree at unmodified pH. The effect of pH (3–8) andmicrobial growth on green color degradation of blanchedbroccoli during storage was investigated by Gunawan andBarringer (2000). Color degradation accelerated whenbroccoli was submerged inMcIlvaine’s buffer at decreasingpH. Pheophytinization followed first-order reaction kinet­ics. The logarithmic values of reaction rate constants werelinearly correlated with the ambient pH up to pH 7. Acidscontaining abenzene ringwere found to cause a faster colorchange than acids with simple chains due to their hydro­phobicity. There is synthesis of olive-brown pheophytinsunder the acidic conditions, which interchanged the mag­nesium in the chlorophyll with the two hydrogen ions(Mangos and Berger, 1997; Van Boekel, 1999, 2000).When the process temperatures are high and the pH of

the tissue is low, pheophytins are formed in the processedvegetables (La and Von Elbe, 1990). The excessive heatingleads to the formation of pyropheophytins as a result ofremoval of the carbomethoxy group from the pheophytins(Schwartz andElbe, 1983; Canjura et al., 1991;Mangos andBerger, 1997). The treatment of broccoli juice at tempera­tures more than 60 °C leads to the degradation of chloro­phylla and b to pheophytin a andb (Weemaes et al., 1999a,1999b). Pheophorbides are formed by the loss of magne­sium from the chlorophyllides under the acidic environ­ment (Heaton et al., 1996). The hydrolysis of the phytolchain in pheophytins produced olive-brown colored pheo­phorbides (Heaton et al., 1996). Higher temperature sen­sitivity was found in mustard leaves than in spinach inrelation to color (Ahmed et al., 2004).The thermal degradation of chlorophyll pigment in

thermally processed green vegetables is an aspect of vitalimportance for the food processing sector. The colordegradation of green peas occurred when green peaswere subjected to temperatures of 70, 80, 90, and 100°C to analyze the effect of heat treatment (Erge et al.,2008). It was reported that chlorophyll a and chlorophyll b

279

followed a kinetics model of the first order during degra­dation, which was recorded using a tristimulus colorime­ter (Baardseth and Von Elbe, 1989). The blanching ofgreen peas was studied under temperatures of 70, 80, 90,and 100 °C in solutions of buffers with pH of 5.5, 6.5, and7.5. The pH effect on the degradation of chlorophyll wasnoted, and the results showed the rise in degradation rateof chlorophyll a and chlorophyll bwith the decrease in pH(Koca et al., 2007). The constant degradation rate ofchlorophyll a and chlorophyll b were highly related tothe changes in the green pea CIE a∗ color values, when theproducts were at a pH of 5.5 or 6.5. The a∗ parameter isrelated to the greenish color of the food; thus the degra­dation of chlorophylls was followed by a reduction in theCIE LAB parameter. Moyano et al. (2008) reported acorrelation between the chlorophyll content of a broadrange of virgin olive oils and their lightness.The degradation of green color of broccoli (Brassica

oleracea) by blanching at a storage temperature of 7 °Cwas carried out; the broccoli was then submerged in McIl­vaine’s buffer at a pH of 3–8, and it was observed thatchlorophyll degradation increased as the pH decreased(Gunawan and Barringer, 2000). The thermal degradationof chlorophyll and chlorophyllides in a puree of spinach attemperatures of 100 to 145 °C (2–25min) and 80 to 115 °C(2.5–39min), respectively, led to the formation of deriva­tives pheophorbides, pyropheophorbides, pheophytins, andpyropheophytins (Canjura et al., 1991). Ahmed et al. (2000)studied the effect of particle size on chlorophyll content andtotal colorof greenchili puree.Bothchlorophyll content andgreen color varied significantlywith processing temperatureand mesh size. Chlorophyll content increased withincreased mesh fineness, but decreased linearly with proc­essing temperature. Up to a 60% loss in chlorophyll wasnoted after heating at 100 °C for 15 minutes.Chlorophyll degradation and yellowing of peel in lime

(Citrus latifolia Tan.) is one of the dominant problemsworldwide. Application of UV-B treatment at 8.8 kJ/m2

led to effective delay in the reduction of chlorophyllcontent. The pheophorbide content decreased, followedby an increase in the pheophytin a content during laterstages of storage. Therefore, the study concluded thatthere was mitigation of degradation of chlorophyll by theapplication of UV-B irradiation (Srilaong et al., 2011).Pistachio (Pistacia vera L.) kernels are green in color due

to the presence of chlorophyll, which appeals to the foodindustry for its diverse uses. The degradation of the greencolor in pistachio is one of the unacceptable features. Theheat treatments applied to pistachio during roasting lead todegradation of chlorophyll a and b to pheophytins andpyropheophytins,whichultimately impacts the quality andmarket value of pistachio kernel (Pumilia et al., 2014).In the case of Thai lime (Citrus aurantifolia Swingle

‘Paan’) the postharvest hot water treatment delays

14 Chlorophylls: Chemistry and Biological Functions

chlorophyll degradation and maintains quality duringstorage. It is reported that the enzyme activities degradingchlorophylls, i.e. chlorophyllase, chlorophyll-degradingperoxidase, pheophytinase, andMg-dechelation activities,were mitigated by hot water treatment (Kaewsuksaenget al., 2015). The processing of vegetables like broccoliflorets, spinach leaves, and green peppers under highpressure does not degrade chlorophylls, whereas high-pressure, high-temperature processing leads to the degra­dation of both chlorophylls, and chlorophyllawas found tobe unstable at 70 °C compared to chlorophyll b. At 117 °Cboth chlorophylls were degraded (Sanchez et al., 2014).The blanching treatment of vegetables followed by freez­ing led to chlorophyll retention in frozen green asparagus,French bean, and peas (Lisiewska et al., 2010).A previous study related the effect of microwave and

conventional cooking on the chlorophyll pigment in peas,leek, squash, broccoli, spinach, and green beans. Except inpeas, the reported results showed chlorophyll a to bemoreresistant than chlorophyll b in five of the six vegetables.There was an increase in the pheophytins of all thevegetables after cooking. Boiled leek showed loss in chlo­rophyll a and chlorophyll b, whereas in the case of boiledandmicrowaved peas, there was retention of chlorophyll aand chlorophyll b. The formation of pheophytin a and bwas reported at higher levels in boiled squash and boiledgreen beans. Themaximum amounts of pheophytins werereported to be formed in boiled vegetables, and the mini­mum levels in microwaved vegetables (Turkmen et al.,2006). HPLC (reverse phase gradient) was used tomonitorthe effect of freezing (–22 °C) on chlorophyll a and bcontent in Padrón peppers and green beans. In the caseof unblanched beans there was considerable reduction inthe pigments within 1month, whereas after 11months thepigments remained stable; almost similar results werereported in blanched beans. Chlorophyll reductions inblanched beans were reported to be induced by blanching.The reductionof chlorophyllawas also reported inPadrónpeppers (Oruna-Concha et al., 1997).The quantitative determination of chlorophyll a and b

and pheophytins a and b under refrigerated storageconditions, followed by industrial processing of spinach,showed that pheophytins a and b were the prominentchlorophyll derivatives formed under refrigerated condi­tions at 8 °C for 3 weeks (Lopez-Ayerra et al., 1998). Theheating of spinach leaves showed degradation of chloro­phylls a and b, and the effect was found more undermicrowave cooking or blanching than under steaming orbaking. Pheophytins a and b were formed under steamedconditions, whereas under the effect of microwave cook­ing pyrochlorophylls a and b were formed (Teng andChen, 1999).The effect of salt on the degradation of visual green

color “-a” in spinach puree under a temperature range of

280 Fruit and Vegetable Phytochemicals

50–120 °C, open pan cooking, and pressure cooking, wasstudied. Salt showed a protective action against the deg­radation of the green color (Nisha et al., 2004). In cannedslices of kiwi fruit, chlorophyll was found to degrade whenheated at 100 °C for 5 minutes (Robertson, 1985).Therefore, in conclusion, chlorophyll pigments in horti­

cultural crops require very specific conditions to avoiddegradation under various processing environments. Thecongenial temperature conditions during thermal and non-thermal processing should be optimized to mitigate thedegradation of chlorophyll pigments in different vegetablesand fruits. However, under other processing steps likeblanching, the pH plays an important role in relation tothe degradation of chlorophyll pigments. The susceptibilityof degradation of chlorophyll during processing of differentvegetables is largely dependent on the different processesemployed with a particular horticultural crop. The process­ing methodology along with its intensity during processingof vegetables or fruits is also a crucial factorwhich affects thedegradation of chlorophylls in horticultural crops.

14.6 Conclusions and ResearchNeeds

Chlorophyll plays a vital role in the photosyntheticprocesses in plants universally. Its biological role for

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