Heat-induced changes in the ultrasonic properties of whey proteins

The physical aggregation of commercial whey protein isolate (WPI) and purified beta-lactoglobulin was studied by ultrasound spectroscopy. Protein samples were dialyzed to achieve constant ionic strength backgrounds of 0.01 and 0.1 NaCl, and gelation was induced in situ at constant temperatures (from 50 to 75 degrees C) or with a temperature ramp from 20 to 85 degrees C. Changes in the ultrasonic properties were shown in the early stages of heating, at temperatures below those reported for protein denaturation. During heating, the relative ultrasound velocity (defined as the difference between sample velocity and reference velocity) decreased continuously with temperature, indicating a rearrangement of the hydration layer of the protein and an increase in compressibility of the protein shell. At temperatures <50 degrees C the ultrasonic attenuation decreased, and <65 degrees C both velocity and attenuation differentials showed increasing values. A sharp decrease in the relative velocity and an increase in the attenuation at 70 degrees C were indications of "classical" protein denaturation and the formation of a gel network. Values of attenuation were significantly different between samples prepared with 0.01 and 0.1 M NaCl, although no difference was shown in the overall ultrasonic behavior. WPI and beta-lactoglobulin showed similar ultrasonic properties during heating, but some differences were noted in the values of attenuation of WPI solutions, which may relate to a less homogeneous distribution of aggregates caused by the presence of alpha-lactalbumin and other minor proteins in WPI.     

Size, stability, and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials

The effect of lipid composition [phosphatidylcholine (PC), phosphatidylglycerol (PG), and cholesterol] on size, stability, and entrapment efficiency of polypeptide antimicrobials in liposomal nanocapsules was investigated. PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30) liposomes had entrapment efficiencies with calcein of 71, 57, and 54% with particle sizes of 85, 133, and 145 nm, respectively. Co-encapsulation of calcein and nisin resulted in entrapment efficiencies of 63, 54, and 59% with particle sizes of 144, 223, and 167 nm for PC, PC/cholesterol (70:30), and PC/PG/cholesterol (50:20:30), respectively. Co-encapsulation of calcein and lysozyme yielded entrapment efficiencies of 61, 60, and 61% with particle sizes of 161, 162, and 174 nm, respectively. The highest concentration of antimicrobials was encapsulated in 100% PC liposomes. Nisin induced more calcein release compared to lysozyme. Results demonstrate that production and optimization of stable nanoparticulate aqueous dispersions of polypeptide antimicrobials for microbiological stabilization of food products depend on selection of suitable lipid-antimicrobial combinations.     

Anhydrous goat's milk fat: thermal and structural behavior. 1. Crystalline forms obtained by slow cooling

The thermal and structural behaviors of anhydrous goat's milk fat (AGMF) have been determined as a function of temperature using a powerful technique allowing simultaneous time-resolved synchrotron X-ray diffraction as a function of temperature (XRDT) and high-sensivity differential scanning calorimetry (DSC) measurements from the same sample. This first paper, aiming at the characterization of the physical properties of AGMF, we examine crystalline organizations made by triacylglycerols (TG) upon slow cooling at /dT/dt/ = 0.1 degrees C/min from 45 to -20 degrees C in order to approach system equilibrium. Three overlapped exotherms were observed by DSC upon cooling, whereas four endotherms were found on the subsequent heating at 1 degrees C/min. XRDT evidenced that AGMF crystallizes under four different lamellar structures, two with double-chain length packings at 41.5 and 38.2 angstroms and two with triple-chain lengths of 72 and 64.7 angstroms stacking. Simultaneous wide-angle XRDT has shown that initial nucleation mainly occurs in a packing of beta' type from approximately 26 degrees C, although some transient presence of alpha was detected. The absence of polymorphic transition, on heating, until final melting (approximately 40 degrees C) demonstrated the relative stability of the structures formed.     

Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil

During repeated deep-fat frying of potato slices at 163 degrees C in yellow or red palm olein of comparable fatty acid profiles, the oxidative stability (peroxide value and anisidine value) of the palm oleins was similar, and in yellow palm olein, the rate of antioxidant depletion decreased in the order gamma-T3 > alpha-T3 > delta-T3 (T3, tocotrienol). In red palm olein, which had a total tocopherol/tocotrienol content of 1260 vs 940 ppm in yellow palm olein and a corresponding longer induction period in the Rancimat stability test at 120 degrees C, only depletion of gamma-T3 was significant among the phenols during frying and slower as compared to that in yellow palm olein. The carotenes in the red palm olein were depleted linearly with the number of fryings, apparently yielding an overall protection of the phenols. In antioxidant-depleted palm olein and in phospholipid liposomes with added increasing concentrations of phenols, gamma-T3 was found to be a better antioxidant than alpha-T3. alpha-T3 and alpha-T (T, tocopherol) had a similar antioxidant effect in antioxidant-depleted palm olein in the Rancimat stability test, while in the liposomes the ordering as determined by induction period for the formation of conjugated dienes was gamma-T3 > alpha-T3 > alpha-T. The addition of 100-1000 ppm beta-carotene to antioxidant-depleted palm olein or liposomes (lycopene also tested) did not provide any protection against oxidation. In the liposomes, synergistic interactions were observed between beta-carotene or lycopene and alpha-T, alpha-T3, or gamma-T3 for carotene/phenol ratios of 1:10 and 1:2 but not for 1:1. In chloroform, carotenes were regenerated by tocopherols/tocotrienols from carotene radicals generated by laser flash photolysis as shown by transient absorption spectroscopy, suggesting that carotenes rather than phenols are the primary substrate for lipid-derived radicals in red palm olein, in effect depleting carotenes prior to phenols during frying. Regeneration of carotenes by the phenols also explains the synergism in liposomes. In the laser flash photolysis experiments, gamma-T3 was also found to be faster in regenerating carotenes than alpha-T3 and alpha-T.     

Ultrasonic determination of chicken composition.

An ultrasonic technique has been developed for measuring the composition of chicken meat. The relationship between the composition and ultrasonic velocity of chicken meat was determined using chicken analogues of different composition, prepared from dried chicken powder, corn oil, and distilled water. The ultrasonic velocity of chicken analogues was measured at temperatures from 5 to 35 degrees C using an ultrasonic spectrometer. The ultrasonic velocity increased with solids-nonfat (SNF) content at all temperatures but had a more complex dependence on fat content. Around 15 degrees C the ultrasonic velocity was independent of fat content; however, at lower temperatures it increased with fat content, and at higher temperatures it decreased. Semiempirical equations were developed to describe the relationship between ultrasonic velocity and chicken composition. To determine the usefulness of these equations, the ultrasonic velocities of various chicken meats were measured. The compositions of the chicken meats predicted on the basis of ultrasonic measurements were in good agreement with those determined by using standard methods (r(2) > 0. 97). The ultrasonic technique could also be used to measure the solid fat content of chicken fat. This study shows that ultrasonic velocity measurements can be used to characterize chicken composition. This method has great potential for application in the food industry because it is simple, fast, nondestructive, and reliable.     

Use of differential scanning calorimetry to study lipid oxidation. 1. Oxidative stability of lecithin and linolenic acid

The oxidation of linolenic acid (LNA) and soy lecithin was studied by differential scanning calorimetry (DSC) with linear programmed heating rates (non-isothermal mode). The interpretation of the shape of DSC curves is discussed, and it has been concluded that temperatures of the extrapolated start of heat release are the most reliable data for the rapid estimation of the oxidative stability of lipid materials. The Ozawa-Flynn-Wall method was used to calculate the kinetic parameters of the process: for LNA autoxidation the activation energy, Ea, and pre-exponential factor, Z, are 66 +/- 4 kJ/mol and 1.5 x 10(7) s(-1), respectively, and the autoxidation of lecithin is described by Ea = 98 +/- 6 kJ/mol and Z = 9.1 x 10(10) s(-1). Values of Ea and Z can be applied for calculation of the overall first-order rate constant of autoxidation at various temperatures, k(T). For the two studied lipids the comparison of k(T) values shows the inversion of their oxidative stabilities; that is, below 167 degrees C lecithin is more stable than LNA, k(T)lecithin < k(T)LNA, and above that temperature (termed the isokinetic temperature) k(T)lecithin > k(T)LNA. The calculated inversion of oxidative stabilities can be an explanation of similar observations for other pairs of lipids if the results of accelerated tests measured at temperatures above 100 degrees C are compared with the results obtained at temperatures below 100 degrees C.     

Conformational study of globulin from common buckwheat (Fagopyrum esculentum Moench) by Fourier transform infrared spectroscopy and differential scanning calorimetry

Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.     

Effects of glycerol and moisture gradient on thermomechanical properties of white bread

The thermomechanical properties of breadcrumb were investigated using dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC). The main transition (T(1), near 0 degrees C) shifted to lower temperature with added glycerol due to freezing point depression. The low-temperature transition (T(3), approximately -50 degrees C), found only in high-glycerol (8.8%) bread, suggested that of excess or phase-separated glycerol. The high-temperature transition (T(2), 60-85 degrees C) appeared only in aged breadcrumbs; its temperature range was correlated well with the amylopectin melting transition (DSC) but its tan delta amplitude did not correlate well with the amylopectin melting enthalpy (r(2) = 0.72). On the other hand, the change of E' ' (viscous behavior) suggested that T(2) might be related to the change in the amorphous region. Domain-to-domain (amorphous) and crumb-to-crust moisture migrations are two critical phenomenological changes associated with aging and could lead to significant local dehydration of some amorphous regions contributing to mechanical firming during storage.     

Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin

The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another. Their alpha-helical contents determined by CD were low compared to values reported before for other species. On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80%. The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion.     

Comparative study of the retrogradation of intermediate water content waxy maize, wheat, and potato starches

The retrogradation of extruded starches from three different botanical sources was studied in concentrated conditions (34 +/- 1% water) at 25 degrees C using differential scanning calorimetry (DSC) and isothermal calorimetry, Fourier transform infrared spectroscopy (FTIR), and wide-angle X-ray scattering. Potato starch showed the highest rate of retrogradation (approximately 0.17 h(-1)) followed by waxy maize (approximately 0.12 h(-1)), while the retrogradation of wheat starch was the slowest (approximately 0.05 h(-1)). In addition to the kinetics, the extent of molecular order in the retrograded samples was studied in detail in terms of "short-range" (helical) and "long-range" (crystalline) distance scales. The amylopectin crystallinity indices were essentially the same (approximately 47-51% amylopectin basis) for the three starches. However, significant differences were found in the enthalpy of melting measured by DSC after "full" retrogradation (potato, 11.6 +/- 0.7; waxy maize, 9.0 +/- 0.5; and wheat, 6.1 +/- 0.3 J/g of amylopectin). The degree of short-range molecular order in the retrograded state determined by FTIR was waxy maize > potato > wheat. The effect of amylopectin average chain length and the polymorphism of the crystalline phase were taken into account to explain the differences in the retrogradation enthalpies.     

Differences in polymer formation through disulfide bonding of recombinant light meromyosin between white croaker and walleye pollack and their possible relation to species specific differences in thermal unfolding

Fast skeletal light meromyosins (LMMs) of white croaker and walleye pollack were prepared in our expression system using Escherichia coli and determined for their polymer-forming ability and thermodynamic properties by using sodium dodecyl sulfate polyacrylamide gel electrophoresis and differential scanning calorimetry (DSC), respectively. White croaker LMM formed dimer by heating at 80 degrees C and showed only a single peak at 32.1 degrees C of temperature transition in DSC. On the other hand, walleye pollack LMM hardly formed polymer and showed four peaks at 27.7, 30.5, 35.8, and 43.9 degrees C. When Cys525 of white croaker LMM was replaced by alanine, this point-mutated LMM showed no change in its DSC profile but formed no dimer upon heating, suggesting a possible role of Cys525 in dimer formation. On the other hand, walleye pollack LMM where Cys491 was substituted by alanine changed its DSC profile, showing four peaks at 27.9, 29.1, 38.4, and 43.9 degrees C. However, this point-mutated LMM formed no dimer upon heating as in the case of native LMM. These results suggest that cysteine residue(s) participates in thermal gel formation of LMM when it locates in a suitable position of the sequence.     

Comparison on the effect of high-methoxyl pectin or soybean-soluble polysaccharide on the stability of sodium caseinate-stabilized oil/water emulsions

The interactions of high-methoxyl pectin (HMP) and soybean-soluble polysaccharide (SSPS) with sodium caseinate-stabilized emulsions were investigated using a multitechnique approach, including dynamic light scattering (DLS), electrophoretic mobility measurements, transmission diffusing wave spectroscopy (DWS), and ultrasonic spectroscopy (US). At pH 6.8, both polysaccharides are negatively charged and did not adsorb onto caseinate-coated droplets due to electrostatic repulsion; however, SSPS showed a different behavior compared to HMP in the turbidity parameter 1/l* and sound attenuation parameters measured by DWS and US, respectively. The present study brought the first evidence of the stabilization effect of SSPS in acidified sodium caseinate-emulsions. While destabilization occurred at low polysaccharide concentrations, probably via bridging flocculation, acid-induced aggregation of the oil droplet was completely prevented by 0.2% SSPS or HMP. However, the interaction behavior of SSPS during acidification was different from that of HMP. This was demonstrated by the different development of the parameter 1/l*, droplet sizes, sound attenuation, and velocity.     

Green tea polyphenols react with 1,1-diphenyl-2-picrylhydrazyl free radicals in the bilayer of liposomes: direct evidence from electron spin resonance studies

Free radical scavenging reactions of green tea polyphenols (GTP) were investigated with electron spin resonance (ESR) spectroscopy in the phospholipid bilayer of liposomes, using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical as a model. The results showed that (1) GTP reacts with DPPH radicals in the bilayer of liposomes of both 1-hexadecanoyl-2-[(cis,cis,cis,cis,cis,cis)-4,7,10, 13,16,19-docosahexaenoyl]-sn-glycero-3-phosphocholine (DHAPC) and 1, 2-di[cis-9-hexadecenoyl]-sn-glycero-3-phosphocholine) (DPPC); and (2) GTP protects DHAPC liposomes effectively from the oxidation initiated by DPPH radicals. These results provide direct evidence that GTP reacts with free radicals in the model membrane and support the hypothesis that GTP protects unsaturated phospholipids from oxidation by reacting directly with the radicals.     

Enzyme-aided modification of chicken-breast myofibril proteins: effect of laccase and transglutaminase on gelation and thermal stability

The effect of laccase and transglutaminase (TG) on cross-linking, gelation, and thermal stability of salt-soluble chicken-breast myofibril proteins was investigated at pH 6. Both enzymes modified the protein pattern detected by SDS-PAGE. Identification of proteins by peptide mass mapping showed that myosin heavy chain (MHC) and troponin T were the most affected proteins. These proteins faded or disappeared as a function of the incubation time with both enzymes on SDS-PAGE. The molecular weight of actin was not, however, affected by either enzyme. The effects that the enzymes had on the gel formation of chicken-breast myofibrils were studied in 0.35 and 0.60 M NaCl solutions at 3% protein content and a constant temperature of 40 degrees C by using a small deformation viscoelastic measurement. TG substantially increased the storage modulus (G') of 3% protein in 0.35 M NaCl. Without the enzymes, gelation was insignificant in 0.35 M NaCl. The increased solubility of the proteins at 0.60 M NaCl intensified gelation with TG. G' increased 32 and 64% at dosages of 10 and 100 nkat of TG, respectively. Also, laccase increased G' of the gel in 0.60 M salt concentration. However, a high laccase dosage decreased the magnitude of G' below the control level. Differential scanning calorimetric (DSC) measurements indicated slightly reduced myosin heat stability after TG pretreatment and increased actin heat stability with both enzymes. Maximum transition temperatures did not alter with either enzyme.     

Thermal denaturation of myofibrillar proteins of striated and smooth adductor muscles of scallop (Zygochlamys patagonica). A differential scanning calorimetric study

Denaturation of proteins from striated and smooth muscles of scallop (Zygochlamys patagonica) was studied with differential scanning calorimetry (DSC) by monitoring maximum temperatures of transition and denaturation enthalpies. DSC thermograms of both striated and smooth whole muscles showed two transitions: Tmax 55.0, 79.2 degrees C; and Tmax 54.7, 78.7 degrees C, respectively. The DSC thermograms of myofibrils and actomyosin were similar to those corresponding to their respective whole muscles. As pH and ionic strength increased, the thermal stability of whole muscles decreased. The pH increase (5.0-8.0) significantly (p < 0.01) decreased the denaturation enthalpies (deltaH total, deltaH peakI, and deltaH peakII) of whole striated muscles. A significant decrease (p < 0.05) in the deltaH total and the deltaH peakI was also observed in DSC thermograms of smooth muscles at pH 8.0. Denaturation enthalpies (deltaH total and deltaH peakI) significantly decreased (p < 0.01) when the ionic strength increased from 0.05 to 0.5 in both types of muscles. Striated muscles were more affected than smooth muscles by changes in the chemical environment.     

Determination of the molecular and structural characteristics of okenia, mango, and banana starches

Starches were isolated from nonconventional sources (banana, mango, and okenia) and their characteristics were examined using polarized light microscopy, X-ray diffraction pattern, Fourier transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). Banana starch granules were of an ellipsoidal shape with size between approximately 8 and 20 microm; okenia had the smallest granule size, between approximately 2 and 5 microm. The three starches showed the Maltese cross, indicative of an intact granule structure. Okenia and mango starches had the A-type X-ray diffraction pattern, common to native cereal starches, whereas banana starch showed a mixture between A- and B-type pattern. Banana starch had the highest temperature (77.6 degrees C) and enthalpy (23.4 J/g) of gelatinization in excess water conditions; okenia had the lowest temperature (71.2 degrees C) and enthalpy (15 J/g), which may be related to the X-ray diffraction pattern and its small granule size. Both the okenia and mango starches had a higher molar mass and gyration radius than banana starch, which may be related to the differences determined in their crystalline structures.     

Capillary electrophoresis analysis of orange juice pectinesterases

Pectinesterase (PE) was extracted from orange juice and pulp with 1 M NaCl, desalted, and separated using capillary electrophoresis (CE) gel procedures (CE-SDS-CGE) and isoelectric focusing (CE-IEF). PE resolved as a single peak using noncoated fused silica columns with CE-SDS-CGE. CE-IEF separation of PE required acryloylaminoethoxyethanol-coated columns, which had limited stability. Thermal stability of PE extracts before and after heating at 75 degrees C for 30 min and at 95 degrees C for 5 min established heat labile and heat stabile fractions with identical PE migration times by CE-SDS-CGE or CE-IEF. Peak magnitude decreased to a constant value as heating time increased at 75 degrees C. Regression analysis of CE-SDS-CGE peak migration times of molecular weight (MW) standards estimated both heat labile and heat stable PE at MW approximately 36 900. Traditional SDS-PAGE gel separation of MW standards and active PE isolated by IEF allowed estimation of MW approximately 36 000. CE-SDS-CGE allowed presumptive, but not quantitative, detection of active PE in fresh juice.     

Glass transition and water effects on sucrose inversion by invertase in a lactose-sucrose system

Enzymatic changes are often detrimental to quality of low-moisture foods. In the present study, effects of glass transition and water on sucrose inversion in a lactose-sucrose food model were investigated. Amorphous samples were produced by freeze-drying lactose-sucrose (2:1)-invertase (20 mg invertase/49.4 g of carbohydrate) dissolved in distilled water. Sorption isotherms were determined gravimetrically at 24 degrees C. Sucrose hydrolysis was determined by monitoring glucose content using a test kit and the amounts of fructose, glucose, and sucrose using HPLC. The glass transition temperatures, T(g), at various water contents were measured using differential scanning calorimetry (DSC). The BET and the GAB sorption models were fitted to experimental data up to a(w) 0.444 and 0.538, respectively. Water sorption and DSC results suggested time-dependent crystallization of sugars at a(w) 0.444 and above. Significant sucrose hydrolysis occurred only above T(g), concomitantly with crystallization. Sucrose hydrolysis and crystallization were not likely in glassy materials.     

Influence of lipid physical state on the in vitro digestibility of emulsified lipids

The objective of this study was to investigate the influence of the physical state of emulsified lipids on their in vitro digestibility by pancreatic lipase. A 10 wt % tripalmitin oil-in-water emulsion stabilized by sodium dodecyl sulfate (0.9 wt % SDS) was prepared at a temperature (>70 degrees C) above the melting point of the lipid phase (T(m) approximately 60 degrees C). A portion of this emulsion was cooled to a temperature (0 degrees C for 15 min) well below the crystallization temperature of the emulsified lipid (T(c) approximately 22 degrees C) and then warmed to 37 degrees C so as to have completely solid lipid particles. Another portion of the emulsion was directly cooled from 70 to 37 degrees C (which is above the T(c)) to have completely liquid (supercooled) lipid particles. Pancreatic lipase (8 mg/mL) and bile extract (5.0 mg/mL) were then added to each emulsion at 37 degrees C, and the evolution of the particle charge, particle size, appearance, and free fatty acid release were measured over a period of 2 h. It was found that the rate and extent of lipid digestion were higher in the emulsion containing liquid particles but that appreciable lipid digestion still occurred in the emulsion containing solid particles (i.e., >35% lipid digestion after 2 h). These results may have important consequences for controlling the digestion rate of lipids or for developing solid lipid particle delivery systems for lipophilic functional components.     

Encapsulation of olive leaf extract in beta-cyclodextrin

Olive leaf extract, rich in oleuropein, formed an inclusion complex with beta-cyclodextrin (beta-CD) upon mixing of the components in aqueous media and subsequent freeze-drying. Inclusion complex formation was confirmed by differential scanning calorimetry (DSC). DSC thermograms indicated that the endothermic peaks of both the olive leaf extract and the physical mixture of olive leaf extract with beta-CD, attributed to the melting of crystals of the extract, were absent in DSC thermogram of inclusion complex. Moreover, DSC studies under oxidative conditions indicated that the complex of olive leaf extract with beta-CD was protected against oxidation, since it remained intact at temperatures where the free olive leaf extract was oxidized. Phase solubility studies afforded A L type diagrams, 1:1 complex stoichiometry, a moderate binding constant ( approximately 300 M (-1)), and an increase of the aqueous solubility by approximately 50%. The formation of the inclusion complex was also confirmed by nuclear magnetic resonance (NMR) studies of beta-CD solutions in the presence of both pure oleuropein and olive leaf extract. The NMR data have established the formation of a 1:1 complex with beta-CD that involves deep insertion of the dihydroxyphenethyl moiety inside the cavity from its secondary side.