Theory and applications of fluorescence spectroscopy in food research

Review Fluorescence spectroscopy is a rapid, sensitive method for characterizi,tg molecular environments and events. In ~ of its utility, food researches have been slow to adopt fluorescence methodology, partly because its value has gone unrecognized. This article presenls a brief overview of the theon/ of fluorescence spectroscopy, together with some examples of applications of this tc~.hniclue to illustrate its potential for addressing key problems in food science.

The history of scienlific x e s e a ~ is replete with examp~s of discoveries and technological advances in one discipline that lay unrecognized or underufilized by related disciplines until their significatr~e was gradually renlized. Flumesccncc s F - ~ is onc such technique, whose theory and methodology have been extensively exploited for studies of molecular structure and function in the disciplines of both chemistry and biochemistry. However, in food science, a discipline based on chemical principles, the utility of f l ~ n c ~ for molecular studies has not been fully recognized. Fluorcscence spectroscopy has enjoyed widespread use in investigations of the stngtute, function and ree~vity of small moleculest, synthetic polymers2, proteins and other biological molecules~ , and in studies of cell structure and functions. Although fluorescence microscopy and other fluorescence analytical methodologies have been employed as empirical ~ . ~ , ' ~ d tools for disceming food quality~, few specifically molecular applications have been pursued. H ~ n c e spectroscopy has the same potential to addrcss molecular woblems in food science as in the biochemical science field, because the scientific questions that need to be answered are closely related. For example, those meflmds that have been _applied to the m~dy of protein denalxwation or of protein inten~em w i ~ figands in biomedical applications are applicable to the study of food protein detmturation or of the interacti~m of a food protein with other ingredients. Fluorescence spectroscopy off~'~ sevcral inhercnt advantages for" the c ~ o n of molecular teatfiom and intera~iom. Fn~t, it is 100-I0~0 times mete ~n~i~ive than ~ techniqees. S e c o ~ f l u o ~ e n t compounds are exquisitely semifive to environment. Tryptophan residues that are beried in the hydrophobic interi~ of a ~ for example, have different fluot~cent ptopetti~ than re,idu~ that are on a hydrcphilic surface. This environmental sensitivity enables a researche~ to c ~ coefmmalioual changes such as those attnbutable to the thermal, Gal~ ~A Serm~mllis at the L'~,4~ranmof Fexl Sc~ce and Huron Nu~F'~, Michi~ 51~ Uni~mi~, FastLam~ MI 48024-D24, USA (fax: +]-517-353-7~0;e-r~h s~allaleemsu.,.,du).~ D. is at the Dep~hnem~ FoodScience,Ru~rs Univenity,FO Box 231, CookColic, NewBrunswick,NJ08~03-0231,USA{fax:+1-9~-932-6776; e-mail:rickeal,caltlv~x.n~Ees,edu). Trendsin FoodScience& TechnologyMarch 1995 [Vol. 61

Theory and applicatiom of fluorescencespectroscopy in food research Gale M. Strasburg and RichardD. Ludescher solvent or surface denaturation of ~ as well as the ~ e r a e t m of p r o t e ~ w~h other food compmea~ Tnkd, most aumescence mcthods me te,~vely rapid: thin, a m b s t a n ~ amoent of infommioa ¢ ~ be qeiddy obuamJ, in ~ h ~ of ~ a~ica~ for ~o~escence specuoscopy, this t~'view will mmmmize some aspects of the technique tha~ we believe have womise f ~ providing novel infonn~ea on problems that Ke pecaliar to fe¢~ science.

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absorption and emission The absorption of light of visible aud UV wavelengths induces an elemm~ transitionthat involves a redhtribution of the electron density within a ,~.~omophme (see GI~'~,'S.). Typi~lly, the absorbed energy is converted into inmunolccular vibra~m that am dissipated to the local environmeat; the enerl~ is lost as ~az. In some o r g a ~ ~ especially throe coamining a system of conjugaml double beads, the excita~ca ~ ~ be eaitml m a pbm~ Ptm~t mi~sica from a singlet state is fluorescence; delayed emission from a triplet state generated by inmsystem amsing is phmphenm:enceL The e n e r ~ difference between ~.be absorbed and the emitml pho(om (which is referred to as the Stok~ shiftfor fluorescence) reflectsthe ene~ lostto the e a s t as heat. The special value of luminescence, and f l ~ for molecular studies is the exuaoMinary sensitivity o~"the emitting chromephore to the chemical and phToical proi~miea of the local e n ~ Luminescent molecules, chosen for their specific spectral properties, can be used as molecular "reporters' to provide information on ceafennatie~ changes, hyd~ty, ~ binding, and various other envircameutal effects5.~. Such molecular wobes an~ either intrinsic of exuinsic; exurinsic gobes are classifted as being either covalent or dispersible, lnerinsic tm~bes me p m of the molecule or system under study, such as a trypcq~ms residue in goteins. Covalent exuinsic probes a ~ ~ that have been chemically modified to include a OJnctional group that r e a ~ with a specif~ chemical group on the macmmol~ule to be labeled. Fluor--~,cein-S-isolhioeyanate (Fig. la). for examp~ c o m p s = ~ ~uor=scein chromopho~ coup~d

3-(p-(6-phenyl)- 1,3,5-hexatrienyl)phenylpropionic acid (I,6-DPH propionic acid; Fig. 10, is more restricted because the charged substitaent is anchored at the membrane surface whereas the lipophilic tail of the probe is localized in the hydrophobic interior of the membrane. Luminescence is able to provide molecular information because the energy distribution, the intensity and the polarization of the emitted light me related to the physical and/or chemical properties of the molecule and its immediate molecular environment4. 1Ile energy distribution of fluorescenc¢ emission, typtcally expressed in the form of wavelength, can provide significant information about the chemical and physical state of the matrix (solvent. macromnlecule or other condensed phase) in which the chromophore residess. Because the energy of emission is proportional to the energy difference between the ground and the excited states of the probe, differential interactions between the matrix and the two states modulate the emission energy; when the excited state is stabilized more than the ground state, for example, the energy difference, and thus the emission energy, decreases (the emission wavelength increases). In the 'general solvent effect', the dipolar properties of the solvent, measured by the dielectric constant and the refractive index, mediate the interaction of the solvent with the chromophore. In the 'specific solvent effect', distinct chemical interactions between the solvent and the chromophora, such as hydrogen bonds, mediate the interaction. The intensity of fluorescence emission is proportional to the probability that the excited chromophore will emit a photon; this probability is termed the quantum yield for emission, and is directly related to the measured emission lifetime of the chromopbore. Several factors in addition to the chemical structure of the probe influence the quantum yield: the polarity, viscosity (related to the frequency of molecular collisions) and chemical structure of the immediate solvent environment, and the presence of small molecules that quench the emission through either collisions (Stern-Volmer quenching9) or resonance interactions (Forster quenchingt°). Their relative importance varies from one chromophore to another. Measurements of emission intensity can be related to, for example, the extent of solvent exposure of a chromophore, its proximity to a specific site or sites, or the molecular dynamics of the immediate environment surrounding the chromophore. with the isothincyanate moiety, which reacts with unprotonated amino groups of proteins. Distersible extrinsic probes have chemical properties that allow them, once physically dispersed throughout a system, to partition into and report on the molecular properties of a specific "phase' of that system (such as a protein surface or a lipid phase). The fipophilic solubility properties of the dispersible probe 1,6-diphenyl-l,3,5-hexatriene (I,6-DPH; Fig. le), for example, enable it to partition into the hydropbobic environment of a membrane where it becomes fluorescent. The orientation in the membrane of the propionic acid derivative of this compound,

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Fluorescence polarization and anisotropy The absorption of electromagnetic energy (light) involves an interaction between the oscillating electric field and the transition dipole of the fluorescent molecule. This transition dipole is a preferred direction in the molecule that acts as an 'antenna' to resonate with the electric field. Excitation with polarized fight excites only those molecules whose transition dipoles me oriented parallel (on average) to the polarization direction. When the excitation polarization is vertical, this photoselection process generates a vertically oriented popuiatinn of excited molecules. The light emitted by an

Trendsin FoodScience& TechnologyMarch 1995 Wol. 6]

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F'lg.t Somecommonlyusedextrinsicfluo~'escenceprobes:laL tluo~,cein-S-isothiecyanate;(b),dxxtamine-5-iodoacetamide; (c),54(((2-iodoacetyl)amino)ethyl)amino)naplghalene-1 -sulfonicacid (1,5-1AEDANS);(d), 1-anilinon~lmm-8-sulfonic acid(1,8-?uNS); (e), 1,G-diphenyl-l,3,S-hexatriene (1,6-DPH);and (f), 3-(p-(6-phenyl)-!,3,S-Imxatfienyl)phenylptopionic acid(I,G-DPHpn~oionicacid). excited molecule is also oriented parallel to the transition dipole. If some of the molecules rotate (owing to molecular motion) before emission, the polarization of the emitted light will be different from the original polarization direction; that is, the emitted light will he pratially horizontally polarized. By measuring the intensifies of the vertically and horizontally polarized components of the emission (I, and lu), it is possible to directly monitor the rotation that occurs while a molecule is excited m. The aniso~opy (r) provides a direct measure of those rotations that occur during the lifetime of the molecule's excited state. The anisotropy varies between a maximum value of 0.4 for immobile molecules and 0 for rapidly rotating molecules '°. If the probe is rigidly attached to a larger macromolecule (covalently attached to a protein sulffwdryl group, for example), tile motions of the probe will, to some extent, reflect the motions of the macromolecule in much the same way that the motions of a tennis racket reflect the motions of a tennis player. The timescale of the detected motions depends critically on the lifetime of the chromophore: tryptophan fluorescence (with a lifetime of - 4 n s ) can be used to monitor the rapid internal of segmental motions of a small protein, whereas erythrosin phosphorescence (with a lifetime of ~0.3ms) can be used to monitor the slow rotational motions of a large macromolecular complex. Luminescence, like all techniques employing molecular probes, is site specific; this is one of its strengths. Probes can he used to investigate the properties of either a single compound or a class of molecules in a complex mixture (e.g. lipids in a membrane), or the properties

Trendsin FoodScience& TechnologyMarch1995Wol.6]

of a single site in a macromolecule. However, as the measurement involves fight absorption and emission, the turbidity of the sample imposes special constraints; polariT~tion measurements, for example, ate problematic and often hnpossible in the case of highly tmbid or sofid samples where depolarization due to scalledng dominates the fluon~conce signal. Althoegh fluornscence measurements are relatively straightforwazd, measurements of phosphoRscence are complicated by the absolute requirement for the removal of oxygen (an efficient quencher of the triplet state) and also by the low quantum yield for phosphotesconcet.

Intrinsic probes Tryptophan is the most widely used inlrlnsic wobe; because it is the rarest amino acid found in g l ~ l a r proteins (with a frequency of ~1%), precise, site-.~pecific molecular interpretations of luminescence data are often possible. The photophysics of the indole side chain of u-yptophan are complex~".'2. The ncar-UV absorp~on spectrum, centered at 280um, displays two overlapping absorbance transidons. In contrast, the fluorescence emission spectrum is broad and featureless because it results from only one of the absorbance transitions. Emission is characterized by a large Stokes shift, which varies with the polarity of the environment (Fig. 2); the fluurescence emission peak is at -350mo in water but the peak shifts to ~315nm in nonpolar media, such as within the hydrophobic regions of folded proteins. The emission spectrum thus provides an estimate of the chemical natm~ of the side chain environmentt~. Tryptophan fluorescence (with a quantum yield typically

71

Because the excitation spectra of these two fluorophores overlap, only tryptephan can be selectively studied, by using excitation at 295 am, a wavelength at which tyrosine does not absorb. o

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Extrinsic probes Extrinsic Wobes are used to characterize molecular events when intrinsic flnorophores are absent or are so numerous that the interpre~tiou of the data becomes ambiguous. Ex~-insic probes may also be used to obtain additional or complementary information from a specific macromolecnlar domain - one that does not contain an intrinsic reporter group. Extrinsic probes may bind covalently to a particular functional group or may partition into specific phases. In either case, the distinct spectral properties of exu~_nsic probes offer the possibiEty of using fluorescence spectroscopy to resolve motecnlar events associated with one species from those of other species in a complex mixture. Covalent fluorescent probes vary in specffal properties and chemical reactivity. The availability of various combinations of fluoropberes with particular spectral properties and functional groups with specific chemical reactivity enables an investigator to select the desired chromophore to suit a particular need, and then to conjugate the probe specifically to a single or limited number of sites in a maeromolecnle7. Two examples, described below, illustrate the type of information that it is possible to obtain through the judicious selection and use of covalent fluorescent molecules. One caution regarding the use of covalent probes must be noted: as with any chemical modification, appropriate tests should be performed to detenmne the stoiet~omelry of protein labeling, to identify the site(s) that ate labeled, and to demonstrate that defivatizatiou has not sig=dfieanfly altered the s~acture or function of the molecule under study. Dispersible probes axe exploited for their abifity m partition into particular sites in a macromolecnle or organelle based on soluhifity or unique affinity. The binding of a dispersible probe is usually accompanied by a subslantial increase in quamum yield and hence fluorescence intensity. Numerous dispersible probes arc available; two of the best known, 1-unilinonaphthalene8-sulfouic acid (1,8-ANS; Fig. ld) and 1,6-DPH (Fig. le), will be discussed below.

in the range 0.1-0.5) is readily quenched by protonated amino and carboxyl groups and amide bonds - functional groups that are common in proteins. As this quenching occurs on contact or at close profanity (-0.1-0.3 rim), changes in fluoreseenc~ intensity due to changes in quencher proxhnlty provide a sensitive (albeit often arbi~'ary) inddcator of changes in protein conformation, such as that resulting from protein~gan~ binding, protein-proteln associations or protein denatcratiou~a.~s. The fluorescence emission for ~Tptnphen is polarized with an in~asie aniso~opy (ro, which is the anlso~ropy in the absence of chromophore motion) of 0.3; this value., which is less than the usual value of 0.4, reflects h~te~octious between the two overlapping encRed states. The v~u~ of r o varies with the exei~tion wavelength and only a ~ its limiting value at >3~Onm (Ref. 16). With a l i f e ~ e of just nanoseconds, ~ryptophan mds ~ p y is sensitive to the fast ~ntemal and segmental ~icatbr~ of in,into fluorescence motions of p~teL~s or the overall rotation of s n ~ l - Protein denaturation and binding sites in 13-1actoglobulin to m~l~nm.sized p~oteins (<40~0Da). Thus, R is poss[~-lactoglobulin, an 18 300 Da globular protein, which ible to use obse~ed changes in a n ~ s ~ p y to monitor constitutes -50% of total whey protein, contains two shap~ changes associated with protein unfol~ng, mass h'Tptephen residues at positions 19 aad 61 in all genetic changes resulting from aggrcgatinn, or couformatiouul v~xiants. Molecular studies of this pro~in may provide changes associated with a change in a particular protein specific tools for understan~ng and modulating the domain. functional prope~as of this protein in ~ and in other T y r o s ~ , anolber ~nino acid, is aLso an h~Irinsie foods 17. A review of the, unfm~uaately scanL literaflu~esccnt chromopbo~e, and is potentia~y u~ful for ture on inlfinsic [3-1actoglobnlin fluorescence will help fluorescence studies of proteins lacking tryptophans,n. to iUusa'ate soma of the potential of ~'yptophan fluorHowever, it should be no~ed tha~ most prote~ns that pos- escence for molecular studies of fund prmeins. The two sess v:yptophan residues also cou~fin Wrosine residues. O~ptophan residues are iaaecassible to water in

72

Trendsin FoodScience& TechnologyMarch 1995 IVol. 6]

hydrophobic environments in the protein interior Is.19. Changes in the fluorescence intensity and emission wavelength (energy) have been used to monitor the denaturation of ~-Inctoglobulin by urea and organic solvents2° and also by temperature19. At pH 6.5, the protcin (variant B) undergoes a reversible conformational change at 50°C, which affects only one of the tryptophan residues, and a second irreversible conformati~nal change at 70°C, which affects the other. Changes in pH do not affect the intensity or energy of emissinnIs but do affect the anisouepy2t; the relaxation times for rotational motion are ia quite good agreement with those expected for the monomer, dimer and octamer forms of the protein. The binding of retinol to [3-1actoglobulin has been measured using retinol fluorescence, excited directly and through energy transfer from the inuinsic uyptophan residues22. In addition, complex formation of [~-lactoglobulin with sodium dodncyl sulfatets and with phosphatidylcholine~ has been monitored using measure: ments of intensity and energy; at least one of the tryptophan residues is involved in phosphatidylcholine bindingu.

Appl~tions of extrinsic fluorescence Molecular basis of pale, soft exudative pork: receptor-ligand binding studies Recently, it has been shown that the incidence of pale, soft, exudative pork (PSE pork) is associated, in part, with an inheritable defect in stress-susceptible pigs, resulting in an abnormal CaZ*-release mechanism owing to a single amino acid.mutstion in the CaZ+-releasechannel of the sarcoplasmlc reticulum (SR)24. SeVelul factors alter CaZ+-release activity in normal skeletal muscle, including binding of the Ca2+-binding protein cabnodulin (CAM) to the channel, which partially inhibits channel-protein activityu. Studies were initiated to define the binding equilibria of CaM for both the normal and the mutant SR channel proteins, and thereby to determine whether CaM regulation of the stress-susceptible channel protein is altered. SR vesicles were prepared from genetically defined normal and stress-susceptible pigs. Crosslinking studies emplnying radiolabeled CaM established that the most abundant receptor for CaM in SR vesicles was the channel protein. Thus, it was possible to use fluorescence spectroscopy to characterize the binding of labeled CaM to the channel protein, which is embedded, along with the myriad of other proteins, in the SR membrane. Rhodamine maleintide was selected for CaM labeling because of several advantages it offered for use in characterizing receptor-ligaud interaction. First, the quantum yield of rhodamine is relatively high, making it useful for experiments even at concentrations as low as I riM. Second, the probe possesses relatively long excitation and emission wavelengths (-570nm and -60Ohm, respectively), making it useful in slightly turbid samples: because light scattering is inversely proportienai to the forum power of the wavelength, the contribution of scattering of the excitation beam by turbid

Trendsin FoodScience& TechnologyMarch1995IVol.6]

samples, such as membrane vesicles, is ~ as one moves towards the red end o f t b e spectouu. Third, because thedami~ is relatively insensitive to its environment compared with other probes, ~ n e f i ~ is less likely to vary with cenfecrmational changes. These characteristics ~ to make rhodamine an ideal probe for detecting binding e~ents via flum~cence pularization. Upon binding of the rbedamit~labeled CaM to the channel wotein embedded in SR vesicles, the anismmpy increased, reflecting the increased mokculag ma~ of the complex and slower rotational motiens of the probe. As the fraction of the ligand (CAM) that is bomld is g o portional to anisotmpy, it was possible to estimate the affiulty and stulchinmetxy of binding of CaM to tbe chmnel t ~ - i n f~m Ix~h nonml~ and stress-sascepe~ t ~ ~ under different metal ion conditions that to the contractile and resting states of muscle. The results indicted that the altered stoichionmry of CaM binding to the channel protein in stre,-msceptible pigs may reflect altered cooperative imerectio~ between channel-pstnein subunits. These results further support tbe hypotbesis that abnomud Ca~÷homeostasis in mmele cells, resulting in part from altered CaM regulation of Ca2÷release, results in PSE meat. Confonnationai changes in woteins Covalent extrinsic probes, like intrinsic probes, may be used to provide infommtion on changes that are induced in proteins. The IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1sulfen/c acid) label (Fig. lc) has enjoyed wide usage, especially in the muscle protein field. It is highly specific for free sulfbydryl groups because of its iodoacetemido fimctinnal group, but in the absence of available sulfhydryl groups in a protein, it may label methionine residueszs. In the case of tmpenin C, the Ca2+=binding subunit of the tmpenm complex in muscle, the sole cysteine (Cys98) is located between the two globular domains of this dumbbell-shaped molecule. each domain binds two Ca2+. When IAEDANS-inbeled apo.u-cponin C binds tbe tint two Ca2* in the C-terminal domain, the f l ~ n c c intensity increases by ~15%, reflecting a confonnafional change in the environment of Cys98, which results in increased h y ~ c i t y of the environment of Cys98 (Ref. 29). As the u~rd and fourth atoms of Ca 2+ bind to the N-tetminul domain, the f l ~ increases by an additional 5-10%, ~ggestins a further conformationai change that is sensed by the probe at Cys98 (Ref. 29). In addition to identifying structural changes in the protein, the probe has been used to define pCa~ values (or -log[Ca2+] at which 50% of the Ca2*-binding sites are occupied) that produce the confotmafional changes induced in the Cterminal domain (pCa~=7.5) and the N.terminai domain (pCa~=6.2) of troponin C (Ref. 29). These results indicate the utility of fluorescence spectroscopy in detecting figand-induced conformatinnal changes in proteins when characterizing stngtute-fingtion relationships.

73

Cotructural studies on the casein micelle The s ~ c t u r e of the casein rnlcelle and the role of phosphate asters, of calcium and of protein conformafioual changes in stabilizing the micellar structure have long eluded biochemists and food scientists. Javor et a l ? ° have combined fluorescence approaches with light severing, turbidity measurements and primary structural information to characterize thermally induced structural changes of phosphorylated human a-casein in the presence and absence of Ca 2". As triply phosphorylated a-casein was heated over the temperature range of 5-40°C, the fluorescence intensity of added 1,8-ANS (Fig. ld) increased only slightly in the range 5-20°(2, but a sharp escalation in fluorescence intensity occurred in the range 25-40°C. As 1,8-ANS binds to hydrophobic areas on a protein, the increase in fluorescence intensity suggests that a conformational change has occurred either to increase the number of hydrophobic sites on the protein or to change the affinity of the hydrophobic sites for I,g-ANS. The analysis of the data strongly suggested that thermally induced conformational changes just below physiological temperature (37°C) led to an increase in the number of hydrophobic sites that prorooted protein aggregation. Complementary dam obtained from turbidity and fight-scattering measuremems further suggest a role for ion-bridge formation between Ca 2÷and inorganic phosphate in addition to hydrophobic interactions stabilizing the raiceilar structure.

be a useful method to probe the peroxidation of membranes in studies on the mechanism of action and efficacy of food anfioxidents. Cond~ns We have attempted in this review to summarize those aspects of fluorescence spectroscopy that may have value for solving problems in food science and technology. The techniques described, which depend on the measurement of the intensity, energy and polarization of fluorescence emission, have been illustrated by examples taken from the food science and related literature. We hope that this coverage will introduce a novel class of techniques to some, and broaden the range of techniques available to others. We must emphasize, however, that the field of luminescence is not limited to the specific techniques detailed herein. Space limitations have prevented full discussion of the current applications of fluorescence microscopy and analytical techniques to food research~ or the wealth of applications of fluorescence to biomedical science33, for example. We have also been unable to discuss any of the technical aspects of the fluorescence measuremen!s themselves. For the reader who is interested in conducting fluorescence experiments, we strongly recommend consultation of the text by Lakowicz4 for a very readable presentation of fluorescence theory and instrumentation, the article by Bentley et al. ~* for more information on fluorescence anlsotropy measuremems, and the publication by Hangland? for information on the variety of probes available a ~ ~ complete set of references on the applications of each probe. It must suffice here to add that fluorescence measurements ate rapid, accurate and require only very small quantifies of sample (nanomoles or less). Fluorescence instrumentation is also relatively inexpensive (instruments are priced in the range ~U3515000-50000) and easy to use. In general, fluorescence experiments am relatively easy to perform; as in many fields, it is the planning of appropriate experiments and the analysis and accurate interpretation of the data that require more extensive experience.

Membrane stability and the mechanisms of rancidity development The integrity o f biological membranes is closely associated with the quality of plant and animal foods. Peroxidation of membrane phospholipids disrupts membrane structure and function3~. These changes may be manifested by dec~lsed membrane fluidity, altered membrane-protein activity and the failure of the membrane to act as a semipermeable barrier, resulting in the development of off-flavors in foods, as well as physical effects such as fluid exudation of meats. Monahan et a l ? 2 used fluorescence anlsolropy to examine the effect of dietary supplementation with vitamin E on membrane stability, l~crosomes were wepated from pigs fed a low level of vitamin E, from pigs supplemented with References vitamin E and from pigs fed a diet containing oxidized 1 Parker,C.A. (1968)Photoluminescenceof Solutions, Elsevier oil. The dispersible probe 1,6-DPH (Fig. le) was incor2 Guillet,J.(198S)Polymer Photophysics~nd fhofncl~mistJy, porated into the membranes, and peroxidation was inCambridgeUniversityPress itiated by the addition of ferrous chloride. Both the con- 3 Lakewicz, l.R.(1983)l~inciplesofFluorescenceS~y, PlenumPress Sol microsomes and those obtained from pigs fed vol. 3: 4 Lakowicz,LR.,ed. (1992)Topicsin Fluorescence5 ~ o p y , oxidized oils showed an inunediate rise in membrane Biochemical Applications, PlenumPress anis~ropy, reflecting increased membrane rigidity reFlumescent Probes in Cellular and Molecular Biolosy, $ Slavik, J. (1994) suiting from tlm presence of peroxidation products. CRCPress However, microsomes from the vitamin E-supplemented 6 Munck,L. and de Francisco,A.,eds(1989)FluorescenceAnalysis in animals showed an increase only after a long lag period. Foods,Longman~lentific andTechnical The lag period was presumably associated with the ac7 Hausland,R.P.(1992)Handbook of FluorescentProbesand Research tivity of vitan~n E in scavenging free radicals. The inChemicals,Mo~'culerProbesInc, Eusene,OR, USA crease in anisotropy was closely associated with the pro8 Demchen~,KP. (1986)Essays8iod~m. 22,120-157 duction of ghioharbi~dc acid.reactive s u b s ~ c e s and 9 Eftink,M.R.(1991)inTopicsinFluor~cenceSpectl~T.,copy, Vol.2: with deteriorative changes associated with the exudation Principks (Lakowicz,I.R.,ed.),pp. 53-127, PlenumPress Vol. 2: of meats. These results indicate that fluorescence could 10 Cheuns, H.C. (1991) in Topicsin Fluorescence~ y o 74

Trends in Food Science & Technology March 1995 IVol. 6]

Frindp,~s(Lakowicz,I.R.,nd.),pp. 128-176, PlenumPress 11 Beechem,J.M.and Brand,L (1985)A,,-mu.Rev.B/ochem.54, 43-71 12 Demchen~,A.P.(1986)U/~'av/o/et. ~ of Pro~/ns, Spdn~er-Veda8 13 Burstein,E~,.,Vedenldna,N~S.andIvkova,M.N. (1973)R~a~ac/~m. /V~ofob[o/.18,263-276 14 Talbot,J.C.,Dufourcq,J.,de Bong,J.,Fauo0n,J.R.and Lurson,C (1979)FEBSLet~102,191.-193 15 Effink,M.R.(1994)B/aphys.1.66, 482-501 16 Valeur,B.andWeber,G. (1977)F/~.,~ochem.Pho~ob~o/.25, 441-444 17 Bat~,C.A., Brady,J. and Sawyer, L (1994) TmndsFood ~ci. Technol. 5, 261-265 18 Kaptanas,R., Bukolova,T.G.andBurshtein,~A. (1973)Mo/. B/o/. (Mo~ow)7, 753-759 [in Russian] 19 Mills, O.E. (1976) Biochim. Biophys. Acta 434, 324-332 20 Kaplanas,R., BuEolova-Odova,T.G.and BursMein,E.A.(1975)Mo/. B/o/.(Moscow)9, 795-804 [in Russian] 21 Wahl,P.,Timasheff,S.N.andAuchet,I.C (1%9) BiochemistryS, 2945.-2949 22 Fusate,R.D.and Long.P-S.(1980)B/ochhn.B/op/p/s.Ac~ 625, 28-42 23 Brown,E.M.,C.arroll,RJ.,Ffeff~',P.L andSampugna,J.(1983)L/p/ds

18,111-118 24 Mad.eriCh,D.H. andFnigips,M.~.(1992)$d~ce 256,789-794 2S Mebsem,G. (1986)B / o c / ~ 2 5 , 236-244 26 Yar~,H.C, Rendy,MM., Burke,C andS~d~& GM. (1994) e[oc/~m/say33, 518--525 27 Yans,H.C.,Pzedy,M.M.,Mickeh~, J.R.,Louis,CF. andSI~ G.M.(1993)B/opt. I. 64,A.303 28 LaPode,D.C.,Kc~,r,CH.~Ohvin,B.B.andS~otm,D.B.(19~1) B / o d ~ 20, 3%5-3972 29 Graham,Z., Le~is, P.C , ~ 1 G e ~ , I. (1~) I. e/o/.O~.,. 261,608-613 3~ l,wor,G.T.,Sond,S.M.,(3~an~,P.andSb~y, C.W.(1991),q.-ck. e~. e/op/~. 289,39-46 31 s~n~, v.w. (1991)c ~ xev.~0odsci. Ted~d. 3O,~¢;-533 32 D.).andMenbse~,P~ (i 994)I. A~r/c.FaodC/~m.41, 59-63 33 Taylor, D.L, Wal~oner, A.S.,~ , R.F.,Lanm, F. and B~rl~, R.R. (1986) A p p l ~ of F ~ in ~heBiomedical~iences, Alan R. Liss 34 Be~'ey,r,J..,Thomman,~K., rdebe,RJ. andHamwi~,P.~. (1~5) e/a~d~/~s 3, 356-3~6

Review

been isolated from conventional food sources, such as tea

The contribution of plant food antioxidants to human health

(green and black), sesame and wild rice, and also from other plant sources, such as rice hulls, and crude plant drugs. Data on new types of water-soluble and lipid-soluble plant antioxidants are provided, and the biolegical activity and functionality of these antioxidants are discussed.

NarasimhanRamarathnam,ToshihikoOsawa, HirotomoOchi and ShunmKawakishi

We have been actively involved in the isolation and characterization of endogenous plant anfioxidants that are believed to inhibit lipid peroxidation and offer protect~z a~ainst oxidative damage to membrane functions. Antioxidants have

It is well known that humans, as they grow older, become less active, have an increased probability of illness, and generally experience a loss o f optimum function o f all physiological systems. Evolutionary

processes have engendered the su,~ival of individuals of all living species until the time that they are able to rep~luce and pass on their genetic codes to their offspring. At sexual maturity, an age-dependent process sets in, progressing gradually, and leading eventually to the death of an individual, if death has not occun'cd from another means before thent. Nm.mim~mRm~r~he~ and Hbe4temoOd~ are at the ~pan Ir~u~ fcr the ConU'old Agin~ NikkenRx~s Co., Lid, 723-I, Hamoka,FukumiCity, Shizuoka - 437-01, ]apan. T e d ~ Osmu ( ~ n g author)and S~we ~ E 4 ~ i areat the Depa~m~,:of AppI~I and Biok~al Sciences, School of A ~ k u m , NagoyaUnive~i(y, Chikusa-Ku,Nagoya- 464-01, Japan(fax:+81-52-789-41201. Trends in FoodScience& Technology March 1995 IVol. 6]

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f m ' m ~ e n in

Oxygen, a vital component for the survival of the human species, is present in the atmosphere as a stable tdptet biradical (302), in the ground state. Once inside the h,nmn body, it can be ~-~usformed, by a fourelectron reduction pve~ess, to water, producing a superoxide radical (Oz.), a hydxoxyl radical (.OH) and hydxogen pezoxide (l~zOz) as the reactive immnedia~ (Fig. 1). Singlet oxygen ('0~) is formed from the ©x¢itcd s ~ e of various seusifizers such as chlorophyll, acridine and other pign~nts. Among the nmjor cellular and extracellular components, proteins, enzymes, lipids, DNA and RNA form the primary targets for these w.active oxygen ~pccics. However, o"x~ation of the unsaturated fatty ~ componenm or" cell membranes is the oxidative event that occuxs most z~equently inside the human bodyz. ©i595,E~ia.sci~ceudogz4.224,e~s~sog.so

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