Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Evaluation of a low-resource soy protein production method and its products.

Introduction: One key approach to achieve zero hunger in Sub-Saharan Africa (SSA) is to develop sustainable, affordable, and green technologies to process nutritious food products from locally available sources. Soybeans are an inexpensive source of high-quality protein that may help reduce undernutrition, but it is underutilized for human consumption. This research evaluated the feasibility of a low-cost method developed initially at the United States Department of Agriculture to produce soy protein concentrate (SPC) from mechanically pressed soy cake and thus create a more valuable ingredient to improve protein intake in SSA. Methods: The method was initially tested in the bench scale to assess process parameters. Raw ingredients comprised defatted soy flour (DSF), defatted toasted soy flour (DTSF), low-fat soy flour 1 (LFSF1; 8% oil), and LFSF2 (13% oil). Flours were mixed with water (1:10 w/v) at two temperatures (22 or 60°C) for two durations (30 or 60 min). After centrifugation, supernatants were decanted, and pellets were dried at 60°C for 2.5 h. Larger batches (350 g) of LFSF1 were used to examine the scalability of this method. At this level, protein, oil, crude fiber, ash, and phytic acid contents were measured. Thiobarbituric acid reactive substances (TBARS), hexanal concentration and peroxide value were measured in SPC and oil to evaluate oxidative status. Amino acid profiles, in vitro protein digestibility, and protein digestibility corrected amino acid score (PDCAAS) were determined to assess protein quality. Results: Bench scale results showed accumulation of protein (1.5-fold higher) and reduction of oxidative markers and phytic acid to almost half their initial values. Similarly, the large-scale production trials showed high batch-to-batch replicability and 1.3-fold protein increase from initial material (48%). The SPC also showed reductions in peroxide value (53%), TBARS (75%), and hexanal (32%) from the starting material. SPC's in vitro protein digestibility was higher than the starting material. Conclusion: The proposed low-resource method results in an SPC with improved nutritional quality, higher oxidative stability, and lower antinutrient content, which enhances its use in food-to-food fortification for human consumption and is thus amenable to address protein quantity and quality gaps among vulnerable populations in SSA.

Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities.

Protease inhibitors of protein nature are rich in seeds of legume crops. There are two common types: Kunitz inhibitor, which mainly inhibits trypsin, and Bowman-Birk inhibitor, which inhibits both trypsin and chymotrypsin. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Reported methods for CIA assay vary greatly and are incompletely described. No standardized or official method is available. The present study focused on developing a robust method for accurately measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Since BTpNA is not water soluble, a water-miscible organic solvent must be present. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels (which affected % chymotrypsin inhibition), the sequence of adding reagents, extractant and extraction time, and so forth, an optimized method for CIA measurement was finally developed. It features dimethylformamide as the organic solvent, the enzyme-last sequence, 5 ml total assay volume, and calculation of the inhibitor activity based on 40% chymotrypsin inhibition. The method can also be slightly modified for measuring chymotrypsin activity. The robust performance of the method was verified by measuring 11 assorted protein products, paving a way for standardization. PRACTICAL APPLICATION: With an increasing use of plant proteins, there is an urgent need to measure chymotrypsin inhibitor activity in various protein products with accuracy. After thoroughly investigating several factors, an optimized method for measuring chymotrypsin inhibitor activity in various protein products was developed. The proposed method is sensitive and robust, providing a basis for standardization. It can also be used for measuring chymotrypsin activity.

Simplified Analysis and Expanded Profiles of Avenanthramides in Oat Grains.

Uniquely, oats contain avenanthramides (AVAs), a group of phenolic alkaloids, exhibiting many health benefits. AVA analysis involves extraction with alcohol-based solvents and HPLC separation with UV and/or mass spectrometer detectors. There are many reported methods to extract AVAs. Almost all entail multiple extractions. The whole procedure is time- and labor-intensive. Furthermore, most quantifications are limited to three common AVAs (2f, 2p, 2c). The present study compared three extraction methods (all at 50 °C) for their effects on AVA concentrations and composition (% relative to total AVA) of oat grains. These included triplicate extractions with 80% ethanol containing 10 mM phosphate buffer (pH 2.0) (A), triplicate extractions with 80% ethanol (B), and a single extraction with 80% ethanol (C), while keeping solid/total solvent ratio at 1/60 (g/mL) and total extraction time of 60 min. Results showed that 80% buffered ethanol gave significantly lower AVA contents than 80% ethanol, while single and triplicate extractions with 80% ethanol produced the same extractability. However, the extraction method had no effect on AVA composition. Using 0.25 g sample size instead of 0.5 g saved extractants by half, without affecting AVA measurements. Consequently, a simplified method of extraction was developed, featuring Method C. The present study also expanded profiling individual AVAs beyond AVA 2c, 2p and 2f. Other AVAs identified and semi-quantified included 5p, 4p, 3f/4f, and 2pd. The simplified analysis was validated by measuring 16 selected oat grain samples. Some of these grains had relatively high contents of 4p, 3f/4f and 2pd, which have been considered minor AVAs previously.

Distributions of nutrients and avenanthramides within oat grain and effects on pearled kernel composition.

Hulled Reins and hulless Lamont oats were dehulled and/or sequentially abraded to produce ten pearling fines and corresponding pearled kernels. Contents of nutrients (protein, oil, starch, beta-glucan, ash and other carbohydrates) and avenanthramides (AVA) 2p, 2c, 2f, and 5p in processing fractions and starting grains were measured. Results show that distribution patterns of nutrients varied with individual nutrients, but those of AVAs varied with variety and individual AVAs. In both varieties, from the surface to inner endosperms, protein and oil increased then decreased; ash and other carbohydrates decreased; starch increased; and beta-glucan unchanged except for the surface area. In Lamont oat, the four AVAs decreased, but in Reins oat, AVA 2p decreased while 2c, 2f and 5p increased, then decreased. Compared to whole grain, pearled oats not only contained lower AVAs, protein, oil, ash, and other carbohydrates and higher beta-glucan and starch but also had a different AVA composition.

Changes in phenolic acid content during dry-grind processing of corn into ethanol and DDGS.

BACKGROUND: Nine fractions (1, ground corn; 2, cooked slurry; 3, liquefied slurry; 4, fermented mash; 5, whole stillage; 6, thin stillage; 7, condensed distillers soluble (CDS); 8, distillers wet grains (DWG); and 9, distillers dried grains with solubles (DDGS)) were collected at different steps from three commercial dry-grind bioethanol processing plants. Samples were analyzed for individual and total phenolic acid content by HPLC and the antioxidant capacity by ferric reducing antioxidant power (FRAP) assay. RESULTS: There were significant differences in phenolic acid (individual and total) content and the antioxidant capacity in the nine fractions collected from the three processing plants, but the changing trends in all three plants were very similar. The four phenolic acids identified in all fractions were caffeic, p-coumaric, ferulic and sinapic acids. Vanillic acid was present in all fractions except fractions 2 and 3. All fractions collected following fermentation, except fractions 6 and 7, had higher concentrations of phenolic acids than fractions before fermentation, with DWG having the highest phenolic acids content. CONCLUSION: The increased concentration of phenolic acid content after fermentation in four fractions (4, 5, 8 and 9) was primarily due to depletion of starch during dry-grind processing. Further research is needed to investigate the influence of enriched phenolic acid concentration in DDGS on diet palatability (sensory property) and animal health.

Methods to recover value-added coproducts from dry grind processing of grains into fuel ethanol.

Three methods are described to fractionate condensed distillers solubles (CDS) into several new coproducts, including a protein-mineral fraction and a glycerol fraction by a chemical method; a protein fraction, an oil fraction and a glycerol-mineral fraction by a physical method; or a protein fraction, an oil fraction, a mineral fraction, and a glycerol fraction by a physicochemical method. Processing factors (ethanol concentration and centrifuge force) were also investigated. Results show that the three methods separated CDS into different fractions, with each fraction enriched with one or more of the five components (protein, oil, ash, glycerol and other carbohydrates) and thus having different targeted end uses. Furthermore, because glycerol, a hygroscopic substance, was mostly shifted to the glycerol or glycerol-mineral fraction, the other fractions had much faster moisture reduction rates than CDS upon drying in a forced air oven at 60 °C. Thus, these methods could effectively solve the dewatering problem of CDS, allowing elimination of the current industrial practice of blending distiller wet grains with CDS for drying together and production of distiller dried grains as a standalone coproduct in addition to a few new fractions.

Compositional equivalence of barleys differing only in low- and normal-phytate levels.

Recent breeding advances have led to the development of several barley lines and cultivars with significant reductions (50% or greater) in phytate levels. Low-phytate (LP) grain is distinguished by containing not only a reduced level of phytate P but also an increased level of inorganic P, resulting in greater bioavailability of P and mineral cations in animal diets. It is important to determine whether other nutritional characteristics are altered by breeding for the low-phytate trait. This study was designed to investigate if breeding for reduced phytate content in barleys had any effect on the contents of other attributes measured by comparing mean and range values of the levels of protein, oil, ash, total carbohydrate, starch, and ß-glucan, fatty acid composition, and levels of tocopherols and tocotrienols between five LP and five normal-phytate barleys grown in three Idaho locations. Results show that only the phytate level in the LP group was substantially lower than that of the normal-phytate group and that all other attributes measured or calculated were substantially equivalent between the two groups of barleys. Therefore, the phytate level had little effect on the levels of protein, oil, ash, total carbohydrate, starch, and ß-glucan, fatty acid composition, and levels of tocopherols and tocotrienols in barley seeds.

Enzymatic method for measuring starch gelatinization in dry products in situ.

An enzymatic method based on hydrolysis of starch by amyloglucosidase and measurement of d-glucose released by glucose oxidase-peroxidase was developed to measure both gelatinized starch and hydrolyzable starch in situ of dried starchy products. Efforts focused on the development of sample handling steps (particle size reduction of dry samples followed by a unique mechanical resolubilization step) prior to the enzymatic hydrolysis using native and fully gelatinized flours of corn and rice. The new steps, when optimized, were able to maximize resolubilization of gelatinized/retrograded starch while minimizing solubilization of native starch in dried samples, thus effectively addressing issues of insusceptibility of retrograded starch and susceptibility of native starch to enzymatic attacks and eliminating the need to isolate starch from dry samples before using an enzymatic method. Various factors affecting these and other steps were also investigated, with the objectives to simplify the procedures and reduce errors. Results are expressed as the percentage of the total starch content. The proposed method, verified by measuring mixed samples of native and fully gelatinized flours of five grain species (corn, rice, barley, oat, and wheat) at different ratios, is simple, accurate, and reliable, with a relative standard deviation of less than 5%.

Comparison of lipid content and fatty acid composition and their distribution within seeds of 5 small grain species.

UNLABELLED: Barley, oats, rice, sorghum, and wheat, each with two genotypes, were sequentially abraded by an electric seed scarifier. The pearling fines (PF) and pearled kernels (PK) at each cycle were analyzed for lipid (mostly nonpolar) content and fatty acid (FA) composition. The oil content in whole or dehulled grains ranged from 2.18% of a wheat variety to 6.38% of an oat line. Compared with barley and wheat, rice, oat, and sorghum had higher relative % of C18:1 (31.60 to 36.64 compared with 12.15 to 15.61) and lower % of C18:2 (35.69 to 45.44 compared with 50.79 to 61.50). The relationship between oil content in PF and the cumulative level of surface removal essentially describes the distribution pattern of oil content within a seed. Barley, rice, and sorghum had a similar distribution pattern, characterized by a rapid rate of decreasing for the first few outer layers and then by gradual decrease to a flat value toward the inner core. In contrast, distribution within oats was characterized by a gradual reduction in oil content across the seed. The distribution of oil within wheat fell between the former 2 types. For all 10 grains, from seed surface to inner core, C16:0 and C18:0 increased, C18:1 and C18:3 decreased, and C18:2 changed slightly, providing a new reason for improved oxidative stability for pearled kernels. The differences in the changing intensity of FA composition among grain species correspond to those in oil distribution within a seed, while varietal difference in distribution patterns of content and FA composition of lipids within a species was insignificant. PRACTICAL APPLICATION: This study was the first to document fatty acid distribution across a grain seed. Results provide 2 major reasons for improved oxidative stability of pearled grains: reduced oil content and shift of fatty acids toward more saturated and less unsaturated composition.

Chemical composition of distillers grains, a review.

In recent years, increasing demand for ethanol as a fuel additive and decreasing dependency on fossil fuels have resulted in a dramatic increase in the amount of grains used for ethanol production. Dry-grind is the major process, resulting in distillers dried grains with solubles (DDGS) as a major coproduct. Like fuel ethanol, DDGS has quickly become a global commodity. However, high compositional variation has been the main problem hindering its use as a feed ingredient. This review provides updated information on the chemical composition of distillers grains in terms of nutrient levels, changes during dry-grind processing, and causes for large variation. The occurrence in grain feedstock and the fate of mycotoxins during processing are also covered. During processing, starch is converted to glucose and then to ethanol and carbon dioxide. Most other components are relatively unchanged but concentrated in DDGS about 3-fold over the original feedstock. Mycotoxins, if present in the original feedstock, are also concentrated. Higher fold of increases in S, Na, and Ca are mostly due to exogenous addition during processing, whereas unusual changes in inorganic phosphorus (P) and phytate P indicate phytate hydrolysis by yeast phytase. Fermentation causes major changes, but other processing steps are also responsible. The causes for varying DDGS composition are multiple, including differences in feedstock species and composition, process methods and parameters, the amount of condensed solubles added to distiller wet grains, the effect of fermentation yeast, and analytical methodology. Most of them can be attributed to the complexity of the dry-grind process itself. It is hoped that information provided in this review will improve the understanding of the dry-grind process and aid in the development of strategies to control the compositional variation in DDGS.

Changes in mineral concentrations and phosphorus profile during dry-grind processing of corn into ethanol.

For determining variation in mineral composition and phosphorus (P) profile among streams of dry-grind ethanol production, samples of ground corn, intermediate streams, and distillers dried grains with solubles (DDGS) were obtained from three commercial plants. Most attributes (dry matter concentrations) increased significantly from corn to cooked slurry but fermentation caused most significant increase in all attributes. During centrifugation, more minerals went into thin stillage than wet grains, making minerals most concentrated in the former. Mineral increase in DDGS over corn was about 3 fold, except for Na, S, Ca, and Fe. The first three had much higher fold of increase, presumably due to exogenous addition. During fermentation, phytate P and inorganic P had 2.54 and 10.37 fold of increase over corn, respectively, while relative to total P, % phytate P decreased and % inorganic P increased significantly. These observations suggest that phytate underwent some degradation, presumably due to activity of yeast phytase.

Changes in composition and amino acid profile during dry grind ethanol processing from corn and estimation of yeast contribution toward DDGS proteins.

Three sets of samples, consisting of ground corn, yeast, intermediate products, and DDGS, were provided by three commercial dry grind ethanol plants in Iowa and freeze dried before chemical analysis. On average, ground corn contained 70.23% starch, 7.65% protein, 3.26% oil, 1.29% ash, 87.79% total carbohydrate (CHO), and 17.57% total nonstarch CHO, dry matter basis. Results from Plant 1 samples showed that compared to ground corn, there was a slight but significant increase in the contents of protein, amino acids (AA), oil, and ash before fermentation, although starch/dextrin decreased sharply upon saccharification. After fermentation, starch content further decreased to about 6.0%, while protein, oil, and ash contents increased over 3-fold. AA increased 2.0-3.5-fold. Total CHO content decreased by 40%, and the content of total nonstarch CHO increased over 2.5-fold. Concentrations of these attributes fluctuated slightly in the remaining downstream products, but oil and ash were concentrated in thin stillage, while protein was concentrated in distiller grains upon centrifugation. When AA composition is expressed in relative % (protein basis), its changes did not follow that of protein concentration, but the influence of yeast AA profiles on those of downstream products became apparent. Accordingly, a multiple linear regression model for the AA profile of a downstream product as a function of AA profiles of ground corn and yeast was proposed. Regression results indicated that, with an r(2) = 0.95, yeast contributed about 20% toward DDGS proteins, and the rest came from corn. Data from Plants 2 and 3 confirmed those found with Plant 1 samples.

Dry fractionation methods to produce barley meals varying in protein, beta-glucan, and starch contents.

UNLABELLED: Barley contains several valuable nutrients including beta-glucan (BG), protein, and starch. Each has additional value when concentrated. Dehulled and hulless barleys were sequentially pearled for 1 to 6 cycles, each with 8% removal. The 6 pearled kernels and the initial kernel were subjected to impact or abrasive milling, followed by sieving with a series of U.S. standard sieves. Results of pearling fines show that protein was most concentrated in the outer area, and decreased all the way toward the core area (near 100% surface removal). Starch showed an opposite trend. BG followed the starch trend, but reached a peak at about 60% surface removal. Upon milling and sieving of kernel samples, genotype and particle size had significant effects on nutrient contents in sieved fractions. The pearling cycle had significant effects on protein and starch contents but little effect on BG content, while the milling method had significant effects on protein and BG contents but little on starch content. Abrasive milling produced sieved fractions with much higher variation in protein content than impact milling, but the opposite effect was observed for shifting BG content. Mass frequency influenced more on recovery rates of nutrients than their concentrations in individual fractions. When the recovery rate was also taken into consideration, pearling alone was found to be the most effective way to enrich protein in barley kernels. However, a combination of pearling with the method of milling and sieving was needed for maximally shifting BG and starch contents. PRACTICAL APPLICATIONS: Although dry fractionation is the method of choice for separating barley into fractions with varying levels of protein, BG, and/or starch, selection of a specific single or combined method is needed for achieving maximum shifts of a particular nutrient. Such information is significant to those who use dry fractionation methods to enrich protein, BG, and/or starch.

Fractionation of distillers dried grains with solubles (DDGS) by sieving and winnowing.

Four commercial samples of distillers dried grains with solubles (DDGS) were sieved. All sieved fractions except for the pan fraction, constituting about 90% of original mass, were then winnowed with an air blast seed cleaner. Sieving was effective in producing fractions with varying composition. As the particle size decreased, protein and ash contents increased, and total carbohydrate (CHO) decreased. Winnowing sieved fractions was also effective in shifting composition, particularly for larger particle classes. Heavy sub-fractions were enriched in protein, oil and ash, while light sub-fractions were enriched for CHO. For protein, the combination of the two procedures resulted in a maximum 56.4% reduction in a fraction and maximum 60.2% increase in another fraction. As airflow velocity increased, light sub-fraction mass increased, while the compositional difference between the heavy and light sub-fractions decreased. Winnowing three times at a lower velocity was as effective as winnowing one time at a medium velocity. Winnowing the whole DDGS was much less effective than winnowing sieved fractions in changing composition, but sieving winnowed fractions was more effective than sieving whole DDGS. The two combination sequences gave comparable overall effects but sieving followed by winnowing is recommended because it requires less time. Regardless of combinational sequence, the second procedure was more effective in shifting composition than the first procedure.

Effects of particle size distribution, compositional and color properties of ground corn on quality of distillers dried grains with solubles (DDGS).

Oftentimes, corn processors believe that ground corn (raw material) and distillers dried grains with solubles (DDGS) are interrelated in certain quality parameters. Yet, previous studies, although rather limited, have not established this relationship. In this study, six ground corn samples and their resulting DDGS were analyzed for particle size distribution (PSD), using a series of six selected US standard sieves: Nos. 8, 12, 18, 35, 60, and 100, and a pan. The original sample and sieve sized fractions were measured for contents of moisture, protein, oil, ash and starch, and surface color. Total carbohydrate (CHO) and total non-starch CHO were also calculated. Results show that the geometric mean diameter (d(gw)) of particles varied with individual corn and DDGS samples, and that d(gw) of DDGS was larger than that of corn (0.696 vs. 0.479 mm, average values), indicating that during conversion of corn to DDGS, certain particles became enlarged. For d(gw) and mass frequency of individual particle size classes, the relationship between ground corn and DDGS varied, but PSD of the whole sample was well correlated between them (r=0.807). Upon conversion from corn to DDGS, on an average, protein was concentrated 3.59 times; oil, 3.40 times; ash, 3.32 times; and total non-starch CHO, 2.89 times. There were some positive correlations in contents of protein and non-starch CHO and in L value between corn and DDGS. Yet, variations in nutrients and color attributes were larger in DDGS than in corn. For either corn or DDGS, these variations were larger in sieved fractions than in the whole fraction. Raw material, processing method and addition of yeasts are among major factors considered for causing larger variations in these attributes among DDGS. The study partially supports the common belief by processors that quality attributes of corn affect those of DDGS.

Particle size distribution of distillers dried grains with solubles (DDGS) and relationships to compositional and color properties.

Eleven distillers dried grains with solubles (DDGS), processed from yellow corn, were collected from different ethanol processing plants in the US Midwest area. Particle size distribution (PSD) by mass of each sample was determined using a series of six selected US standard sieves: Nos. 8, 12, 18, 35, 60, and 100, and a pan. The original sample and sieve sized fractions were measured for surface color and contents of moisture, protein, oil, ash, and starch. Total carbohydrate (CHO) and total non-starch CHO were also calculated. Results show that there was a great variation in composition and color among DDGS from different plants. Surprisingly, a few DDGS samples contained unusually high amounts of residual starch (11.1-17.6%, dry matter basis, vs. about 5% of the rest), presumably resulting from modified processing methods. Particle size of DDGS varied greatly within a sample and PSD varied greatly among samples. The 11 samples had a mean value of 0.660mm for the geometric mean diameter (dgw) of particles and a mean value of 0.440mm for the geometric standard deviation (Sgw) of particle diameters by mass. The majority had a unimodal PSD, with a mode in the size class between 0.5 and 1.0mm. Although PSD and color parameters had little correlation with composition of whole DDGS samples, distribution of nutrients as well as color attributes correlated well with PSD. In sieved fractions, protein content, L and a color values negatively while contents of oil and total CHO positively correlated with particle size. It is highly feasible to fractionate DDGS for compositional enrichment based on particle size, while the extent of PSD can serve as an index for potential of DDGS fractionation. The above information should be a vital addition to quality and baseline data of DDGS.

Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems.

Soy protein, mixed with gluten and starch, was extruded into fibrous meat analogues under high-moisture and high-temperature conditions. The protein solubility of samples collected at different extruder zones and extrudates made with different moistures was determined by 11 extraction solutions consisting of 6 selective reagents and their combinations: phosphate salts, urea, DTT, thiourea, Triton X-100, and CHAPS. Protein solubility by most extractants showed decreasing patterns as the material passed through the extruder, but the solution containing all 6 reagents, known as isoelectric focus (IEF) buffer, solubilized the highest levels and equal amounts of proteins in all samples, indicating that there are no other covalent bonds involved besides disulfide bonds. With regard to relative importance between disulfide bonds and non-covalent interactions, different conclusions could be made from protein solubility patterns, depending on the type of extracting systems and a baseline used for comparison. The observation points out pitfalls and limitation of current protein solubility methodology and explains why controversy exists in the literature. Using the IEF buffer system with omission of one or more selective reagents is considered to be the right methodology to conduct protein solubility study and thus recommended. Results obtained with this system indicate that disulfide bonding plays a more important role than non-covalent bonds in not only holding the rigid structure of extrudates but also forming fibrous texture. The sharpest decrease in protein solubility occurred when the mix passed through the intermediate section of the extruder barrel, indicating formation of new disulfide bonds during the stage of dramatic increase in both temperature and moisture. After this stage, although the physical form of the product might undergo change and fiber formation might occur as it passed through the cooling die, the chemical nature of the product did not change significantly.

Comparison of the phosphorus and mineral concentrations in bran and abraded kernel fractions of a normal barley (Hordeum vulgare) cultivar versus four low phytic acid isolines.

Phytic acid consists of 65-80% of the total phosphorus (P) in cereal grains. Its salts are concentrated in the germ and aleurone layers, which are typically removed during milling. We hypothesize that concentrations of different types of P and minerals in milled products will be greatly altered in low phytic acid (lpa) barleys. Seeds of cv. Harrington (control) and four lpa isolines-lpa1-1, lpa2-1, lpa3-1, and M955-were abraded by a laboratory method into five surface layer and four remaining kernel fractions. Results show that phytic acid in the four lpa lines ranged from 75% to 5% of the control. The decrease in phytic acid P concentration was matched almost equally by an increase in inorganic P, so that the rest of P (the sum of all P-containing compounds other than phytic acid P and inorganic P) and total P levels remained relatively unchanged among the five genotypes. These trends were also observed for the processed fractions. The major mineral elements in barley seeds were P, K, Mg, S, and Ca, while minor ones were Fe, Zn, Mn, Cu, and Ba. All types of P and other minerals measured were generally concentrated in the outer layers of the grain. Although there were substantial differences in mineral contents of bran fractions among genotypes, the level of phytic acid P had little effect on mineral contents in whole or abraded kernels. One major exception was Fe, which had the highest level in all tissues of M955 genotype. The above findings were all confirmed by analyzing another set of barley samples grown in a different environment. Thus, in general, breeding lpa barleys does not lead to reduced mineral contents in whole grains or elevated mineral levels in milled products.

Glyphosate-tolerant soybeans remain compositionally equivalent to conventional soybeans (Glycine max L.) during three years of field testing.

Previous studies have shown that the composition of glyphosate-tolerant soybeans (GTS) and selected processed fractions was substantially equivalent to that of conventional soybeans over a wide range of analytes. This study was designed to determine if the composition of GTS remains substantially equivalent to conventional soybeans over the course of several years and when introduced into multiple genetic backgrounds. Soybean seed samples of both GTS and conventional varieties were harvested during 2000, 2001, and 2002 and analyzed for the levels of proximates, lectin, trypsin inhibitor, and isoflavones. The measured analytes are representative of the basic nutritional and biologically active components in soybeans. Results show a similar range of natural variability for the GTS soybeans as well as conventional soybeans. It was concluded that the composition of commercial GTS over the three years of breeding into multiple varieties remains equivalent to that of conventional soybeans.