Beta-lactoglobulin molten globule induced by high pressure.

Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.     

Gelling properties of heat-denatured beta-lactoglobulin aggregates in a high-salt buffer

Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.     

Rheological properties of concentrated skim milk: influence of heat treatment and genetic variants on the changes in viscosity during storage

Heat treatment during manufacturing of milk powder is one of the most important tools for manipulation of its functional properties, and it is the basis of the classification of these proteins into low-, medium-, and high-heat types. Slight differences in the sequences of the major proteins in milk (genetic variants) seem to have also a significant effect in milk powder processing (U.S. patent). Therefore, the effects of high-temperature storage and heat treatment on skim milk of defined genetic variants of beta-lactoglobulin (beta-LG) were measured. The samples had 45% total solids, the temperature of aging was 50 degrees C, and the heat treatment was 90 degrees C for 10 min prior to evaporation. Measurements on shear rate and on apparent viscosity were determined for each sample. During storage of the concentrated milk, the apparent viscosity and yield values increased markedly, and the age-dependent increase in viscosity in heat-treated concentrated skim milks was much more pronounced than in those prepared from unheated skim milks. The increase in apparent viscosity and yield value with storage time was notably different for milks containing different genetic variants. Unheated concentrated milks containing the B variant of beta-LG showed the most rapid increase in apparent viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the A variant. In contrast, heat-treated concentrated milks containing the A variant of beta-LG showed the most rapid increase in viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the AB variant. The changes in apparent viscosity of concentrated milk were largely reversible under high shear during the early stages of storage, but samples stored for a long time showed irreversible changes in apparent viscosity. Particle size analysis confirmed irreversible aggregation and fusion of casein particles during storage.     

Phospholipid/fatty acid-induced secondary structural change in beta-lactoglobulin during heat-induced gelation

Effects of phosphatidylcholine (PC) and the predominant fatty acids (FAs) in milk, butyrate, oleate, and palmitate, on secondary structural changes in beta-lactoglobulin (beta-LG) during heat-induced gelation were analyzed on the basis of circular dichroism (CD) spectra. Small-strain oscillatory measurements were carried out to characterize viscoelastic properties of the heat-induced gels. In the absence of added salt, PC and FAs induced helix formation of beta-LG on heating to 80 degrees C and increased the storage moduli (G') of heat-induced gels. In the presence of 500 mM NaCl, PC did not change the CD spectrum of beta-LG but decreased G'. In contrast, butyrate substantially unfolded beta-LG in 500 mM NaCl on heating, forming very elastic gels with increased G' values. Palmitate and oleate induced beta-LG gel formation at 25 degrees C without heating; heating to 80 degrees C almost completely unfolded beta-LG in 500 mM NaCl.     

Influence of binding of sodium dodecyl sulfate, all-trans-retinol, palmitate, and 8-anilino-1-naphthalenesulfonate on the heat-induced unfolding and aggregation of beta-lactoglobulin B

Heat treatment of bovine beta-lactoglobulin B (beta-LG) causes it to partially unfold and aggregate via hydrophobic association and intra- and interprotein disulfide bonds. The first stage, which involves a "loosening" of the native structure, is influenced by the environmental conditions, such as pressure, pH, and added solutes. In the present study, four potential beta-LG ligands [palmitate, sodium dodecyl sulfate (SDS), 8-anilino-1-naphthalenesulfonate (ANS), and all-trans-retinol (retinol)] were added to beta-LG solutions prior to heat treatment for 12 min at temperatures between 40 and 93 degrees C. The extent of the changes in secondary and tertiary structures, unfolding, and aggregation at 20 degrees C were determined by circular dichroism, fluorescence, and alkaline- and SDS-polyacrylamide gel electrophoresis (PAGE). Both palmitate and SDS stabilized the native structure of beta-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation. Retinol was less effective, probably because of its lower affinity for the calyx-binding site, and ANS did not stabilize beta-LG, suggesting that ANS did not bind strongly in the calyx. It was also noted that holding a beta-LG solution with added SDS or ANS promoted the formation of a hydrophobically associated non-native dimer.     

Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions

Whey protein concentrate solutions (12% w/v, pH 6.65 +/- 0.05) were pressure treated at 800 MPa for 20-120 min and then examined using size exclusion chromatography (SEC), small deformation rheology, transmission electron microscopy, and various types of one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). The pressure-treated samples showed a time-dependent loss of native whey proteins by SEC and 1D PAGE and a corresponding increase in non-native proteins and protein aggregates of different sizes. These aggregates altered the viscosity and opacity of the samples and were shown to be cross-linked by intermolecular disulfide bonds and by noncovalent interactions using 1D PAGE [alkaline (or native), sodium dodecyl sulfate (SDS), and SDS of reduced samples (SDS(R))] and 2D PAGE (native:SDS and SDS:SDS(R)). The sensitivity of the major whey proteins to pressure was in the order beta-lactoglobulin B (beta-LG B) > beta-LG A > bovine serum albumin (BSA) > alpha-lactalbumin (alpha-LA), and the large internal hydrophobic cavity of beta-LG may have been partially responsible for its sensitivity to high-pressure treatments. It seemed likely that, at 800 MPa, the formation of a beta-LG disulfide-bonded network preceded the formation of disulfide bonds between alpha-LA or BSA and beta-LG to form multiprotein aggregates, possibly because the disulfide bonds of alpha-LA and BSA are less exposed than those of beta-LG either during or after pressure treatment. It may be possible that intermolecular disulfide bond formation occurred at high pressure and that hydrophobic association became important after the high-pressure treatment.     

Heat-induced gelation of chicken Pectoralis major myosin and beta-lactoglobulin

The denaturation, aggregation, and rheological properties of chicken breast muscle myosin, beta-lactoglobulin (beta-LG), and mixed myosin/beta-LG solutions were studied in 0.6 M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, during heating. The endotherm of a mixture of myosin and beta-LG was identical to that expected if the endotherm of each protein was overlaid on the same axis. The maximum aggregation rate (AR(max)) increased, and the temperature at the AR(max) (T(max)) and initial aggregation temperature (T(o)) decreased as the concentration of both proteins was increased. The aggregation profile of <0.5% myosin was altered by the presence of 0.25% beta-LG. Addition of 0.5-3.0% beta-LG decreased storage moduli of 1% myosin between 55 and 75 degrees C, but increased storage moduli (G') when heated to 90 degrees C and after cooling. beta-LG had no effect on the gel point of > or =1.0% myosin, but enhanced gel strength when heated to 90 degrees C and after cooling. After cooling, the G' of 1% myosin/2%beta-LG gels was about 1.7 times greater than that of gels prepared from 2% myosin/1% beta-LG.     

Effect of milk concentration on the irreversible thermal denaturation and disulfide aggregation of beta-lactoglobulin

The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.     

Changes in volatile flavor components of guava juice with high-pressure treatment and heat processing and during storage.

The changes in volatile flavor components of guava juice during pressure processing (25 degrees C, 600 MPa, 15 min), heat processing (95 degrees C, 5 min), and storage at 4 and 25 degrees C were evaluated by purge and trap/gas chromatography/mass spectrometry. Esters were the major volatile fraction in guava juice, and alcohols were the second. Pressure processing could maintain the original flavor distribution of the juice. Heat processing (95 degrees C, 5 min) caused decreases in the majority of flavor components in the juice when compared with freshly extracted juice. High-pressure treatment at 600 MPa for 15 min can effectively sterilize microbes but partially inactivate enzymes of guava juice; therefore, volatile components in pressure-treated juice gradually changed during storage periods. Pressure-treated guava juice showed increases in methanol, ethanol, and 2-ethylfuran with decreases in the other components during storage period. Nevertheless, the volatile distribution of 600 MPa treated guava juice was similar to that of freshly extracted juice when stored at 4 degrees C for 30 days.     

Denaturation of beta-lactoglobulin in pressure-treated skim milk

The kinetics of beta-lactoglobulin (beta-LG) denaturation in pressure-treated reconstituted skim milk samples over a wide pressurization range (100-600 MPa) and at various temperatures (10-40 degrees C) was studied. Denaturation was extremely dependent on the pressure and duration of treatment. At 100 MPa, no denaturation was observed regardless of the temperature or the holding time. At higher pressures, the level of denaturation increased with an increasing holding time at a constant pressure or with increasing pressure at a constant holding time. At 200 MPa, there was only a small effect of changing the temperature at pressurization. However, at higher pressures, increasing the temperature from 10 to 40 degrees C markedly increased the rate of denaturation. The two major genetic variants of beta-LG (A and B) behaved similarly to pressure treatment, although the B variant appeared to denature slightly faster than the A variant at low pressures (< or =400 MPa). The denaturation could be described as a second-order process for both beta-LG variants. There was a marked change in pressure dependence at about 300 MPa, which resulted in markedly different activation volumes in the two pressure ranges. Evaluation of the kinetic and thermodynamic parameters suggested that there may have been a transition from an aggregation-limited reaction to an unfolding-limited reaction as the pressure was increased.     

Hydrophobic probe binding of beta-lactoglobulin in the native and molten globule state induced by high pressure as affected by pH,KIO(3) and N-ethylmaleimide

High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.     

Gelation of chicken pectoralis major myosin and heat-denatured beta-lactoglobulin

Thermal, rheological, and microstructural properties of myosin (1 and 2% protein) were compared to mixtures of 1% myosin and 1% heat-denatured beta-lactoglobulin aggregates (myosin/HDLG) and 1% myosin and 1% native beta-lactoglobulin (myosin/beta-LG) in 0.6 M NaCl and 0.05 M sodium phosphate buffer, pH 6.0, 6.5, and 7.0 during heating to 71 degrees C. Thermal denaturation patterns of myosin and myosin/HDLG were similar except for the appearance of an endothermic peak at 54-56 degrees C in the mixed system. At pH 7.0, 2% myosin began to gel at 48 degrees C and had a storage modulus (G') of 500 Pa upon cooling. Myosin/HDLG (2% total protein) had a gel point of 48 degrees C and a G' of 650 Pa, whereas myosin/beta-LG had a gel point of 49 degrees C but the G' was lower (180 Pa). As the pH was decreased, the gel points of myosin and myosin/HDLG decreased and the G' after cooling increased. The HDLG was incorporated within the myosin gel network, whereas beta-LG remained soluble.     

Effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible thermal denaturation of beta-lactoglobulin and alpha-lactalbumin in skim milk

The effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible denaturation of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) in reconstituted skim milk samples was studied over a wide temperature range (75-100 degrees C). The irreversible thermal denaturation of beta-LG had a reaction order of 1.5 and that of alpha-LA had a reaction order of 1.0 in all systems and under all conditions. The rates of irreversible denaturation of beta-LG and alpha-LA were markedly dependent upon the composition of the milk. At all temperatures, the irreversible denaturations of beta-LG and alpha-LA were enhanced at a higher protein concentration and were retarded when the nonprotein-soluble components and lactose concentrations were increased. The effects of increasing the concentrations of lactose and nonprotein-soluble components were interpreted using the preferential hydration theory and allowed for the interpretation of the changes in the denaturations of beta-LG and alpha-LA when the milk total solids concentration was increased.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with acidic oligosaccharides

Bovine beta-lactoglobulin (beta-LG) was conjugated with the acidic oligosaccharides, alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides (POs) by the Maillard reaction to reduce the immunogenicity of beta-LG. The molar ratios of beta-LG to ALGO and POs in the conjugates were 1:6 and 1:8. The carbohydrate-binding sites in the beta-LG-ALGO conjugate were partially identified to be (60)Lys, (77)Lys, (100)Lys, (138)Lys, and (141)Lys. The isoelectric point of each conjugate was lower than that of beta-LG. CD spectra indicated that the secondary structure of beta-LG was almost maintained after conjugation. The results of fluorescence studies indicated that the conformation around Trp had not changed in each conjugate and that the surface of each conjugate was covered with a saccharide chain. Structural analyses with monoclonal antibodies indicated that the conformation around (8)Lys-(19)Trp (beta-sheet, random coil, short helix) in the conjugates had changed, whereas the native structure was maintained around (15)Val-(29)Ile (beta-sheet) and (125)Thr-(135)Lys (alpha-helix). The beta-LG-ALGO and beta-LG-POs conjugates maintained 77 and 70% of the retinol binding activity of beta-LG. Conjugation with ALGO and POs substantially enhanced the thermal stability of beta-LG. The anti-beta-LG antibody response was markedly reduced after immunization with both conjugates in BALB/c, C57BL/6, and C3H/He mice. B cell epitopes of beta-LG and the conjugate recognized in these mice were determined with 15-mer multipin peptides, and the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, whereas the antibody response to each epitope was dramatically reduced. In particular, effective reduction of the antibody response was observed in the vicinity of the carbohydrate-binding sites. Conjugation of beta-LG with these acidic oligosaccharides was effective in reducing the immunogenicity of beta-LG. The conjugates obtained in this study are edible, so they would be very useful for food application.     

Effects of thermal treatments and storage on pectin methylesterase and peroxidase activity in freshly squeezed orange juice

A specific indicator of freshness, allowing routine distinction between freshly squeezed orange juices (FSOJs) and FSOJ-like products, was to be identified. Using the Actijoule unit of a tubular heater at a flow rate of 60 L/h, FSOJs from Citrus sinensis (L.) Osbeck cv. Valencia Late were continuously heated on a pilot plant scale at six different temperatures (42-92 degrees C), followed by continuous cooling to ambient temperature and subsequent filling into sterilized glass jars. The cloud stability and residual activities of pectin methylesterase (PE) and peroxidase (POD) were monitored over the storage at 4 degrees C for up to 62 days, thus considering the storage conditions of FSOJs in retail markets. As shown by the viable microbial counts throughout storage, microbial activity was insignificant due to good sanitary practice, thus proving that the enzyme activities detected were of plant origin. The juices processed at temperatures > or =62 degrees C were characterized by minor residual activities. When exposed to temperatures <62 degrees C in the genuine acidic matrix of the juices, the heat stability of PE exceeded that of POD. Compared with the aforementioned samples, the juice processed at 52 degrees C with a residual PE activity of 33.8% was hardly inferior in terms of cloud stability within the first 14 days. After the juice was processed at 42 degrees C, rapid clarification occurred within the first 8 days, consistent with undetectable PE deactivation. Hence, only the range of approximately 50-60 degrees C is relevant in minimal heat-processing for the retention of cloud stability within the short turnover period of FSOJ-like products, with partial PE and POD deactivation being already sufficient to distinguish those juices from FSOJs. Irrespective of the previous thermal treatment, the total PE activity remained nearly constant during storage, whereas the POD activity rapidly declined to minor levels after 20 days. Consequently, as to the future analysis of samples with unknown processing history, PE was suggested as an indicator enzyme for the freshness of FSOJs, allowing their unambiguous distinction from minimally heat-processed juices.     

Commercial scale pulsed electric field processing of tomato juice

Effects of commercial scale pulsed electric field (PEF) processing on the quality of tomato juice were studied and compared with those of thermal processing. Tomato juice was prepared by hot break at 88 degrees C for 2 min or by cold break at 68 degrees C for 2 min and then thermally processed at 92 degrees C for 90 s or PEF processed at 40 kV/cm for 57 micros. Thermally processed, PEF processed, and unprocessed control juices were packed into 50 mL sterilized polypropylene tubes in a sanitary glovebox and stored at 4 degrees C for 112 days. Both thermally and PEF processed juices showed microbial shelf life at 4 degrees C for 112 days. The lipoxygenase activities of thermally and PEF processed juices were 0 and 47%, respectively. PEF processed juice retained more ascorbic acid than thermally processed juice at 4 degrees C for 42 days (p < 0.05). No significant differences were observed in the concentration of lycopene, degrees Brix, pH, or viscosity between thermally and PEF processed juices during the storage (p > 0.05). Sensory evaluations indicated that flavor and overall acceptability of PEF processed juice were preferred to those of thermally processed juice (p < 0.05).     

Influence of critical storage temperatures on degradative pathways of pigments in green beans (Phaseolus vulgaris cvs. Perona and boby)

In this work a study of critical storage temperatures on pigment degradation of green beans (Phaseolus vulgaris, cvs. Perona and Boby) was conducted. In this way, green beans kept better quality at 4 degrees C than either 8 or 12 degrees C, maintaining a bright green color and good texture. Nevertheless, temperatures of 4 degrees C induced chilling injury (CI) after eight days of storage, which became evident when the pods were transferred to 20 degrees C. Cold storage temperatures, 12, 8, and 4 degrees C, produced different changes on the green beans chlorophyll profile. Green beans of both cultivars, Perona and Boby, stored at 4 and 12 degrees C showed a continuous degradation of chlorophyll pigments during storage, while samples stored at 8 degrees C showed an increase of chlorophyll content at the first 15 days. Carotenoid pigments also suffered different changes during cold storage. Perona was the green beans cultivar which maintained the higher level of lutein, mainly when samples were stored at the most suitable temperature (8 degrees C).     

Ethanol effect on the structure of beta-lactoglobulin b and its ligand binding

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.     

Effects of heat processing and storage on flavanols and sensory qualities of green tea beverage

This research was conducted to understand the effects of heat processing and storage on flavanols and sensory qualities of green tea extract. Fresh tea leaves were processed into steamed and roasted green teas by commercial methods and then extracted with hot water (80 degrees C) at 1:160 ratio (tea leaves/water by weight). Green tea extracts were heat processed at 121 degrees C for 1 min and then stored at 50 degrees C to accelerate chemical reactions. Changes in flavanol composition and sensory qualities of green tea extracts during processing and storage were measured. Eight major flavanols (catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, catechin gallate, epigallocatechin gallate, and gallocatechin gallate) were identified in the processed tea extract. Among them, epigallocatechin gallate and epigallocatechin appeared to play the key role in the changes of sensory qualities of processed green tea beverage. The steamed tea leaves produced a more desirable quality of processed green tea beverage than the roasted ones.     

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran differing in molecular weight

To reduce the immunogenicity of beta-lactoglobulin (beta-LG), two beta-LG-carboxymethyl dextran (CMD) conjugates (Conj. 40 and Conj. 162) were prepared by using water-soluble carbodiimide (EDC). The molar ratios of beta-LG to CMD in Conj. 40 and Conj. 162 were 8:1 and 7:1, respectively. Each conjugate maintained approximately 50% of the retinol binding activity of beta-LG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface of beta-LG in each conjugate was covered by CMD without great disruption of native conformation. By conjugation with CMD, the antibody response to beta-LG was reduced in BALB/c, C3H/He, and C57BL/6 mice, which was eminent in Conj. 162. The results of B cell epitope scanning using overlapping synthesized peptides showed that the linear epitope profiles of the conjugates were similar to those of beta-LG, whereas the antibody response to each epitope was reduced, which was eminent in Conj. 162. It was concluded that conjugation with CMD of higher molecular weight is effective in reducing the immunogenicity of beta-LG and that masking of epitopes by CMD is responsible for the reduced immunogenicity.