和饮料有关的英文文献
Eur Food Res Technol (2005)221:382–386DOI 10.1007/s00217-005-1184-6
O R I G I N A L P A P E R
Eleonora Miquel Becker ·Daniel R. Cardoso ·Leif H. Skibsted
Deactivation of riboflavin triplet-excited state
by phenolic antioxidants:mechanism behind protective effects in photooxidation of milk-based beverages
Received:10January 2005/Revised:24February 2005/Published online:19May 2005 Springer-Verlag 2005
Abstract The water-soluble plant polyphenols rutin, (+)-catechin, and ( )-epigallocatechingallate were each found to be efficient quenchers for the triplet-excited state of riboflavin in aqueous solution using nano-second laser flash photolysis. The deactivation followed second-order kinetics with rates close to the diffusion control:( )-epigallocatechin gallate (1.70€0.2 109l mol 1s 1), (+)-catechin (1.44€0.04 109l mol 1s 1), and rutin (9.7€0.2 108l mol 1s 1) at 25 Cand pH =6.8. No synergetic effects for mixtures of the phenolic compounds were found. Hydrogen atom transfer is calculated to be more exergonic than electron transfer, suggesting the hydrogen atom transfer mechanism to be operative. The efficient deactivation by the plant polyphenols makes this type of compounds of importance as protectors against light-in-duced oxidation in flavored milk-based beverages. Keywords Plant polyphenols ·Photooxidation ·Triplet riboflavin ·Real-time kinetics
Exposure to light is known to result in development of off-flavors in dairy products [1].Riboflavin (VitaminB 2) present in milk is an efficient photosensitiser, which upon absorption of visible light forms excited-singlet states, which by intersystem crossing yields the very reactive triplet-excited state [2].In milk and cheeses, the triplet
E. M. Becker ·D. R. Cardoso ·L. H. Skibsted () ) Food Chemistry, Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, Frederiksberg C, Denmark e-mail:[email protected].:+45-35283221Fax:+45-35283344
D. R. Cardoso
Department of Chemistry and Molecular Physics, Chemistry Institute of S¼oCarlos, University of S¼oPaulo,
C.P. 780S¼oCarlos, SP, Brazil
riboflavin may oxidise proteins forming low-molecular weight sulphur compounds like dimethyldisulfide result-ing in a “burnt-feather”off-flavour of the product [1,3].Water-soluble antioxidants such as uric acid present in milk have been shown experimentally and by ab initio calculations to quench triplet riboflavin by electron-transfer rather than by the hydrogen-atom transfer mechanism in competition with protein oxidation [4].New types of milk-based beverages are being intro-duced in the market with natural flavourings based on chocolate, various herbs and fruits. Such products will contain plant polyphenols like catechin from chocolate, rutin from fruits and ( )-epigallocatechingallate (EGCG)from tea, which are known as antioxidants in lipid oxi-dation [5,6].Such compounds could also be effective as quenchers of triplet riboflavin and protect milk whey proteins against degradation and consequently against formation of off-flavours. We have accordingly selected three plant polyphenols important in such flavourings and studied their reaction with triplet riboflavin alone and in combination. The results presented are based on real-time kinetic methods using nano-second laser flash photolysis combined with transient absorption spectroscopy set up to generate the excited-state of riboflavin and for detection of the reaction intermediates.
Chemicals
Riboflavin, rutin hydrate, (+)-catechinand ( )-(EGCG)of analytical grade were purchased from Sigma-Aldrich (Steinheim,Germany) and used as received. Coumarin 120, spectroscopic grade, was obtained from Lambda Physic (Göttingen,Germany). All solvents were of HPLC grade supplied by Lab Scan Analytical Sciences (Dublin,Irland) and used without further purification.
Table 1Second-order rate constant (k ), one-electron redox potential (Eq ), calculated D G q ET , D G ¼, and D G q HT for the triplet riboflavin deactivation by plant polyphenols at 25€0.5 C,N 2-saturated acetonitrile:phosphatebuffer 1:1solution adjusted to pH =6.8(I=0.01M) Plant Polyphenol
( )-epigallocatechingallate (EGCG)(+)-catechinrutin
rutin:EGCG(1:1)rutin:catechin(1:1)
(+)-catechin:EGCG(1:1)
k (Lmol 1s 1) 1.70€0.2 1091.44€0.04 1090.97€0.02 1091.52€0.1 1091.44€0.2 1091.42€0.05 109
E q vs. NHE a (V)0.430.360.40–––
D G q ET (kJ/mol)– 142 145-––
D G q HT (kJ/mol)– 367 354–––
D G ¼
(kJ/mol)200.8204.9214.6–––
a E q
vs. NHE data from reference
[8]
Sample preparation
Riboflavin and the antioxidants were dissolved in a solu-tion (1:1,v/v)of acetonitrile:phosphatebuffer (I =0.01M; pH 6.8). The samples consisted of a mixture of 125m M riboflavin and 125, 250, 500, and 1000m M of the anti-oxidant. For antioxidant combinations equimolar concen-trations of each pair of antioxidants were added to the riboflavin solution obtaining the same final concentration of 125, 250, 500, and 1000m M. The samples were purged with high purity nitrogen in curvets closed by rubber septa for 10min. All solutions were protected from light prior to experiments.
Laser flash photolysis kinetic experiments
Laser flash photolysis experiments were carried out with an LKS.50spectrometer from Applied Photophysics Ltda (Leatherhead,UK). The third harmonic at 355nm of a pulsed Q-switched Nd-YAG laser was used to pump a dye laser Spectron Laser System (Rugby,UK) using Coumarin 120which has an emission peak at 440nm. The intensity of the laser pulse was approximately 2.7mJ cm 2. A R928photomultiplier tube from Hama-matsu (Hamamatsu,Japan) was used to detect the tran-sient absorption (300–800nm). Appropriate UV cut-off filters were used to minimise the sample degradation by the monitoring light. The samples were excited in 1.0cm 1.0cm fluorescence curvets from Hellma (Mul-heim, Germany). All samples were prepared using fresh solutions thermostated at 25€0.5 Cand purged with N before experiment.
2Following excitation with 440nm light-pulses of 8ns, aqueous solution of riboflavin were found to form triplet riboflavin as evidenced by the transient absorption spectra recorded after 1m s of the laser-pulse in the 300–800nm spectral region. The transient spectra were similar to those previously reported using the same experimental set up and with a comparable half-life time of 15m s in the ab-sence of quencher [3].
Repeating the experiments with rutin present together with riboflavin gave the transient spectra reported in Fig. 1. Immediately after excitation, 0.1m s, a riboflavin ground state bleaching centered around 445nm was ev-ident together with the triplet-triplet absorption with a maximum at 720nm. After 50m s an absorption band with a maximum around 480nm appears which is not seen in the absence of rutin. This absorption maximum was found to increase in intensity concomitant with the decay of the triplet riboflavin absorption monitored at 720nm. Based on the transient absorption spectra of rutin obtained by pulse radiolysis [8],this transient spectrum was assigned to the rutin neutral radical. From the time profile up to 90m s observed for the riboflavin triplet-triplet absorption at 720nm and presented in Fig. 2, the decay rate constant was calculated using exponential fitting to the absorption time traces. The decay of triplet riboflavin was in each experiment fully described by single exponential decay as tested statistically, and the pseudo-first order rate constant is plotted in Fig. 3as a function of varying rutin con-centration. From Fig. 3it is further seen that the pseudo-first order rate constant for triplet riboflavin deactivation is linearly dependent on rutin concentration for excess of
Fig. riboflavin 2Absorption trating the decay exponential in time the traces fitting presence monitored to the and absorption absence at 720time
of nm quencher for the triplet traces
illus-rutin, and the second-order rate constant for the bimo-lecular deactivation of triplet riboflavin by rutin was calculated by linear regression and is presented in Ta-ble 1. It should be noted that only a small fraction of the total riboflavin concentration is in the triplet state, how-ever, the observed linearity confirms the pseudo-first or-der condition for the triplet decay.
Similar procedures were used for (+)-catechinand ( )-EGCG. The transient absorption spectra obtained for (+)-catechin are presented in Fig. 4, which are similar to the spectra obtained for ( )-EGCG(datanot shown). For (+)-catechin and ( )-EGCGthe spectral resolution is less clear due to an overlap of the phenoxyl radical absorption and triplet riboflavin absorption, as was evident from a lower total change in transient absorption. The concen-tration dependence for (+)-catechinand ( )-EGCGis also linear as is seen in Fig. 3. The second-order rate constant for the three plant polyphenols studied is reported in Table 1and each is a mean value of the slope from two or three plots like those shown in Fig. 3.
In order to explore a synergistic effect among these three plant polyphenols, they were combined two and two in three independent series of experiments as those de-scribed above. The apparent second-order rate constant for these combinations are also included in Table 1and it is seen that values are merely interpolations between the values of the specific rate constants for the individual compounds, indicating a lack of synergism [6].In addi-tion, the transient spectra, Fig. 5, obtained for the com-bination of (+)-catechinand rutin did not show the for-mation of the rutin radical which is expected in case of a synergic regeneration of the (+)-catechin.
Aiming to establish a detailed mechanism for the triplet deactivation, changes in free energy for an electron
transfer, D G q ET , and for hydrogen atom transfer, D G q
calculated from riboflavin triplet-state excited HT , were en-
ergy, D E 0,0, and E q and p K a for the involved species,
respectively:
Fig. 0.05solution and 4Transient at 25€0.5containing 80m s after difference C
125the 440m M nm absorption riboflavin laser pulse spectra recorded between and (8500ns) m of M a of deoxygenated (+)-catechinD G q ET ¼96:48 E q phenolic ÀE q
riboflavin ÀD E 0; 0D G q HT
¼96:48 E q phenolic ÀE q
riboflavin ÀD E 0; 0
À2:303RT Âp K a ðRibH ÞÀp K a ðphenolic þÞ
Ã
The values for the two types of deactivation mechanism for rutin and catechin calculated using the Rehm-Weller equation given above [9],are reported in Table 1inviting further speculation. All thermodynamic parameters for a similar calculation for ( )-EGCGare not available.
385
Discussion
The finding of an almost diffusion controlled deactivation of triplet riboflavin by (+)-catechin,rutin and ( )-EGCGis important when considering the resistance of several types of beverages and foods against light exposure. Riboflavin absorbs light forming the very reactive triplet-excited state by intersystem crossing from the initially populated sin-glet-excited state [1,2].Riboflavin is present in a wide variety of products, where it accordingly may act as a photosensitiser inducing protein oxidation as in milk and beer or inducing lipid oxidation as in cheese [1,2, 7].Riboflavin is a water-soluble vitamin and long lived triplet-excited state reacts with others compounds present in the aqueous phase by electron transfer (Eqs.(3),(4))or by hydrogen-atom transfer (Eqs.(5),(6))following ex-citation and intersystem crossing (Eqs.(1),(2))as shown in the following reaction sequence valid for aerobic conditions and which includes subsequent generation of superoxide anion radical (Eq.(6)):Rib þh n ! 1Rib Ã
ð1Þ1Rib Ã! 3Rib Ã
ð2Þ3Rib ÃþROH ! 2Rib Àþ2ROH þð3Þ2ROH þ! 2RO þH þ
ð4Þ3Rib ÃþROH ! 2RibH þ2RO ð5Þ2
RibH þO 2! 1Rib þH þþO 2 À
ð6Þ
ROH represent a phenolic compound which may re-duce the triplet 3riboflavin, 3Rib*,as shown in Eq. (3)or deactivate Rib*by transfer of a hydrogen atom to for-m 2RibH •, as shown in Eq. (5).For anaerobic conditions the reaction of Eq. (6)will be replaced by reaction with another electron acceptor. For the aqueous solution used in the present study the change in free energy both for the electron transfer Eq. (3)and for the H-atom transfer Eq. (5)mechanism were calculated based on E q , the standard reduction potential for one-electron reduction of the plant phenoxyl radical (RO•), and the bond energy for oxygen-hydrogen found in the phenolic compound, re-spectively. Establishment of a linear free energy rela-tionship (LFER)in order to distinguish between various mechanism normally requires larger series of compounds as in the study of reduction of ferrylmyoglobin by 14structurally related flavanoid aglycones and the glycoside rutin [8]or if only fewer compounds are being studied, in very large energy differences as for purine derivatives deactivation of triplet riboflavin [4].However, the mag-nitude for the D G q and D G q
ET for electron transfer HT for the
hydrogen atom transfer with D G q
HT being more negative, points toward a hydrogen atom transfer mechanism, Ta-ble 1, with the same ordering of the free energy of acti-vation, D G ¼, calculated from the rate constants deter-mined in the present study as for D G q
HT . More
compounds
386
of similar structure will have to be included in the studies before more definitive mechanistic assignment can be made. Such studies should, moreover, include the effect of pH, as a shift in the mechanism from the electron transfer under basic conditions to hydrogen atom transfer in acidic medium was observed for triplet riboflavin de-activation by cystein [3].It should also be noted, that ( )-EGCG is the more efficient reactant towards triplet ex-cited state riboflavin followed by (+)-catechinand the glycosid rutin. A similar ordering of polyphenols has been observed for reaction with free radicals like the super-oxide anion radical [6].
In conclusion, the plant polyphenol present in various flavourings used for milk-based beverages are efficient quenchers of triplet riboflavin responsible for light-in-duced off-flavours. Independent of the detailed mecha-nism such compounds may be important for flavour-sta-bility under light-exposure. Notably, any superoxide an-ion radical subsequently formed from the reduced dou-blet-state of riboflavin in Eq. (6)may also react with the plant polyphenols showing a dual role of these polyphe-nols both as triplet quenchers and as radical scavengers.
Acknowledgments New This research is part of the research programme Health Antioxidants and (FOODANTIOX)Strategies supported for by Food Quality and Consumer Research Development PDEE fellowship Foundation. in Öresund(BEXDaniel region 2476/01-9)
R. Cardoso (Öforsk)the Committee for Research thanks and the CAPES Danish for Dairy the References
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