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In the present study emulsions were made with various potato protein preparations, which varied in protease inhibitor and patatin content. These emulsions were characterized with respect to average droplet size, plateau surface excess, and the occurrence of droplet aggregation. Droplet aggregation occurred only with potato protein preparations that contained a substantial amount of protease inhibitors and could be prevented only at pH 3. The average droplet size of the emulsions made with potato proteins appeared to be related to the patatin content of the preparation used. Average droplet size was found to be dominated by the patatin-catalyzed lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. Addition of monoglycerides and especially fatty acids, at concentrations representative of those during emulsification, was shown to cause a stronger and much faster decrease of the interfacial tension than that with protein alone and to result in a drastic decrease in droplet size. The patatin used was shown to have a lipolytic activity of 820 units/g with emulsified tricaprylin as the substrate. Because of the droplet aggregating properties of the protease inhibitors, the patatin-rich potato preparations seem to be the most promising for food emulsion applications over a broad pH range, provided the lipolytic activity can be diminished or circumvented.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.

Foam properties of a sunflower isolate (SI), as well as those of helianthinin and sunflower albumins (SFAs), were studied at various pH values and ionic strengths and after heat treatment. Less foam could be formed from helianthinin than from SFAs, but foam prepared with helianthinin was more stable against Ostwald ripening and drainage than foam prepared with SFAs. Foams made with SFAs suffered from extensive coalescence. The formation and stability of foams made from reconstituted mixtures of both proteins and from SI showed the deteriorating effect of SFAs on foam stability. Foam stability against Ostwald ripening increased after acid and heat treatment of helianthinin. Partial unfolding of sunflower proteins, resulting in increased structural flexibility, improved protein performance at the air/water interface. Furthermore, it was observed that the protein available is used inefficiently and that typically only approximately 20% of the protein present is incorporated in the foam.

Emulsions were made with sunflower protein isolate (SI), helianthinin, and sunflower albumins (SFAs). Emulsion formation and stabilization were studied as a function of pH and ionic strength and after heat treatment of the proteins. The emulsions were characterized with respect to average droplet size, surface excess, and the occurrence of coalescence and/or droplet aggregation. Sunflower proteins were shown to form stable emulsions, with the exception of SFAs at neutral and alkaline pH values. Droplet aggregation occurred in emulsions made with SI, helianthinin, and SFAs. Droplet aggregation and subsequent coalescence of emulsions made with SFAs could be prevented at pH 3. Calcium was found to cause droplet aggregation of emulsions made with helianthinin, at neutral and alkaline pH values. Treatments that increase conformational flexibility of the protein molecule improved the emulsion properties of sunflower proteins.

The structure and solubility of helianthinin, the most abundant protein of sunflower seeds, was investigated as a function of pH and temperature. Dissociation of the 11S form (hexamer) into the 7S form (trimer) gradually increased with increasing pH from 5.8 to 9.0. High ionic strength (I = 250 mM) stabilizes the 11S form at pH > 7.0. Heating and low pH resulted in dissociation into the monomeric constituents (2-3S). Next, the 7S and 11S forms of helianthinin were isolated and shown to differ in their secondary and tertiary structure, and to have denaturation temperatures (T(d)) of 65 and 90 degrees C, respectively. Furthermore, the existence of two populations of the monomeric form of helianthinin with denaturation temperatures of 65 and 90 degrees C was described. This leads to the hypothesis that helianthinin can adopt two different conformational states: one with T(d) = 65 degrees C and a second with T(d) = 90 degrees C.

In the present study, foam-forming and -stabilizing properties of potato proteins were studied using whipping and sparging tests. The soluble potato proteins are mainly composed of patatin and protease inhibitors. The whipping tests showed that less foam was formed from untreated patatin than from the protease inhibitors, but patatin foam was much more stable. The foam-forming properties of patatin could be strongly improved by partial unfolding of the protein. Whipping tests, at both low (0.5 mg/mL) and high (10 mg/mL) protein concentration, also indicated that foams made with an ethanol-precipitated protein isolate were more stable than those made with beta-casein and beta-lactoglobulin. More generally, it can be concluded that when proteins are used as a foaming agent, a high concentration is required, because the protein available is inefficiently used. Also, there are several variables that may all, in different ways, affect both foam formation (amount of foam, bubbles size distribution) and foam stability. These variables include the type and concentration of protein, solvent conditions (pH, I), and the method used to make the foam.

In this study, a protein isolate with a high solubility at neutral pH was prepared from industrial potato juice by precipitation at pH 5 in the presence of ethanol. The effects of ethanol itself and the effects of its presence during precipitation on the properties of various potato protein fractions were examined. The presence of ethanol significantly reduced the denaturation temperature of potato proteins, indicating that the preparation of this potato protein isolate should be performed at low temperature in order to retain a high solubility. In the presence of ethanol, the thermal unfolding of the tertiary and the secondary structure of patatin was shown to be almost completely independent. Even at 4 degrees C, precipitation of potato proteins in the presence of ethanol induced significant conformational changes. These changes did, however, only result in minor changes in the solubility of the potato protein fractions as a function of pH and heat treatment temperature.

A method for obtaining sunflower protein (SFP) isolate, nondenatured and free of chlorogenic acid (CGA), has been developed. During the isolating procedure, the extent of CGA removal and protein denaturation was monitored. The defatted flour contained 2.5% CGA as the main phenolic compound. Phenolic compounds were removed by aqueous methanol (80%) extraction, before protein extraction at alkaline pH and diafiltration. Differential scanning calorimetry and solubility tests indicated that no denaturation of the proteins had occurred. The resulting protein products were biochemically characterized, and the presence of protein-CGA complexes was investigated. SFPs of the studied variety were found to be composed of two main protein fractions: 2S albumins and 11S globulins. In contrast to what has been previously reported, CGA was found to elute as free CGA, not covalently associated to any protein fraction.

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.

This study describes the relationship between the solubility of glycinin, a major soy protein, and its structural properties at a quaternary, tertiary, and secondary folding level under conditions representative for food products. When the ionic strength is lowered from 0.5 to 0.2 or 0.03, the basic polypeptides shift more to the exterior of the glycinin complex, as determined at pH 7.6 by labeling solvent-exposed lysines, supported by the study of the proteolytic action of clostripain on glycinin. This structural reorganization caused the pH of minimal solubility to shift to higher values. Ultracentrifugational analysis shows that at pH 7.6 and an ionic strength of 0.5 glycinin forms hexameric complexes (11S), whereas at pH 3.8 and at an ionic strength of 0.03 glycinin exists as trimers (7S). Intermediate situations are obtained by modulation of pH and ionic strength. The observed quaternary dissociation correlates with an increased amount of nonstructured protein at a secondary level and with changes in tertiary folding as determined using circular dichroism. Tryptophan fluorescence shows no significant structural changes for different ionic strengths but demonstrates a more tightly packed fluorophore environment when the pH is lowered from 7.6 to 3.8.

In this work dynamic light scattering was used to study the thermal aggregation of patatin in situ, to elucidate the physical aggregation mechanism of the protein and to be able to relate the aggregation behavior to its structural properties. The dependence of the aggregation rates on the temperature and the ionic strength suggested a mechanism of slow coagulation, being both diffusion and chemically limited. The aggregation rate dependence on the protein concentration was in accordance with the mechanism proposed. The aggregation rates as obtained at temperatures ranging from 40 to 65 degrees C correlated well with unfolding of the protein at a secondary level. Small-angle neutron scattering and dynamic light scattering results were in good accordance; they revealed that native patatin has a cylindrical shape with a diameter and length of 5 and 9.8 nm, respectively.

Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented. Patatin could be separated in four isoform pools, denoted A, B, C, and D, representing 62%, 26%, 5%, and 7% of the total amount of patatin, respectively. These isoforms differed in surface charge, resulting in a different behavior on anion exchange chromatography, isoelectric focusing, native polyacrylamide gel, and capillary electrophoresis. All isoforms of the patatin family contained proteins with two molecular masses of approximately 40.3 and 41.6 kDa, respectively. The size of this difference in the molar mass (1300 Da) is on the order of one carbohydrate moiety. Despite the biochemical differences given above, no variations in the structural properties nor in the thermal conformational stability could be observed using far-ultraviolet circular dichroism, infrared, and fluorescence spectroscopy.

A kinetic model of the thermal aggregation of patatin is presented based on chromatographic analysis of the proportions of nonaggregated and aggregated patatin. It was observed that the decrease of the amount of nonaggregated patatin proceeded initially quickly and was followed by slower aggregation at longer incubation times. It was shown that this behavior was not due to heterogeneity of the starting material. It was noted that overestimation of the amount of native molecules after a heat treatment, caused by refolding of the unfolded protein during the cooling step prior to the analysis, was significant and could not be neglected. Hence, corrections based on information on the structural properties of patatin were applied. Taking this into account, a model was proposed consisting of a first-order formation of reactive particles, followed by a second-order aggregation reaction. This model described the thermal aggregation of patatin rather accurately and was confirmed by experiments at various protein concentrations.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an alpha-helical and 46% a beta-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 degrees C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the alpha-helical contributions unfold in the 45-55 degrees C region, whereas the beta-stranded parts unfold more gradually at temperatures of 50-90 degrees C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60 degrees C, most likely reflecting the unfolding of alpha-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55 degrees C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly alpha-helical structures were formed.