Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

Подождите немного. Документ загружается.

characterized by bad smells, dripping out, and

hygienic and preparation problems at the consumer

level. For processed fish (smoked, salted, pickled),

vacuum packaging is commonly utilized because it

limits the spoilage factors of this kind of product

(mainly fat oxidation and mold growth). The storage

life of vacuum-packaged processed fish depends upon

the water activity level, ranging between 2 and 3

weeks for slightly salted fish and a few months for

highly salted fish.

Prepared Foods

0078 Vacuum packaging is also utilized for a wide range of

prepared foods (delicatessen products, cooked meats

and poultry, prepared meals, salads).

0079 The spoilage mechanism and perishability of these

products are related to their chemical composition

(pH, additives) and heat treatment (pasteurization),

and therefore, their storage life is very variable,

ranging from 2 weeks to several months.

0080 An interesting application of vacuum packaging to

prepared foods is the cook-in-the-package technique,

which implies packaging under vacuum of raw or

partially cooked foods that are cooked inside the

package, pasteurized (when required), chilled and

stored, and reheated and unpackaged at the time of

consumption. This technology allows the rationaliza-

tion of meal preparation in central units (e.g., in

institutional kitchens and restaurant chains) because

of the storage life of the prepared meal (1 week or

more) and it has also been introduced to food-pro-

cessing plants to prepare meals to be distributed

chilled at the retail level.

See also: Cheeses: Cheeses with ‘Eyes’; Controlled-

atmosphere Storage: Applications for Bulk Storage of

Foodstuffs; Lactic Acid Bacteria; Meat: Structure;

Oxidation of Food Components; Spoilage: Bacterial

Spoilage; Molds in Spoilage

Further Reading

Brody AL (ed.) (1989) Controlled/Modified Atmosphere/

Vacuum Packaging of Foods. Trumball, CT: Food and

Nutrition Press.

Farber JM and Dodds KL (ed.) (1995) Principles of Modi-

fied Atmosphere and Sous Vide Product Packaging.

Lancaster, PA: Technomic.

Kadoya T (ed.) (1990) Food Packaging. San Diego, CA:

Academic Press.

Mathlouthi M (1994) Food Packaging and Preservation.

London: Blackie Academic & Professional.

Renerre M and Labadie J (1993) Fresh Meat Packaging and

Meat Quality. Proceedings of the 39th International

Congress of Meat Science and Technology. Calgara.

Robertson GL (1992) Food Packaging – Principles and

Practice. New York: Marcel Dekker.

CHLOROPHYL

H G Daood, Central Food Research Institute,

Budapest, Hungary

Introduction

0001 The green color in nature results from the presence of

chlorophyll pigments that occur in the chloroplasts of

plant tissues. The chloroplasts hold chlorophylls close

to the cell wall and carry out photosynthesis on bio-

membranes. In coordination with other pigments like

carotenoids, chlorophylls play an important role in

the metabolism of light energy and catalyze synthesis

of carbohydrates, the key metabolites of the metabol-

ism of living cells.

0002 Because of their widespread occurrence in a variety

of plant products, chlorophylls play a vital role in

the acceptability of food commodities. Chlorophyll-

containing products have the potential to be used as a

natural food colorant and nutritional supplement.

The changes in chlorophyll content and composition

can be used as an indication for some physiological

process and orders. Recently, biological studies have

postulated the anticarcinogenic and antimutagenetic

effect of chlorophyll-related compounds. This article

covers topics concerning biosynthesis, biochemical

conversion, response to technological factors and

the antioxidant–oxidant role of food chlorophylls.

Biosynthesis

0003Chlorophylls in plant foods are synthesized from

d-aminolevulinic acid (ALA), whose role is demon-

strated in the biosynthesis of the tetrapyrrole nucleus.

Once ALA is formed, two molecules condense to

form porphobilinogen (PBG) by converting an ali-

phatic compound into an aromatic one. The head-

to-tail condensation of four molecules of PBG results

in the formation of the first tetrapyrrole intermediate,

1196 CHLOROPHYL

a linear hydroxymethylbilane porphyrin. This linear

molecule is enzymatically closed to form the first

cyclic tetrapyrrole, uroporphyrinogen III. By the

action of decarboxylase-catalyzed decarboxylation

of the acetic acid groups on the pyrrole rings A, B,

C, and D, uroporphyrinogen III is converted to copro-

porphyrinogen III. Propionic acid group oxidation

and aromatization of the oxidized intermediate

forming protoporphyrin IX follow the last step.

0004 The next phase of chlorophyll biosynthesis starts

with the chelation of protoporphyrin IX, mediated by

Mg chelatase. This is followed by methylation of one

of the propionic acid residues to form Mg-protopor-

phyrin-n-monomethyl ester (Figure 1). The last phase

is characterized by the conversion of Mg-protopor-

pyrin-Me to protochlorophyllide and the protochlor-

ophyllide to chlorophyllide through reduction of the

vinyl substitutuent in the side chain of the pyrrole ring

B, followed by oxidation and photoreduction of the D

ring. The final step is esterification of the propionate

substituent on pyrrole ring D with geranyl geraniol,

which is then reduced to phytyl.

0005 The first enzyme involved in tetrapyrrole synthesis,

aminolevulinic acid synthetase, regulates chlorophyll

biosynthesis. During maturation of some plant seeds

chlorophyll biosynthesis may be stimulated by

ethylene in the dark.

0006Concerning biosynthesis of chlorophyll b, old and

more recent investigations gave convincing evidence

that chlorophyll b is formed from chlorophyll a. The

kinetics of

C pulse labeling indicate that chloro-

phyll a is the precursor of chlorophyll b. In light

pulse experiments, chlorophyll b formation was

shown to take place stoiciometrically at the expense

of chlorophyll(ide) a. In homogenate of plant leaves,

in the presence of NADPþ , labelled chlorophyll b is

formed from [

C]chlorophyll a. The label locates in

the tetrapyrrole and the phytol portion of the chloro-

phyll b, ruling out the possibility that the only carbon

transfer is by transphytylation reaction.

Biochemical conversion

0007Degradation of chlorophyll usually happens all the

way from ripeness to processing and storage of plant-

derived foods. Although the mechanism of degrad-

ation is fragmentary, chlorophylls are degraded to

colorless products.

0008Initially, chlorophylls are degraded to the phytyl-

free chlorophyllides by the enzyme chlorophyllase

COOH

OCH

HOOC

OCH

HOOC

OCH

Mg-protoporphyrin IX Mg-protoporphyrin IX

monomethyl ester

Mg-2,4-divinyl-pheoporphyrin Protochlorophyllide

COOH

Protochlorophyllide Chlorophyllide

fig0001 Figure 1 Last steps of chlorophyll biosynthesis in higher plants. Adapted from Castelfranco PA and Beale SI (1981) Chlorophyll

biosynthesis. In: Hatch MD and Boardman NK (eds) The Biochemistry of Plants. New York: Academic Press.

CHLOROPHYL 1197

(chlorophyll chlorophyllide hydrolase, EC3.1.1.14).

The enzyme is located in the lipid envelope of the

thylakoid membranes as an intrinsic membrane glyco-

protein. Although chlorophyllase activity increases

during ripening of some fruits and vegetables and

parallels the respiratory climacteric, it is not affected

by the endogenous ethylene. The enzyme activity

remains even during storage and after processing of

some plant products such as green tea. Generally,

enzyme activity reaches a maximum at both the begin-

ning and end of the vegetative growth phase.

0009 Small amounts of chlorophyllide a and b can

be found during the initial growth period, which

coincides with a phase of great chlorophyll synthesis.

Later, the synthetic and degeneration mechanisms

may overlap, making the detection of phytyl-free

compounds impossible. In ripening fruits and vege-

tables, the presence of allomerized chlorophyll de-

rivatives suggests that, in addition to dephytylation

by chlorophyllase, chlorophylls can be degraded by

other oxidative systems involving active oxygen.

0010 A chlorophyllide a-degrading enzyme is present in

green tissues. It catalyzes the breakdown of chloro-

phyllide a in the presence of H

and 2,4-dichloro-

phenol.

0011 Biochemical conversion of chlorophylls to pheo-

phytins or pheophorbides is initiated by the coordin-

ating function of Mg-dechelatase (the enzyme

catalyzes dechelation of Mg ion from chlorophyllins

to form different pheophytins) and decarbometh-

oxylase. It is evident from the occurrence of 13-2-

hydroxy-chlorophylls, hydroxy-lactone-chlorophylls,

and unknown intermediates in fresh or stored green-

colored plant products that more than one enzyme

system is involved in the chlorophyll degradation

mechanism.

0012 Of the biological oxidants, lipoxygenase

(EC1.12.13.11) contributes to the oxidative degrad-

ation of chlorophyll. The enzyme is well known to

have pigment-bleaching activity of its isoenzyme-

1 and isoenzyme-2, with the former being more

active. Lipoxygenase cooxidizes chlorophylls through

the oxidation of 1,2-pentadiene containing unsatur-

ated fatty acids and results in the rapid formation

of free radicals and, in the presence of molecular

oxygen, in the creation of very reactive peroxy rad-

icals. The results of many biochemical studies con-

firm that the chlorophyll-bleaching reaction requires

an intermediate formed during peroxidation of poly-

unsaturated fatty acids by the isoenzyme. This type

of degradation is responsible for the low storage

stability of a variety of plant products. Furthermore,

lipoxygenase-catalyzed chlorophyll bleaching is a

characteristic change in some legumes and cereal

products.

0013Due to various bioconversions of chlorophylls, the

dephytylated polar intermediates, chlorophyllides,

pheophorbides, and probably hydroxy chlorophylls

appear and disappear during the ripening and storage

of food commodities. The results of several investi-

gations suggest that chlorophyll is first degraded to

chlorophyllide by chlorophyllase, followed by oxida-

tive degradation by peroxidase, lipoxygenase, or

chlorophyll oxidase. The simultaneous action of

dechelase may raise the diversity of chlorophyll ex-

tract by producing the corresponding Mg-free deriva-

tives of the aforementioned chlorophyll degradation

products. It should be noted that further degradation

of chlorophylls and their derivatives to colorless com-

pounds of low molecular weight is a function of

oxidation processes which is not yet well described.

Degradation during Processing

0014Except for enzymatic conversions, chlorophylls

undergo light-, heat-, and acid-catalyzed alterations

that lead to a marked shift in the greenness of stored

and processed foods.

0015Acid-catalyzed removal of Mg ion upon release of

endogenous acids (in case of mechanical injuries and

processes), acid formation via fermentation (brining,

pickling, etc.), and dressing with an acidic ingredient

are responsible for chlorophyll-to-pheophytin con-

version. The acids produced in the fermentation that

occurs naturally and spontaneously in brined or

pickled fruits induce degradation of chlorophylls

and chlorophyllides to the corresponding pheophy-

tins and pheophorbides. Although physically acid-

catalyzed conversion occurs inside the chloroplast,

the media in which it takes place is the fermentor,

since it is the diffusion across the membranes by

osmosis that leads to fermentation. The intracellular

pH is thus altered by the pH of the brining solution.

The change in content of chlorophylls and pheophy-

tins as a function of fermentation time and pH of the

brine of green olive fermentation is shown in Table 1.

The kinetic equation describing the degradation of

the pigments is:

dc=dt ¼ k½H



½pigment

ð1Þ

In brining starting with alkaline treatment, when the

interior of the fruit reaches a pH of 8, the concen-

tration of chlorophylls is greater than that of hydro-

gen ions. In this case, if pheophytinization occurs, its

kinetics would be of second order. When the con-

centration of hydrogen ions in the fermentation

media, compared with the existing chlorophyll, is in

excess and can thus be considered to be constant, the

kinetic reaction is of pseudoorder and can be ex-

pressed as:

1198 CHLOROPHYL

dc=dt ¼ k

½pigment

ð2Þ

where k

¼ k½H



0016 At the first step of brining, when the pH is suitable

for chlorophyllase activity a portion of the chloro-

phyll is converted to chlorophyllides, followed by

acid-catalyzed formation of the corresponding Mg-

free derivatives (pheophytins and pheophorbides).

The decrease in the concentration of chloropyll a

and b as well as the accumulation of Mg-free forms

as a function of time follows first-order kinetics. A

rapid accumulation of pheophytins in cooked or

canned foods is a result of the combined action of

heat and acid release after cell rupture.

001 7 At neutral or alkaline pH values, high-temperature

short-time heat treatment, as in blanching, often

results in an epimerization on carbon-10 of chloro-

phyll molecule, giving rise to the formation of the so-

called a

and b

epimers that are a brighter green color

than their original pigments. With an extended period

of thermal treatment the rate and final products of

chlorophyll degradation are changed. In canning the

function of time, temperature, and pressure deter-

mines the kinetics of chlorophyll degradation.

Chlorophyll degradation during thermal processing

is frequently described as a first-order reaction. For

such kinetics, the integrated form of eqn (2) for a

decay process at constant temperature and pressure

is given by:

X=X

¼ expðktÞð3Þ

where X

is the response value at t ¼ 0 (i.e., the

concentration of chlorophyll at t ¼ 0) and X is

the residual response value after treatment (i.e., the

residual concentration of chlorophyll).

0018The temperature dependence of the degradation

rate constant (k) at atmospheric pressure can be

adequately described using activation energy E

,as

given in the Arrhenius relationship:

k ¼ k

ref

exp½E

=Rð1=T

ref

 1=TÞ ð4Þ

where k

ref

is the rate constant at reference tempera-

ture T

ref

is the activation energy, and R is the

universal gas constant.

0019The pressure dependence of the rate constant (k)at

a certain temperature is commonly described as an

activation volume V

, as given in the Eyring relation:

k ¼ k

ref

exp½V

=RTðP  P

ref

Þ ð5Þ

where k

ref

is the rate constant at reference pressure

ref

, V

is the activation volume at a certain tempera-

ture, T is the absolute temperature, and R is the

universal gas constant.

0020In order to interpret the validity of a first-order

reaction of chlorophyll degradation, eqn (3) is linear-

ized using a logarithmic data transformation [ln (X/

) ]. A log linear plot of relative chlorophyll a and

b retention versus degradation time is depicted in

Figure 2. These plots show that the rate of

chlorophyll degradation follows a first-order kinetic

model.

tbl0001 Table 1 Rate constant (k) for degradation of chlorophyll a, chlorophyll b, and total chlorophyll content in broccoli juice due to a

thermal or a combined pressure–temperature treatment

Pressure (MPa) Temperature (



C) Chlorophyll a k (10

2

min

1

) Chlorophyll b k (10

2

min

1

) Total chlorophyll k (10

2

min

1

)

0.1 80 2.51 + 0.26

0.57 + 0.03

1.59 + 0.19

90 4.41 + 0.24 1.20 + 0.05 2.95 + 0.20

100 8.84 + 0.24 2.46 + 0.11 6.15 + 0.31

110 14.13 + 0.95 4.73 + 0.27 10.29 + 0.65

120 23.60 + 0.35 9.07 + 0.78 17.66 + 0.36

200 60 0.45 + 0.02 ND 0.33 + 0.02

70 1.60 + 0.15 0.57 + 0.01 1.13 + 0.09

80 4.11 + 0.43 1.59 + 0.05 2.76 + 0.19

500 60 0.52 + 0.02 0.10 + 0.01 0.38 + 0.02

70 1.48 + 0.09 0.56 + 0.03 1.13 + 0.04

80 4.04 + 0.24 2.91 + 0.07 3.45 + 0.13

700 60 0.64 + 0.03 0.23 + 0.01 0.50 + 0.02

70 1.67 + 0.06 0.84 + 0.06 1.37 + 0.05

80 3.99 + 0.41 2.78 + 0.16 3.36 + 0.23

800 50 0.20 + 0.02 ND 0.16 + 0.02

60 0.57 + 0.04 0.19 + 0.02 0.44 + 0.03

70 1.91 + 0.05 0.96 + 0.04 1.45 + 0.04

80 3.48 + 0.02 3.67 + 0.14 3.94 + 0.27

Asymptotic standard error of regression.

ND, not determined.

Reproduced from Van Loey A, Ooms V, Weemaes C et al. (1998) Thermal and pressure degradation of chlorophyll in broccoli (Brassica oleraecea) juice.

A kinetic study. Journal of Agriculture and Food Chemistry 46: 5289, with permission.

CHLOROPHYL 1199

0021 With regard to the ranking in heat stability of

chlorophylls a and b, it is evident that chlorophyll a

is less heat-stable than chlorophyll b, and hence the

former degrades more quickly. The higher thermal

stability of chlorophyll b is attributable to the

electron-withdrawing effect of its C-3 formyl group.

0022 Chlorophyll exhibits an extreme stability toward

pressure processing at ambient or relatively low

temperatures (not higher than 50



C). This can be

ascribed to the stability of the covalent structure of

chlorophyll to high pressure and to the slight com-

pressibility of covalent bonds.

0023From semilogarithmic plots of chlorophyll reten-

tion in broccoli juice as a function of treatment time

at constant pressure and temperature, it can be seen

that the pressure–temperature-induced degradation

process follows a first-order kinetics. Rate constants

for chlorophyll degradation due to thermal or com-

bined pressure–temperature treatment are shown in

Table 2.

0024Severe heat processing enhances the decarbo-

methoxylation reaction, leading to formation of

pyro derivatives of chlorophylls. Heat treatment of

121



C for more than 15 min is required for the

appearance of pyropheophytin a and b. The rate

of decarbomethoxylation reaction as a function of

time and temperature follows first-order degradation

kinetics.

0025Chlorophyll to pheophytin conversion has been

recognized for more than 50 years in frozen foods

stored at lower than 18



C. This conversion is re-

sponsible for color change from a bright green to a

dull olive green in freeze-stored fruits and vegetables.

The rate of such a conversion is shown to be a first-

order model with respect to acid concentration. A

linear relationship is expected between the appear-

ance and pheophytin formation for frozen plant

products.

0026In drying processing, the rate of chlorophyll deg-

radation is affected by some factors, including drying

temperature and time, water activity (a

), and the

storage conditions of dried foods. At a

values greater

than 0.32 and ambient storage with nitrogen gas,

pheophytins are expected to be the predominant pig-

ment of green-colored dry vegetables. It is well dem-

onstrated that chlorophyll a undergoes degradation

more rapidly than does chlorophyll b by a factor of

2.5–3.0. A linear relationship between a

and log

time for a 20% loss of chlorophyll is recognized for

dry fruit and vegetables. Recent studies indicate that

the main reason for chlorophyll degradation in heat-

dried foods (sweet peppers, kidney beans, Chinese

onions, etc.) is the free radicals initiated in the food

system, particularly in the presence of unsaturated

fatty acids. It is recommended to increase the content

of effective scavengers in the product before drying

to neutralize free radicals and prevent pigment

degradation.

0027Formation of pheophytins in edible oils during ex-

traction and refining is a well-known photo-catalyzed

degradation. At different temperature and luminous

energies, this conversion follows first-order kinetics.

From the Arrhenius plot display, it appears that the

In(C/Co)

−0.5

0.0

−1.0

−1.5

−2.0

−0.25

0.00

−0.50

−0.75

−1.00

0 180150120906030

(a)

(b)

Time (min)

fig0002 Figure 2 First-order thermal degradation of (a) chlorophyll a

and (b) chlorophyll b in broccoli juice at (

) 80, ( ) 90, ( ) 100, ( )

110, and (

) 120



C. Reproduced from Weemaes CA, Ooms V, Van

Loey M, Handrickx ME (1999) Kinetics of chlorophyll degradation

and color loss in heated broccoli. Journal of Agriculture and Food

Chemistry 47: 2404, with permission.

1200 CHLOROPHYL

incident luminous energy does not change activation

energy, but increases the reaction frequency factor.

Effect of Controlled Storage and

Atmosphere

0028 Storage in controlled or ethylene-containing atmos-

phere can affect, to a considerable extent, the chloro-

phyll content of food commodities. Recently, such

storage has been widely applied in food technology

either to speed up chlorophyll loss in some fruit

(citrus fruits, bananas, mangoes, pears, etc.) or to

maintain the greenness of stored crops.

0029 Ethylene can be used directly as gas to treat the

product in the package or in closed stores. Also it is

indirectly applicable either by treating trees or im-

mersing fruit in diluted solutions of ethylene, releas-

ing plant growth regulators such as ethrel and hydrel

(150–250 p.p.m.). To accelerate the ripening of some

pears, a combination of 500 p.p.m. ethylene and

100 p.p.m. acetylene gas is recommended.

0030 Ethylene treatment enhances degreening of the peel

of some fruit via de novo synthesis of chlorophyllase

and chloroplast-dependent enzyme that regulates

chlorophyllase activity. However, in spinach leaves

accelerated color loss by ethylene is not associated

with increased content of dephytylated derivatives.

This difference may be due to differences in the deg-

radation pathway in the different products. It is stated

in the literature that ethylene treatment can also en-

hance the synthesis of chlorophyll-oxidative enzymes

in cotyledons held in the dark. An inverse correlation

between the content of chlorophyll of ethylene-stored

radish cotyledons and the activity of chlorophyll-

oxidizing enzymes indicates that chlorophyll oxida-

tion is an important step in the degreening process.

0031Storage under controlled atmosphere (CA) retards

chlorophyll decomposition in a wide variety of agri-

cultural crops. CA of 10% O

and 10% CO

is very

effective in reducing the rate of chlorophyll degrad-

ation to the extent that the shelf-life would be

extended about 20% longer than that in air-held

samples (Figure 3).

0032Low O

(3%) and high CO

(20%) treatment, as in

CA of tomato storage, maintains firmness, reduces

chlorophyll loss, and slows lycopene and carotenoid

development. In CA storage, ethylene-mediated re-

sponses are impaired by low O

and high CO

and exogenous ethylene and low O

/high CO

con-

centrations act antagonistically. Controlled storage

may include vacuum cooling and prestorage treat-

ment with ethylene-reducing and respiration rate-

lowering agents. Of these agents, ethanol vapor

is used to maintain freshness and keep the chlorophyll

tbl0002 Table 2 Qualitative and quantitative changes in chlorophylls during table olive processing as a function of pH

Time (days) Pigment concentration (mmol kg

1

)

pH a series b series

Fruit Brine Chl Phy Chl þphy Chld Pho Chld þpho Chl Phy Chl þphy Chld Pho Chld þpho

0 6.08 49.97 49.97 12.37 12.37

4 8.44 8.28 40.73 40.73 7.04 7.04 7.62 7.62 4.52 4.52

6 7.28 6.93 33.09 8.47 41.56 4.70 2.70 7.40 7.29 0.37 7.66 3.86 0.85 4.71

8 6.67 5.49 26.18 15.44 41.62 3.18 3.90 7.08 6.61 0.72 7.33 3.28 1.43 4.71

10 5.17 5.49 25.01 17.20 42.21 2.60 4.95 7.55 6.58 1.06 7.64 2.58 2.12 4.70

14 5.40 4.56 15.12 26.52 41.64 1.40 5.61 7.01 5.95 1.43 7.38 2.11 2.35 4.46

19 5.41 4.76 13.91 27.63 41.54 0.70 6.10 6.80 4.86 2.43 7.29 1.41 3.10 4.51

20 5.08 4.65 12.59 29.01 41.60 0.28 6.80 7.08 4.83 2.48 7.31 1.05 3.40 4.45

26 4.83 4.57 11.76 29.53 41.29 7.02 7.02 3.79 2.74 6.53 4.14 5.10

30 4.69 4.60 6.16 35.40 41.56 7.05 7.05 3.99 3.65 7.64 0.35 4.36 4.71

33 4.55 4.52 5.78 35.32 41.10 7.23 7.23 3.66 3.17 6.83 4.61 4.61

40 4.61 4.42 3.69 36.99 40.68 7.16 7.16 3.14 4.42 7.56 4.60 4.60

50 4.53 4.46 1.52 38.50 40.02 7.18 7.18 2.42 5.20 7.62 4.66 4.66

54 4.44 4.35 1.54 38.24 39.78 7.18 7.18 2.24 5.70 7.97 4.70 4.70

60 4.43 4.29 0.90 39.10 40.00 7.15 7.15 1.88 6.18 8.06 4.70 4.70

70 4.41 4.32 0.37 40.53 40.90 7.20 7.20 1.47 6.30 7.77 4.71 4.71

89 4.33 4.21 0.23 40.82 41.05 7.12 7.12 0.95 6.30 7.25 4.78 4.78

104 4.45 4.25 40.77 40.77 7.14 7.14 0.61 6.59 7.20 4.58 4.58

117 4.37 4.26 40.69 40.69 7.15 7.15 6.57 6.57 4.90 4.90

161 4.21 4.10 40.85 40.85 6.94 6.94 6.41 6.41 4.80 4.80

203 4.34 4.19 40.81 40.81 6.97 6.97 6.68 6.68 4.51 4.51

287 4.27 4.08 40.76 40.76 7.17 7.17 6.59 6.59 4.67 4.67

a series: chlorophyll a and derivatives; b series: chlorophyll b and derivatives; Chl, chlorophyllide; Phy, pheophytin; Pho, pheophorbide.

Reproduced from Mı

nguez-Mosquera M, Candul-Rojas B, Mı

nguez-Mosquera J (1994) Mechanism and kinetics of the degradation of chlorophyll during

the processing of green table olives. Journal of Agriculture and Food Chemistry 42: 1089, with permission.

CHLOROPHYL 1201

content at high levels in some horticultural crops. A

prestorage treatment of special interest is the short-

term immersion of broccoli florets in ozonated water

(10 p.p.m.)for 10–50 min. Although the treatment

significantly reduces ethylene formation and total

soluble proteins by the end of cold storage, it causes

the vegetables to lose their green color rapidly.

Antioxidant–Oxidant Role

0033 Due to their chemical nature and hydrophobic prop-

erties, chlorophylls are able to interfere with the

chains of lipid oxidation in foods. From industrial

practices and in vitro experiments, chlorophylls and

their degraded products can play the role of either

antioxidants or prooxidants. Factors which are most

likely to determine the antioxidative or oxidative

activity of chlorophylls include variety, ripeness

stage, and chemical composition of food where they

exist, the presence or absence of effective oxidants

or antioxidant, and the climate.

0034 When tested by ferric thiocyanate and other assays,

chlorophylls and their derivatives exhibit marked

antioxidant activity. Recent studies show that acidic

fractions from plant containing pheophytins and re-

lated compounds have an antioxidant effect which is

higher than that of a-tocopherol and comparable to

that of BHT (butylated hydroxytoluene) (Figure 4).

100

150

200

0246

Days in storage

Chl b

Chl a

Air

Ethylene

Chlorophyll content (mg 100g

−1

f. wt)

fig0003 Figure 3 Change in content of chlorophylls of parsley leaves stored in air with or without 10 p.p.m. C

or controlled atmosphere of

10% O

and 10% CO

. Reproduced from Yamauchi N and Watada AE (1993) Pigment changes in parsley leaves during storage in

controlled and ethylene containing atmosphere. Journal of Food Science 58: 616, with permission.

0246810

OD 500 nm

2.0

1.5

1.0

0.5

Days

fig0004Figure 4 Antioxidative effect of fractionated extracts from

marine algae by the ferric thiocyanate method. Sample concen-

tration 0.02%; BHT concentration 0.01%. h, neutral fraction;

weakly acidic fraction; &, strongly acidic fraction; n, basic frac-

tion; continuous line, BHT; s, control. Reproduced from Cahyana

AH, Shuto Y, Kinoshita Y (1992) Pyropheophytin a as antioxidative

substance from the marine alga, arame (Eisenia bicyclis).

Bioscience Biotechnology Biochemistry 56: 1533, with permission.

1202 CHLOROPHYL

0035 The antioxidant activity of chlorophyll-derived

compounds is attributable to their interference with

free radical cycles as scavengers to neutralize highly

reactive oxygen-free forms such as superoxides. The

porphyrin ring system of chlorophylls seems to be

important for the antioxidant activity.

0036 Treating some foods with oil containing chloro-

phyll between 200 and 800 mg% significantly re-

duces peroxide value during storage in the dark.

This gives convincing evidence on the antioxidant

effect of chlorophylls. In dry, oiled, and toasted

laver, for example, the high lipid oxidative stability

is ascribed to the presence of chlorophylls in the oil

used. A positive correlation between oil stability and

chlorophyll content is recognized in the processing of

virgin oil.

0037 The antioxidant activity of chlorophylls can be

enhanced by ascorbic acid and a-tocopherol which

exists at low level as 10

6

mol l

1

for each in the food

system. This suggests that synergism between chlor-

ophylls and other effective bioantioxidants is possible

and of great importance from the point of view of oil

oxidative stability.

0038 Cooling green colored vegetables modifies their

antioxidative ability due to chlorophylls. In cooked

juices from broccoli, cabbage, green pepper, and leek,

the superoxide scavenging ability is higher than that

of the original raw materials. Moreover, the antioxi-

dant effect of cooked spinach containing considerable

amounts of chlorophyll a and b is much lower than

that estimated for other vegetable extracts having

no chlorophyll remains. It is, therefore, convincingly

evident that Mg-free derivatives of chlorophylls

have greater chemical capacity and ability as oxida-

tion barriers than their origins. Also, as many

recent in vitro studies have indicated, the antioxidant

effect is more marked for chlorophyll a than chloro-

phyll b.

0039 In oil-in-water food emulsions which have been

stored in the dark, the addition of chlorophylls from

plant sources such as dried leaves insures oxidation

prevention equal to that provided by 80 p.p.m. of

propyl gallate. However, the effect of green plant

extract on the stability of oil-in-water emulsions is

not the same when the emulsions are exposed to light.

In contrast, under various conditions the prooxidant

effect of chlorophylls is recognized.

0040 As a practice in the processing of rapeseed oil, the

chlorophyll content of raw oil should not exceed

25 mg kg

1

to maintain top-grade oil and extend

shelf-life. The oxidation stability of the refined oil is

reduced as the preprocessing content of pheopytins

exceeds 30 p.p.m. The prooxidant effect of chloro-

phyll-derived compounds is increased, to a high

extent, when the oil is stored in the light.

0041The degree of unsaturation of oil is another factor

which modifies the prooxidant ability of chloro-

phylls. In highly unsaturated marine oil, the addition

of chlorophylls from green tea extracts at less than

25 p.p.m. impairs oil oxidative stability and, more-

over, removing chlorophylls from the tea extract

gives it an antioxidant effect which is even higher

than that of synthetic antioxidants at the same

concentration. It can therefore be concluded that, in

highly unsaturated oils, the prooxidant capacity of

chlorophyll-related compounds is so high that oxida-

tion is scarcely inhibited by spontaneous antioxidants

like flavonoids and tocopherols.

0042Another type of chemical decay in foods is the

photo-catalyzed oxidation of unsaturated lipids.

Chlorophylls contribute to photooxidation of oils

exposed to light or irradiation via a photosensitiza-

tion reaction. The mechanism of light-induced photo-

sensitization may involve free radical initiation and

incorporation of singlet oxygen.

0043Fat-soluble oxygen quenchers and free radical

scavengers such as carotenoids, ascorbyl palmitate,

and tocopherols can interfere to minimize chloro-

phyll-sensitized oxidation of fatty acids and oils.

Ascorbyl palmitate is recommended to be applied

as an antioxidant with photo-oxidation-preventive

properties (total quenching rate ¼ 10

8

mol s

1

)in

fatty foodstuffs. Tocopherols are efficiently used to

minimize chlorophyll-sensitized oxidation. The activ-

ity of tocopherol analogs to prevent photosensitiza-

tion is in the order of d > g > b > a. Carotenoid-type

pigments actively interfere with photosensitized

oxidation in food systems. The antiphotosensitization

ability of carotenoids is attributed to the rapid trans-

fer of radicals from chlorophyll to carotenoid mol-

ecules as well as the ability of carotenoids to quench

oxygen free forms. In photosensitized reactions,

carotenoid pigments undergo trans-to-cis isomeriza-

tion on their structure. This may explain the presence

of cis carotenoids in photosynthetic tissues.

0044It should be noted that all chlorophyll-derived

compounds are able to induce photoisomerization of

carotenoids in light-exposed food systems. Different

carotenoids vary in their rate and capacity of inter-

action with photosensitizing agents. When tested by

the headspace and rancimat methods, capsanthin has

the highest rate and ability to minimize chlorophyll-

induced photosensitization, followed by b-carotene

and lutein.

Metal–Chlorophyll Interaction

0045Bright green color in drastically heat-processed vege-

tables is associated with the formation of metal–

chlorophyll complexes. Copper and zinc, present as

CHLOROPHYL 1203

contaminants in processing solutions, are the most

familiar metals reacting with chlorophylls during the

thermal processing and storage of processed foods.

Such metal complexes can be found in canned green

beans, Brussel sprouts, spinach and pea pure

es, table

olives, candies, and chewing gum. The formation

of Zn–chlorophyll complexes is of greater interest

because of the toxicity nature of copper complexes.

0046 The formation of green-colored metal complexes

of chlorophyll derivatives is the basis of efforts by

processors to preserve the desired green color of

canned vegetables. This process is called regreening.

Spontaneous regreening of vegetables during process-

ing, which has long been known by canners as a

nonuniform defect, is attributed to Zn-containing

pigments.

0047 Copper–chlorophyll complexes are formed more

rapidly than Zn complexes. Clorophyll a-related

derivatives form metallocomplexes more rapidly

than chlorophyll b derivatives. Furthermore,

pyropheophytins and pheophorbides have a mark-

edly high tendency to react with metal ions. Steric

hindrance due to the lack of a carbomethoxy group

at C-10 is one possible reason for the rapid inter-

action of pyropheophorbides and metals. The other

explanation is that the distribution of charge in the

aromatic system is affected by the carbomethoxy

group, which is strongly electron-withdrawing. In

the absence of a carbomethoxy group, the pyrrole

nucleus could become slightly more negatively

charged, resulting in an increased reaction rate with

positively charged ions. The kinetic parameters of

Zn–chlorophyll reaction are shown in Table 3.

0048Food additives such as sugars, salts, and other pre-

servatives may influence metal complexes of chloro-

phylls. While sucrose, glucose fructose, chlorides,

sulfates, lactates, acetates, and propionates have no

influence on metal–chlorophyll complex formation,

malate, tartarate, citrate phosphate, and other metal-

chelating agents such as ethylenediaminetetraacetic

acid significantly decrease the rate of metal–chloro-

phyll reaction. In contrast, thiocyanate, benzoate,

oleate, and caprylate ions accelerate the chemical

interaction of chlorophyll with Cu and Zn.

0049The presence of active anionic compounds in

canning solutions facilitates the formation of metal

complexes of chlorophyll-derived compounds by

adsorbing on to chloroplast membranes, thereby

increasing the negative surface charge and giving

rise to a higher affinity to bind positively charged

ions. Increasing cation concentration in the food pro-

motes the complex formation of chlorophylls. The

mechanism for this promotion includes a direct

common ion effect, which increases the rate constant

Table 3 Reaction rate constants (k), half-life values (t

1/2

) and energy activation (E

) for the reaction of chlorophyll a and derivatives

with zinc (II) ion at 20, 25, 30, and 35



Pigment Temp. (



C) Slope + SD (10

3

)(min

1

) k + SD (min

1

) Correlation coefficient t

1/2

+ SD (min) E

(kcalmol

1

)

Pheophytin 20 0.99 + 0.02 0.035 + 0.001 0.998 305 + 7 22.09

25 1.91 + 0.05 0.067 + 0.002 0.999 157 + 3

30 3.37 + 0.16 0.12 + 0.01 0.995 89 + 4

35 6.36 + 0.16 0.22 + 0.01 0.998 47 + 1

Pyropheophytin 20 1.73 + 0.01 0.061 + 0.004 0.998 174 + 1 22.60

25 3.39 + 0.01 0.119 + 0.002 0.999 89 + 1

30 6.41 + 0.01 0.23 + 0.01 0.998 47 + 1

35 11.45 + 0.01 0.40 + 0.01 0.999 26 + 1

Ethyl pheophorbide 20 1.76 + 0.01 0.062 + 0.001 0.998 171 + 1 21.40

25 3.12 + 0.01 0.110 + 0.001 0.998 96 + 1

30 5.65 + 0.01 0.20 + 0.01 0.998 53 + 1

35 10.55 + 0.01 0.37 + 0.01 0.998 29 + 1

Methyl pheophorbide 20 1.81 + 0.01 0.064 + 0.001 0.999 166 + 1 21.36

25 3.28 + 0.01 0.115 + 0.001 0.999 92 + 1

30 6.11 + 0.01 0.21 + 0.01 0.998 49 + 1

35 10.72 + 0.01 0.38 + 0.01 0.995 28 + 1

Pheophorbide 20 3.36 + 0.01 0.118 + 0.001 0.998 90 + 1 21.52

25 5.73 + 0.01 0.20 + 0.01 0.999 53 + 1

30 10.31 + 0.01 0.36 + 0.01 0.997 29 + 1

35 20.47 + 0.01 0.72 + 0.02 0.998 15 + 0.5

Pyropheophorbide 20 5.57 + 0.01 0.20 + 0.01 0.998 54 + 1 22.06

25 10.68 + 0.01 0.38 + 0.01 0.998 28 + 1

30 19.33 + 0.01 0.69 + 0.01 0.998 16 + 0.5

35 35.50 + 0.01 1.25 + 0.01 0.998 9 + 0.5

SD, standard deviation for duplicate determination.

Reproduced from Tonucci LH and von Elbe H (1992) Kinetics of zinc complexes of chlorophyll derivatives. Journal of Agriculture and Food Chemistry 40:

2341, with permission.

1204 CHLOROPHYL

of the reaction of cations and chlorophylls, and an

indirect pH-lowering effect of cation.

0050 The pH of the media is an important factor

affecting chlorophyll–metal complex formation. The

rate of Zn–complex formation increases between

pH 4.0 and 8.0, reaching a maximum between 6.0

and 8.0. Raising the pH to values higher than 8.0

decreases the amount of metal–chlorophyll com-

plexes formed. At high pH values, chlorophyll a and

b are very stable and the amount of derivatives avail-

able for the reaction is reduced.

See also: Antioxidants: Natural Antioxidants; Colorants

(Colourants): Properties and Determination of Natural

Pigments; Copper: Physiology; Freezing: Structural and

Flavor (Flavour) Changes; Oxidation of Food

Components; Storage Stability: Mechanisms of

Degradation; Zinc: Physiology