Effects of protein engineering of canola procruciferin on its physicochemical and functional properties

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.     

Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.     

Analysis of grape Vitis vinifera L. DNA in must mixtures and experimental mixed wines using microsatellite markers

Because wine quality highly relies on the varietal composition of the must, the development of methods allowing the authentication of varieties in musts and wines would be of great value as a guarantee of quality. Microsatellite markers have already been applied to the authentication of grape juices (Faria, M. A.; Magalh?es, R.; Ferreira, M. A.; Meredith, C. P.; Ferreira Monteiro, F. J. Agric. Food Chem. 2000, 48, 1096-1100) and to the analysis of experimental wines (Siret, R.; Boursiquot, J. M.; Merle, M. H.; Cabanis, J. C.; This, P. J. Agric Food Chem. 2000, 48, 5035-5040). In the present paper, we accessed the usefulness of this technology for the analysis of must and wine mixtures. The detection limit of DNA mixtures was first estimated on DNA extracted from leaves: 4% of a foreign DNA can be detected. Analysis of must and wine mixtures (Chardonnay B/Clairette B and Syrah N/Grenache N) was performed on experimental fermentations. DNA was extracted along the fermentation process and analyzed using five microsatellite loci. The 70:30 (v/v) mixtures were successfully analyzed until the end of the fermentation. The applications of these results to commercial purposes are discussed.     

Ultrastructure of individual and compound starch granules in isolation preparation from a high-quality, low-amylose rice, ilpumbyeo, and its mutant, G2, a high-dietary fiber, high-amylose rice

The ultrastructures of isolated starch granules from Ilpumbyeo (IP), a low-amylose japonica rice, and its mutant, Goami2 (G2), a high-amylose rice, which have extreme contrasts in physicochemical properties, cooking qualities (Kang, H. J.; Hwang, I. K.; Kim, K. S.; Choi, H. C. Comparative structure and physicochemical properties of Ilpumbyeo, a high-quality japonica rice, and its mutant, Suweon 464. J. Agric. Food Chem. 2003, 51, 6598-6603. Kim, K. S.; Kang, H. J.; Hwang, I. K.; Hwang, H. G.; Kim, T. Y.; Choi, H. C. Comparative ultrastructure of Ilpumbyeo, a high-quality japonica rice, and its mutant, Suweon 464: Scanning and transmission electron microscopy studies. J. Agric. Food Chem. 2004, 52, 3876-3883), and susceptibility to amylolytic enzymes (Kim, K. S.; Kang, H. J.; Hwang, I. K.; Hwang, H. G.; Kim, T. Y.; Choi, H. C. Fibrillar microfilaments associated with a high-amylose rice, Goami2, a mutant of Ilpumbyeo, a high-quality japonica rice. J. Agric. Food Chem. 2005, 53, 2600-2608), were compared. In isolated preparation, IP consisted entirely of well-separated individual starch granules (ISG), whereas G2 consisted of two populations, the large voluminous bodies and the smaller forms, the ISGs. High-voltage electron microscopy revealed that each of the voluminous bodies consisted of tightly packed smaller subunits, the ISGs, indicating that they represent the compound starch granules (CSGs) of G2. This suggests that the structural as well as functional unit of G2 involved in food processing is, unlike IP and other ordinary rices, not ISG but is primarily CSG. ISGs located at the periphery of CSGs were fused to each other with adjacent ones forming a thick band or wall encircling the entire circumference. The periphery of ISGs separated from CSGs of G2 consisted of thin radially oriented filaments arranged side by side along the entire granule surface, whereas no such filaments occurred in ISG of IP. It appears that the thick band and the peripheral filaments surrounding CSGs and ISGs, respectively, function as a structural barrier that limits the entrance of water into the granules and subsequent absorption, causing the low swelling power, incomplete gelatinization, and finally poor quality of cooked rice in G2.     

Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.     

Structure-physicochemical function relationships of soybean beta-conglycinin constituent subunits

Beta-conglycinin, one of the dominant storage proteins of soybean, has a trimeric structure, being composed of three subunits alpha, alpha', and beta. The alpha and alpha' subunits contain the extension regions in addition to the core regions common to all subunits, which are N-glycosylated. Physicochemical functions of recombinant nonglycosylated individual subunits and deletion mutants (alpha(c) and alpha'(c)) lacking the extension regions of the alpha and alpha' subunits were examined at pH 7.6 and 3.7 at low (mu = 0.08) and high (mu = 0.5) ionic strengths. Although individual recombinant subunits exhibited different properties at all conditions, there were some consistencies. Surface hydrophobicities and thermal stabilities of the individual subunits were likely to be conferred by their core regions, and the carbohydrate moieties did not contribute to these properties at any conditions examined here. Solubility at mu = 0.08, heat-induced association, and emulsifying ability remarkably depended on the extension regions and the carbohydrate moieties in addition to the structural features of the core regions. These findings indicate that various end products could be produced by the selection of soybean varieties containing beta-conglycinin with different subunit compositions and suggest a direction for a principle of soybean breeding.     

GC-FID- and acyl carbon number-based determination of characteristic groupings of complex triglyceride (Benefat S and other) mixtures

Techniques for the gas and liquid chromatographic separation of complex mixtures of triglycerides have evolved over the past two decades, as reviewed in detail by Huang et al. (J. Agric. Food Chem. 1995, 43, 1834-1844; J. Agric. Food Chem. 1997, 45, 1770-1778). A novel method for the quantitative partitioning of complex mixtures of triglycerides into functionally related groups is developed and applied to a low-calorie triglyceride mixture [namely, Benefat S or Salatrim plus mid-chain (C(6,8,10,12)) fatty acids]. The method is based on a nonlinear calibration of retention times (RTs) of a suite of standard triglycerides on their acyl carbon numbers [(ACNs), the sum of all the acyl carbon atoms in a given triglyceride] to estimate all of the intermediate ACNs (from 6 to 66). With the calibrated ACN scale and identifications of some components of a complex mixture's composition, ACN-based partitions were established and a Benefat S-triglyceride chromatogram was partitioned into seven functionally related groups. This method is provisional in the sense that it would typically be employed when the identifications of many components of a complex, homologous series were unknown, yet functionally related groups needed to be quantified. This method has proven to be particularly useful in the intercalibration of research laboratories with production facility laboratories during complex ( approximately 50-90 compounds) and large-scale ( approximately 20 ton) syntheses because of the high reproducibility of the ACN-based partitioning of complex chromatograms. This carbon number and statistically based method can be generally applicable to other complex mixtures of organic compounds and is readily adaptable to laboratory intercalibration efforts.     

Hydrophobicity,Solubility, and Emulsifying Properties of Enzyme-Modified Rice Endosperm Protein

Rice endosperm protein was modified to enhance solubility and emulsifying properties by controlled enzymatic hydrolysis. The optimum degree of hydrolysis (DH) was determined for acid, neutral, and alkaline type proteases. Solubility and emulsifying properties of the hydrolysates were compared and correlated with DH and surface hydrophobicity. DH was positively associated with solubility of resulting protein hydrolysate regardless of the hydrolyzing enzyme, but enzyme specificity and DH interactively determined the emulsifying properties of the protein hydrolysate. The optimum DH was 6–10% for good emulsifying properties of rice protein, depending on enzyme specificity. High hydrophobic and sulfhydryl disulfide (SH-SS) interactions contributed to protein insolubility even at high DH. The exposure of buried hydrophobic regions of protein that accompanied high-temperature enzyme inactivation promoted aggregation and cross-linking of partially hydrolyzed proteins, thus decreasing the solubility and emulsifying properties of the resulting hydrolysate. Due to the highly insoluble nature of rice protein, surface hydrophobicity was not a reliable indicator for predicting protein solubility and emulsifying properties. Solubility and molecular flexibility are the essential factors in achieving good emulsifying properties of rice endosperm protein isolates.     

Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric (DSC) and circular dichroism (CD) experiments. At pH 7.6, at which glycinin is mainly present in the 11S form, the disulfide bridge linking the acidic and the basic polypeptides is broken during heat denaturation. At pH 3.8, at which glycinin has dissociated partly into the 7S form, and at pH 5.2 this disruption does not take place, as demonstrated by solubility and gel electrophoretic experiments. A larger exposure of the acidic polypeptides (Lakemond et al., 2000) possibly correlates with a higher endothermic transition temperature and with the appearance of an exothermic transition as observed with DSC. Denaturation/aggregation (studied by DSC) and changes in secondary structure (studied by far-UV CD) take place simultaneously. Generally, changes in tertiary structure (studied by near-UV CD) occur at lower temperatures than changes in secondary structure.     

Development and evaluation of an immunological approach for the identification of novel acetyl coenzyme-A carboxylase inhibitors: assay optimization and pilot screen results

Cyclohexanediones, aryloxyphenoxypropionates, indolizidinediones, and triazinediones are four known structural classes of herbicides that inhibit acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2). An immunological study to determine the potential of ACCase inhibitor-specific monoclonal antibodies as screening tools to identify novel lead chemistry was undertaken. Using two cyclohexanedione-specific monoclonal antibodies (mAb A and mAb B; Webb, S. R.; Hall, J. C. J. Agric. Food Chem. 2000, 48, 1210-1218) and three different cyclohexanedione hapten coating conjugates, competitive indirect enzyme-linked immunosorbent assays (ciELISA) were developed. Cross-reactivity of the monoclonal antibodies with four structural classes of ACCase inhibitors revealed that the ciELISA using mAb A and a modified cyclohexanedione hapten coating conjugate detected analogues from all four known classes of ACCase inhibitors. A pilot screen using this ciELISA format identified two novel ACCase inhibitors, demonstrating the potential for antibodies as rapid and cost-effective screening tools for identifying novel lead chemistry in pesticide discovery programs.     

Authenticity of the traditional cypriot spirit "zivania" on the basis of 1h NMR spectroscopy diagnostic parameters and statistical analysis

A previous publication (Kokkinofta et al. J. Agric. Food Chem. 2003, 51, 6233-6239) discussed the use of inductively coupled plasma spectroscopy to differentiate between the traditional Cypriot alcoholic beverage zivania and other spirits similar in alcoholic content collected from different countries. In the present paper (1)H NMR spectroscopy is applied to confirm the previous conclusions and to obtain additional physical-chemical characteristics that may be used to differentiate zivania from other similar beverages. NMR spectroscopy gave a satisfactory degree of prediction and classification between zivanias and other distillings. The validity of quantification of the method was tested using comparative GC data. It appears that chemical analysis can be very helpful for identifying the unique geological and climatic conditions existing in the island of Cyprus that lead to an authentic product.     

Solid phase microextraction/gas chromatography of Salmonella-infected beef

Eight strains of Salmonellae were incubated in TSB culture medium at 37 degrees C for 24 h. Volatile compounds derived from the bacteria were collected using solid-phase microextraction fibers and then applied to gas chromatography (GC). Similarity analysis of the GC patterns thus obtained could separate these strains on principal component similarity (PCS) scattergrams. Five major food-related pathogenic bacteria and 10 other bacteria (including one Salmonella strain) were also classified by growing in the same medium. It is then proposed to utilize this approach to improve the GC/PCS method of Nakai et al. [Nakai, S.; Wang, Z. H.; Dou, J.; Nakamura, S.; Ogawa, M.; Nakai, E.; Vangerstoep, J. J. Agric. Food Chem. 1999, 47, 576-583] that has been developed for screening safe foods by detecting bacteria contaminated foods. Inoculating food samples pre-enriched through preliminary incubation into a culture medium and then subjecting to the GC/PCS method after secondary incubation enhances the detectability of pathogenic bacteria.     

Crystal structures and structural stabilities of the disulfide bond-deficient soybean proglycinin mutants C12G and C88S

The constituent subunits of seed storage protein 11S globulin have two disulfide bonds that are common among 11S globulins from legume and nonlegume seeds. In the case of the A1aB1b subunit of soybean 11S globulin, glycinin, Cys12-Cys45 and Cys88-Cys298 are observed by X-ray crystallography. The significance of these two disulfide bonds for structural stability was investigated by mutagenesis of Cys12 to Gly and of Cys88 to Ser. The disulfide bond-deficient mutants C12G and C88S could form the correct conformations identical to that of the wild-type proglycinin except in the vicinities of the mutation sites C12 and C88 as shown by their crystal structures. Thermal stability monitored by differential scanning calorimetry of the mutants indicated that the contribution of these disulfide bonds to the thermal stability of proglycinin A1aB1b is low, although there is a small difference in the extent of the contribution between the two disulfide bonds (Cys12-Cys45 > Cys88-Cys298). The contribution of Cys88-Cys298 to the resistance of proglycinin A1aB1b to proteinase digestion is higher than that of Cys12-Cys45. Possible effects of structure on the different properties of C12G and C88S are discussed.     

Taste active compounds in a goat cheese water-soluble extract. 2. Determination of the relative impact of water-soluble extract components on its taste using omission tests

The aim of this work was to determine the relative impact of water-soluble compounds on the gustatory properties of a goat cheese water-soluble extract (WSE). Using a semisynthetic model mixture (MWSE) previously elaborated in physicochemical and gustatory accordance with the cheese WSE (see part 1, Engel et al. J. Agric. Food Chem. 2000, 48, 4252-4259), omission tests were performed. Among the main taste characteristics of the WSE (salty, sour, and bitter), saltiness was explained by an additive contribution of sodium, potassium, calcium, and magnesium cations, whereas sourness was mainly due to a synergistic effect involving sodium chloride, phosphates, and lactic acid and bitterness was found to result from calcium and magnesium chlorides, the impact of which was partially masked by sodium chloride. In contrast, amino acids, lactose, and peptides did not have any significant impact on WSE taste properties. To quantify the contribution of the taste active compounds to bitterness and saltiness, stepwise multiple linear regressions were performed. Those contributions were expressed as a percentage of the considered taste characteristic intensity in the WSE. The model obtained allowed up to 97.4% of the perceived saltiness to be described and approximately 85% of the bitterness.     

Comparison of two dietary folate intake instruments and their validation by RBC folate

An optimal folate nutritional status is important in minimizing developmental and degenerative disease. Therefore, constant monitoring of folate intake and of biomarkers of folate nutritional status is essential. The objective of this research was to compare two folate intake instruments and validate each one against RBC folate measured by a high-throughput liquid chromatography tandem mass spectrometry (HT LC-MS/MS) method described in the companion paper (Owens, J. E.; Holstege, D. M.; Clifford, A. J. J. Agric. Food Chem. 2007, 55, 3292-3297). A food frequency questionnaire (FFQ) and a folate-targeted semiquantitative Block dietary folate equivalents (DFE) screener were compared and individually validated against an HT LC-MS/MS method. RBC folate was 1178 +/- 259 nmol/L (mean +/- SD) in a population of 337 normal adult subjects. Folate intakes were 556 +/- 265 microg/day by the FFQ and 524 +/- 276 microg/day by the DFE screener. Folate intakes by the DFE screener were approximately 34 microg less than by the FFQ (paired t test, p<0.01), but the intake instruments were highly correlated for total folate intake (r=0.608, p<0.01). Correlations between instruments and RBC folate were low (r<0.35) but strong (p<0.01). ROC curve analysis indicates that the measurement of RBC folate by the HT LC-MS/MS method is a better predictive tool than are intake instruments for the evaluation of marginal folate status.     

Identification of a new form of lipid transfer protein (LTP1) in wheat seeds

Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jégou, S.; Douliez, J. P.; Mollé, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced.     

Aroma comparisons of traditional and mild yogurts: headspace gas chromatography quantification of volatiles and origin of alpha-diketones

A quick headspace GC method for quantification of volatiles was developed, involving only minor sample preparation. Yogurt flavor compounds could be quantified in the micrograms per kilogram to milligrams per kilogram range without any difficulty, despite the complex matrix. Volatiles of traditional acidic and mild, less acidic yogurts were compared, and important differences were found for acetaldehyde, 2,3-butanedione, and 2,3-pentanedione. Concentrations of 2,3-butanedione and 2,3-pentanedione increased 2-3-fold in mild, less acidic yogurts compared to traditional acidic ones. This is due to accumulation of the precursors of the diketones, 2-acetolactate and 2-acetohydroxybutyrate, during fermentation in mild, less acidic yogurt. These precursors are subsequently converted to the corresponding diketones during storage. On the contrary, acetaldehyde formation was reduced in the mild yogurt, due to growth differences between the lactic acid bacteria used for fermentation of the milk. The quantitative results presented in this study validate previous GC sniffing conclusions (Ott et al. J. Agric. Food Chem. 1997, 45, 850-858), showing that yogurt aroma is the superposition of impact flavor compounds generated by fermentation on milk compounds.     

Distribution of aflatoxin in pistachios. 7. Sequential sampling

Sequential sampling for aflatoxin testing in pistachios is evaluated using the aflatoxin distribution and Monte Carlo results previously obtained (J. Agric. Food Chem. 1999, 47, 3771-3775). The sequential protocol is modeled on the current EU test protocol by applying a three-step sampling, using 10, 20, and 30 kg sample averages. An acceptance level of 15 ng/g of total aflatoxin, under consideration for U.S. standards, is applied. Optimization leads to indifference regions of 2-30 ng/g for the first two steps. The resulting OC curve approximates that for a single 50 kg sample. The sequential protocol is applied to the results for a set of 1293 lots of the 1998 crop year, each tested with a single 10 kg sample. Ninety-five percent of the lots would have been accepted on the basis of the single test and 1.5% would have been rejected, whereas 3.5% of the lots would have required retesting.