V. EMULSIFYING PROPERTIES OF PHOSPHOLIPIDS

phatidylinositol (PI) and diacylglycerol and glyceroldiphosphate are formed from

diphosphatidylglycerol. Total hydrolysis into each of the component parts of all phospholipids can be accomplished by strong acid (HCl, H2SO4) catalysis in 6 N aqueous

or 5–10% methanolic solutions [23]. Kinetics and mechanism for hydrolysis in 2 N

HCl at 120ЊC have been described by DeKoning and McMullan [24]. Deacylation

occurs first, followed by formation of a cyclic phosphate triester as an intermediate

to cyclic glycerophosphate and choline. Eventually an equilibrium mixture of ␣ - and

␤ -glycerophosphates is formed.

Mild alkaline hydrolysis of ester bonds in phospholipids at 37ЊC (0.025–0.1

M NaOH in methanolic or ethanolic solutions) leads to fatty acids and glycerophosphates. In contrast, phosphosphingolipids are not affected unless subjected to strong

alkaline conditions. Some selectivity is seen in the susceptibility of phosphoglycerides to hydrolyze with diacyl > alk-1-enyl, acyl > alkyl, acyl. With more vigorous

alkaline hydrolysis, the glycerophosphates are apt to undergo further hydrolysis because the phosphoester bond linking the hydrophilic component to the phospholipid

moiety is not stable enough under alkaline conditions and splits, yielding a cyclic

phosphate. When the cycle opens up, it gives a 1:1 mixture of 2- and 3-glycerophosphates.

Both state of aggregation and specific polar group have been shown to affect

the reaction rates for alkaline hydrolysis of phospholipids [25]. Higher activation

energies were observed for hydrolysis of phospholipids in membrane vesicles than

when phospholipids were present as monomers or Triton X-100 micelles. Alkaline

hydrolysis of PC, on the other hand, was three times faster than hydrolysis of PE.

B.



Enzymatic Hydrolysis



Selective hydrolysis of glycerophospholipids can be achieved by the application of

phospholipases. One beneficial aspect to application of phospholipase is improved

emulsifying properties to a PC mixture [26]. Unfortunately, while these enzymes

may be isolated from a variety of sources, in general they are expensive.

Several phospholipases exist differing in their preferential site of attack. The

ester linkage between the glycerol backbone and the phosphoryl group is hydrolyzed

by phospholipase C while the ester linkage on the other side of the phosphoryl group

is hydrolyzed by phospholipase D. Hydrolysis of the acyl groups at the sn-1 and sn2 position of phospholipids is carried out by phospholipases A1 and A 2 , respectively.

While phospholipase A 2 binding to membrane phospholipids has been enhanced 10-fold by the presence of calcium [27], membrane surface electrostatics

dominated phospholipase A 2 binding and activity in the absence of calcium [28]. A

highly cationic enzyme (pI > 10.5), phospholipase A 2 , has a marked preference for

anionic phospholipid interfaces. Thus, phosphatidic acid and palmitic acid promoted

the binding of phospholipase A 2 to the bilayer surface [28,29]. Perturbations and a

loosening of the structure associated with the presence of these hydrolysis products

were suggested as the properties contributing to enhanced binding [30]. The presence

of phospholipid hydroperoxides has also facilitated enhanced binding of phospholipases through a similar mechanism [31].

VII.



HYDROGENATION OF PHOSPHOLIPIDS



Hydrogenation of fats involves the addition of hydrogen to double bonds in the

chains of fatty acids. While hydrogenation is more typically applied to triacylglyc-



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



erols to generate semisolid or plastic fats more suitable for specific applications, it

may also be applied to phospholipid fractions. Hydrogenated lecithins are more stable

and more easily bleached to a light color, and therefore are more useful as emulsifiers

than the natural, highly unsaturated lecithin from soybean oil. These advantages are

exemplified by a report that hydrogenated lecithin functions well as an emulsifer and

as an inhibitor of fat bloom in chocolate [32].

In practice, hydrogenation involves the mixing of the lipid with a suitable

catalyst (usually nickel), heating, and then exposing the mixture to hydrogen at high

pressures during agitation. Phospholipids are not as easily hydrogenated as triacylglycerols; as a result, their presence decreases the catalyst activity toward triacylglycerols [33]. In this situation, phosphatidic acid was the most potent poisoning

agent; however, fine-grained nickel catalyst was more resistant to the poisoning effect

of phospholipids than moderate-grained catalyst. In any event, hydrogenation of

phospholipids requires higher temperatures and higher hydrogenation pressures. For

example, hydrogenation of lecithin is carried out at 75–80ЊC in at least 70 atm

pressure and in the presence of a flaked nickel catalyst [34]. In chlorinated solvents

or in mixtures of these solvents with alcohol, much lower temperatures and pressures

can be used for hydrogenation, particularly when a palladium catalyst is used [35].

VIII.



HYDROXYLATION



Hydroxylation of the double bonds in the unsaturated fatty acids of lecithin improves

the stability of the lecithin and its dispersibility in water and aqueous media. Total

hydroxylating agents for lecithin include hydrogen peroxide in glacial acetic acid

and sulfuric acid [36]. Such products have been advocated as useful in candy manufacture in which sharp moldings can be obtained when the hydroxylated product is

used with starch molds.

IX.



HYDRATION



The amount of water absorbed by phospholipids has been measured by a number of

different methods, including gravimetry, X-ray diffraction, neutron diffraction, NMR,

and DSC [37]. For any measurement, however, Klose et al. [38] cautioned that the

morphology and method of sample preparation can induce the formation of defects

in and between the bilayers, and therefore will influence the water content of lamellar

phospholipids.

The electrical charge on the phospholipid head group does not in itself determine the nature of the water binding. However, it does affect the amount of water

bound. The amount of water absorbed by PC from the vapor phase increased monotonically from 0 water molecules per PC molecule at 0% humidity to between 14

[39] and 20 [40] water molecules per lipid molecule at 100% relative humidity.

Observed differences may be due to the difficulty of exerting accurate control over

relative humidities near 100% when temperature gradients in the system are present.

The results of X-ray diffraction studies indicated that when directly mixed with bulk

water PC imbibed up to 34 water molecules [41,42]. Considerably less water was

imbibed by PE, with a maximum of about 18 water molecules per lipid [43]. From

the saturated vapor phase, however, liquid crystalline egg yolk PE only absorbed

about 10 water molecules per lipid molecule [39], whereas for charged phospholipids,



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



such as PI or phosphatidylserine (PS), the phospholipid imbibed water without limit

[44,45].

Hydration of a phospholipid appears to be cooperative. A water molecule that

initiated hydration of a site facilitated access of additional water molecules, until the

hydration of the whole site composed of many different interacting polar residues

was completed [46]. Incorporation of the first three to four water molecules on each

phospholipid occurs on the phosphate of the lipid head group and is exothermic [47].

The remaining water molecules are incorporated endothermically.

Neutron diffraction experiments on multilayers containing PC [48,49], PE [50],

and PI [51] have revealed that water distributions are centered between adjacent

bilayers and overlap the head group peaks in the neutron scattering profile of the

bilayer. These results imply that water penetrates into the bilayer head group region,

but appreciable quantities of water do not reach the hydrocarbon core. By combining

X-ray diffraction and dilatometry data, McIntosh and Simon [52,53] were able to

calculate the number of water molecules in the interbilayer space and in the head

group region for dilauroyl-PE bilayers. They found that there are about 7 and 10

water molecules in the gel and liquid crystalline phases, respectively, with about

half of these water molecules located between adjacent bilayers and the other half

in the head group region.

The amount of water taken up by a given phospholipid depends on interactions

between the lipid molecules, including interbilayer forces (those perpendicular to the

plane of the bilayer) and intrabilayer forces (those in the plane of the bilayer). For

interbilayer forces, at least four repulsive interactions have been shown to operate

between bilayer surfaces. These are the electrostatic, undulation, hydration (solvation), and steric pressures. Attractive pressures include the relatively long-range van

der Waals pressure and short-range bonds between the molecules in apposing bilayers, such as hydrogen bonds or bridges formed by divalent salts. Several of the same

repulsive and attractive interactions act in the plane of the bilayer, including electrostatic repulsion, hydration repulsion, steric repulsion, and van der Waals attraction.

In addition, interfacial tension plays an important role in determining the area per

lipid molecule [54]. Thus, as the area per molecule increases, more water can be

incorporated into the head group region of the bilayer. Such a situation is found with

bilayers having an interdigitated gel phase compared with the normal gel phase and

with bilayers having unsaturated fatty acids in the phospholipid compared with saturated fatty acids [55,56].

The presence of monovalent and/or divalent cations in the fluid phase changes

the hydration properties of the phospolipids. For example, the partial fluid thickness

˚

between dipalmitoyl PC bilayers increased from about 20 A in water to more than

˚ in 1 mM CaCl2 [57]. In contrast, monovalent cations, such as Naϩ, Kϩ, or Csϩ,

90 A

decrease the fluid spaces between adjacent charged PS or PG bilayers as a result of

screening of the charge [58,59]. In addition, divalent cations have a dehydrating

effect on PS. The most extensively studied divalent cation, Ca2ϩ, binds to the phosphate group of PS [60], liberates water between bilayers and from the lipid polar

groups [61], crystallizes the lipid hydrocarbon chains [59,60], and raises the gel to

the liquid crystalline melting temperature of dipalmitoyl PS by more than 100ЊC

[59]. In response to these changes, one would expect permeability of the membrane

to be altered.



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



The interaction of phospholipids with water is critical to the formation, maintenance, and function of membranes and organelles. It is the low solubility of the

acyl chains in water combined with the strong hydrogen bonding between the water

molecules that furnishes the ‘‘attractive’’ force that holds together polar lipids as

supramolecular complexes (the ‘‘hydrophobic bond’’). These ordered structures are

generated when the phospholipid concentration exceeds its critical micelle concentration (cmc), which is dependent on the free energy gained when an isolated amphiphile in solution enters an aggregate [61]. For diacyl phospholipids in water, the

cmc in general is quite low, but it depends on both the chain length and the head

group. For a given chain length, the solubility of charged phospholipids is higher,

while the cmc of a single-chain phospholipid is higher than that of a diacyl phospholipid with the same head group and the same chain length [61].

X.



COMPLEXATION OF PHOSPHOLIPIDS



A.



Ions



To comprehend ion binding to phospholipid molecules or to phospholipid membranes, it is necessary to understand the behavior of ions in bulk solution and in the

vicinity of a membrane–solution interface. If ion–solvent interactions are stronger

than the intermolecular interactions in the solvent, ions are prone to be positively

hydrating or structure-making (cosmotropic) entities. The entropy of water is decreased for such ions, while it is increased near other ion types with a low charge

density. The latter ions are thus considered to be negatively hydrating or structurebreaking (chaotropic) entities.

When an ion approaches a phospholipid membrane it experiences several

forces, the best known of which is the long-range electrostatic, Coulombic force.

This force is proportional to the product of all involved charges (on both ions and

phospholipids) and inversely proportional to the local dielectric constant. Since phospholipid polar head groups in an aqueous medium are typically hydrated, ion–phospholipid interactions are mediated by dehydration upon binding. Similarly, dehydration of the binding ion may occur. For instance, a strong dehydration effect is

observed upon cation binding to the acidic phospholipids, where up to eight water

molecules are expelled from the interface once cation–phospholipid association has

taken place [62–64].

Various degrees of binding exist between phospholipids and ions. When several

water molecules are intercalated between the ion and its binding site, there was

actually an association between the ion and phospholipid rather than binding. Outersphere complex formation between ion and phospholipid exists when only one water

molecule is shared between the ion and its ligand. On the other hand, complete

displacement of the water molecules from the region between an ion and its binding

site corresponds to an inner-sphere complex. Forces involved in the inner-sphere

complex formation include ion–dipole, ion–induced dipole, induced dipole–induced

dipole, and ion–quadrupole forces, in addition to the Coulombic interaction. Hydrogen bonding can also participate in inner sphere complex formation. Under appropriate circumstances, the outer-sphere complexes may also be stabilized by ‘‘throughwater’’ hydrogen bonding.

Phospholipid affinity for cations appears to follow the sequence lanthanides >

transition metals > alkaline earths > alkali metals, thus documenting the significance



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



of electrostatic interactions in the process of ion–membrane binding. Electrostatic

forces also play a strong role in lipid–anion binding with affinity for anions by PC,

following the sequence ClOϪ > I Ϫ Ն SCNϪ > NOϪ Ն BrϪ > ClϪ > SO2Ϫ. Here anion

4

3

4

size also has an important role in the process of binding, partly as a result of the

transfer of the local excess charges from the anion to the phospholipid head groups

and vice versa. However, strength of anion binding to phospholipid membranes decreases with increasing net negative charge density of the membrane [58].

Results of NMR, infrared spectroscopy, and neutron diffraction studies strongly

imply that the inorganic cations interact predominantly with the phosphodiester

groups of the phospholipid head groups [63–67]. On the other hand, inorganic anions

may interact specifically with the trimethylammonium residues of the PC head groups

[68,69].

Temperature may influence binding of ions to phospholipids. Under conditions

of phase transitions, phospholipid chain melting results in a lateral expansion of the

lipid bilayers, which for charged systems is also associated with the decrease in the

net surface charge density. In the case of negatively charged membranes, this transition leads to lowering of the interfacial proton concentration and decreases the

apparent pK value of the anion phospholipids [70].

B.



Protein



Complexes of PC with soy protein have been demonstrated by Kanamoto et al. [71].

In this study, using a linear sucrose density gradient centrifugation analysis, 14C-PC

was found to be nonspecifically bound to either the 7S or 11S proteins.

C.



Iodine



In the presence of phospholipid micelles, iodine changes color in aqueous solution.

It does not undergo color change in the presence of unassociated molecular species.

The color change coincides with the cmc of the substance and is due to formation

of the triiodide ion, I Ϫ [72]. Based on data from laser Raman studies, the reaction

3

appeared to be related to iodine–phospholipid interaction, as well as to penetration

of iodine into the bilayer membrane, rather than to an ion transport process.

XI.



OXIDATION



Unsaturated fatty acids of phospholipids are susceptible to oxidation through both

enzymatically controlled processes and random autoxidation processes. The mechanism of autoxidation is basically similar to the oxidative mechanism of fatty acids

or esters in the bulk phase or in inert organic solvents. This mechanism is characterized by three main phases: initiation, propagation, and termination. Initiation occurs as hydrogen is abstracted from an unsaturated fatty acid of a phospholipid,

resulting in a lipid free radical. The lipid free radical in turn reacts with molecular

oxygen to form a lipid peroxyl radical. While irradiation can directly abstract hydrogen from phospholipids, initiation is frequently attributed to reaction of the fatty

acids with active oxygen species, such as the hydroxyl free radical and the protonated

form of superoxide. These active oxygen species are produced when a metal ion,

particularly iron, interacts with triplet oxygen, hydrogen peroxide, and superoxide

anion. On the other hand, enzymatic abstraction of hydrogen from an unsaturated



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



fatty acid occurs when Fe3ϩ at the active site of lipoxygenase is reduced to Fe2ϩ.

While the majority of lipoxygenases require free fatty acids, there have been reports

of lipoxygenase acting directly on fatty acids in phospholipids [73,74]. Hence, enzymatic hydrolysis may not always be required prior to lipoxygenase activity.

During propagation, lipid–lipid interactions foster propagation of free radicals

produced during initiation by abstracting hydrogen from adjacent molecules; the

result is a lipid hydroperoxide and a new lipid free radical. Magnification of initiation

by a factor of 10 [75] to 100 [76] may occur through free radical chain propagation.

Further magnification may occur through branching reactions (also known as secondary initiation) in which Fe2ϩ interacts with a hydroperoxide to form a lipid

alkoxyl radical and hydroxyl radical, which will then abstract hydrogens from unsaturated fatty acids.

There are many consequences to phospholipid peroxidation in biological and

membrane systems. On a molecular level, lipid peroxidation has been manifested in

a decreased hydrocarbon core width and molecular volume [77]. In food, the decomposition of hydroperoxides to aldehydes and ketones is responsible for the characteristic flavors and aromas that collectively are often described by the terms ‘‘rancid’’

and ‘‘warmed-over.’’ Numerous studies, on the other hand, have shown that specific

oxidation products may be desirable flavor components [78–81], particularly when

formed in more precise (i.e., less random) reactions by the action of lipoxygenase

enzymes [82–87] and/or by the modifying influence of tocopherol on autoxidation

reactions [88].

Through in vitro studies, membrane phospholipids have been shown to oxidize

faster than emulsified triacylglycerols [89], apparently because propagation is facilitated by the arrangement of phospholipid fatty acids in the membrane. However,

when phospholipids are in an oil state, they are more resistant to oxidation than

triacylglycerols or free fatty acids [90]. Evidence that phospholipids are the major

contributors to the development of warmed-over flavor in meat from different animal

species has been described in several sources [91–94]. Similarly, during frozen storage of salmon fillets, hydrolysis followed by oxidation of the n-3 fatty acids in

phospholipids was noted [95]. The relative importance of phospholipids in these food

samples has been attributed to the high degree of polyunsaturation in this lipid fraction and the proximity of the phospholipids to catalytic sites of oxidation (enzymic

lipid peroxidation, heme-containing compounds) [96]. However, the importance of

phospholipids has not been restricted to animal and fish tissues. In an accelerated

storage test of potato granules, both the amounts of phospholipids and their unsaturation decreased [97]. Moreover, with pecans, a much stronger negative correlation

was found between headspace hexanal and its precursor fatty acid (18:2) from the

phospholipid fraction (R = Ϫ0.98) than from the triacylglycerol fraction (R = Ϫ0.66)

or free fatty acid fraction (R = Ϫ0.79) [98]. These results suggest that despite the

fact that membrane lipid constitutes a small percentage of the total lipid (0.5%),

early stages of oxidation may actually occur primarily within the phospholipids.

The presence of phospholipids does not preclude acceleration of lipid oxidation.

When present as a minor component of oil systems, solubilized phospholipids have

limited the oxidation of the triacylglycerols [99–101]. Order of effectiveness of

individual phospholipids was as follows: SPH = LPC = PC = PE > PS > PI > PG

[102] with both the amino and hydroxy groups in the side chain participating in the

antioxidant activity [103]. It was postulated that antioxidant Maillard reaction prod-



Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.



ucts were formed when aldehydes reacted with the amino group of the nitrogencontaining phospholipid. Alternatively, antioxidant activity occurred when complexes

between peroxyl free radicals and the amino group were formed [104]. The latter

activity is supported by an extended induction period when both tocopherol and

phospholipids were present.

Fatty acid composition is a major factor affecting the susceptibility of a phospholipid to assume an oxidized state, with carbon–hydrogen dissociation energies

decreasing as the number of bisallylic methylene positions increase [105,106]. However, lipid unsaturation also physically affects oxidation. In model membrane bilayers

made from single unilamellar vesicles, lipid unsaturation resulted in smaller vesicles

and therefore a larger curvature of the outer bilayer leaflet. The increased lipid–lipid

spacing of these highly curved bilayers, in turn, facilitated penetration by oxidants

[107,108]. Other functional groups on the phospholipid will also impact their oxidative stability. For example, the presence of an enol ether bond at position 1 of the

glycerol backbone in plasmalogen phospholipids has led to inhibition of lipid oxidation, possibly through the binding of the enol ether double bond to initiating

peroxyl radicals [109]. Apparently, products of enol ether oxidation do not readily

propagate oxidation of polyunsaturated fatty acids. Alternatively, inhibition of lipid

oxidation by plasmalogens has been attributed to the iron binding properties of these

compounds [110]. Variation within the phospholipid classes toward oxidation has

also been ascribed to the iron trapping ability of the polar head group [111]. For

example, PS was shown to inhibit lipid peroxidation induced by a ferrous–ascorbate

system in the presence of PC hydroperoxides [112]. However, stimulation of phospholipid oxidation by trivalent metal ions (Al3ϩ, Sc3ϩ, Ga3ϩ, In3ϩ, Be2ϩ, Y 3ϩ, and

La3ϩ) has been attributed to the capacity of the ions to increase lipid packing and

promote the formation of rigid clusters or displacement to the gel state—processes

that bring phospholipid acyl chains closer together to favor propagation steps [113–

115].

XII.



SUMMARY



This chapter has attempted to highlight the major chemical activities associated with

phospholipids and the relevance of these activities to the function of phospholipids

in foods. When present in oils or formulated floods, phospholipids may have either

detrimental or beneficial effects. As a major component of membranes, phospholipids

may also impact the quality of food tissues to a significant extent. Consequently,

their modifying presence should not be overlooked, even when they represent a small

proportion of the total lipid of a given food tissue.

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