William L. Boatright

Effect of lipids on soy protein isolate solubility

Reduced-lipid soy protein isolate (SPI), prepared from soy flour treated so that most of the polar lipids have been removed, exhibited an increase in protein solubility of 50% over that of the control SPI prepared from hexane-defatted flour. Adding lipids from a commercial SPI during processing of reduced-lipid SPI decreased SPI solubility by 46%. The 19% decreased solubility caused by the lipids (primarily phospholipids) was largely recovered by treating the protein with a reducing agent (2-mercaptoethanol). The balance of protein insolubility, caused by the lipids, was attributed to a smaller lipid fraction (approximately 5% of the total lipids). Adding lipids during SPI processing contributed to both the formation of oxidized protein sulfhydryls, incapable of being reduced by 2-mercaptoethanol, and to oxidative deterioration of protein as determined by protein carbonyl contents.

Soybean protein bodies: Phospholipids and phospholipase D activity

Protein bodies from mature soybeans were isolated by differential centrifugatioin to examine their composition and relationship with phospholipase D. Densities were adjusted by varying mixtures of soybean oil and carbon tetrachloride. The purity of the final isolate was confirmed by electron microscopy. Approximately 4.5% by weight of the protein bodies was lipid and 2.0% by weight phospholipid. Thin-layer chromatography revealed only a trace of phosphatidic acid. Treatment with 60% ethanol/40% sodium acetate buffer (pH 5.6) (vol/vol) resulted in phosphatidyl transferase activity with conversion of both phosphatidylcholine and phosphatidylethanolamine into phosphatidylethanol. It is proposed that protein bodies of soybeans contain phospholipase D.

Oxygen dependency of one-electron reactions generating ascorbate radicals and hydrogen peroxide from ascorbic acid

The effect of oxygen on the two separate one-electron reactions involved in the oxidation of ascorbic acid was investigated. The rate of ascorbate radical (Asc(-)) formation (and stability) was strongly dependent on the presence of oxygen. A product of ascorbic acid oxidation was measurable levels of hydrogen peroxide, as high as 32.5 μM from 100 μM ascorbic acid. Evidence for a feedback mechanism where hydrogen peroxide generated during the oxidation of ascorbic acid accelerates further oxidation of ascorbic acid is also presented. The second one-electron oxidation reaction of ascorbic acid leading to the disappearance of Asc(-) was also strongly inhibited in samples flushed with argon. In the range of 0.05-1.2 mM ascorbic acid, maximum levels of measurable hydrogen peroxide were achieved with an initial concentration of 0.2 mM ascorbic acid. Hydrogen peroxide generation was greatly diminished at ascorbic acid levels of 0.8 mM or above.

Effect of storage conditions on carbon-centered radicals in soy protein products.

Using electron paramagnetic resonance (EPR) spectroscopy, the levels of carbon-centered radicals in retail samples of isolated soy protein (ISP), soy protein concentrate (SPC), and powdered soy milk were estimated to contain from 6.12 x 10(14) to 1.98 x 10(15) spins/g of soy product. Roasted soy nuts contained about 5.70 x 10(15) spins/g. The peak to peak line width of the carbon-centered radicals from soy nuts was about 10 gauss, whereas ISP samples with a similar peak height had a peak to peak line width of about 8 gauss. Retail snack bars containing ISP, SPC, and/or roasted soy nuts with a total protein content of either 13, 21, or 29% contained 5.32 x 10(14), 6.67 x 10(14), and 5.74 x 10(14) spins/g of snack bar, respectively. Levels of carbon-centered radicals in newly prepared samples of ISP were much lower than levels in the retail soy protein products and levels previously reported for commercial ISP and laboratory ISP samples. The levels of radicals in ISP samples increased over a 12-25 week period of storage in the dark at 22 degrees C and exposed to air from about 8.00 x 10(13) spins/g immediately after preparation to 9.95 x 10(14) spins/g of ISP. Storing the ISP samples under nitrogen at 22 degrees C greatly reduced the increase in radical content, whereas storing the ISP in 99.9% oxygen at 40 degrees C accelerated the formation of stable carbon-centered radicals. ISP samples hydrated at either 22 or 92 degrees C, rapidly frozen, and dried lost about 92% of the trapped radicals. The level of carbon-centered radicals in these same ISP samples immediately began to increase during subsequent storage exposed to the air and gradually returned to similar levels obtained before they were hydrated.

Carbon‐Centered Radicals in Isolated Soy Proteins

Solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of isolated soy proteins (ISP) revealed a symmetrical free-radical signal typical of carbon-centered radicals (g= 2.005) ranging from 2.96 x 10(14) to 6.42 x 10(14) spins/g. The level of free radicals in ISP was 14 times greater than similar radicals in sodium caseinate, 29 times greater than egg albumin, and about 100 times greater levels than casein. Nine soy protein powdered drink mixes contained similar types of free radicals up to 4.10 x 10(15) spins/g of drink mix, or up to 6.4 times greater than the highest free-radical content found in commercial ISP. ISP samples prepared in the laboratory contained trapped radicals similar to the levels in commercial ISP samples. When ISP was hydrated in 2.3 mM sodium erythorbate or 8.3 mM L-cysteine, frozen and dried, the level of trapped free radicals increased by about 17- and 19-fold, respectively. The ESR spectrum of defatted soybean flakes contained overlapping signals from the primary free-radical peak (g= 2.005) and a sextet pattern typical of manganese-II. The manganese signal was reduced in the laboratory ISP and very weak in the commercial ISP.

Lipid components that reduce protein solubility of soy protein isolates

A lipid fraction from a commercial soy protein isolate (SPI), previously found to be detrimental to SPI solubility, was analyzed by size-exclusion liquid chromatography, by high-performance liquid chromatography (HPLC), and for chemical composition. The molecular weight of most of this material was greater than 1,100 daltons. This lipid fraction was water-soluble yet required a strong nonpolar solvent mixture to elute it from a C18 HPLC column. The lipid material was alkaline (pH 8.7) and composed of 3.0% nitrogen, 1.6% phosphorus, 17.5% nonvolatile crude fatty acids primarily hydroxylated), 10.4% long-chain bases, 9.9% hexuronic acid, 3.2% hexosamine, and 6.6% total sugar. The molecular weight, chemical composition, and physical characteristics (solubility characteristics, surfactant characteristics, and appearance) of this material were all similar to those reported for phytoglycolipid.

Metastable Radicals and Intrinsic Chemiluminescence from Soy Proteins

UNLABELLED Chemiluminescence from various powdered food proteins were examined without the addition of any external source of free radicals or luminescent agents. In the solid-state, soy and whey proteins produced more intrinsic chemiluminescence than casein, sodium caseinate, or egg albumin. However, when these same food proteins were hydrated, intrinsic chemiluminescence from soy proteins was about 4- to 8-times greater than other source proteins. Quenching the alkyl-radicals in the powdered soy proteins with hydrogen sulfide reduced the typical electron paramagnetic resonance spectra from soy proteins below detectable levels, and reduced the chemiluminescence from the hydrated soy proteins by about 65%. Antioxidants also reduced chemiluminescence in hydrated soy proteins by about 50% to 92%, with ellagic acid being the most effective. The reduction in chemiluminescence from both quenching radicals in the solid state, and by the addition of antioxidants to aqueous mixtures, indicate that the chemiluminescence produced when soy proteins are hydrated is a free radical catalyzed event. Based on the production of chemiluminescence, the radicals from soy protein were largely released within 30 min of hydration at 23 °C. Elevating the hydration temperatures increased chemiluminescence by as much as 280% at 70 °C, and decreased the half-life of the light-emitting reaction by about 9-fold. PRACTICAL APPLICATIONS Levels of metastable radicals in powdered soy proteins typically range from to 10 to 100 times greater than free radicals from other food protein sources. This research focuses on the types of reactions these radicals catalyze when soy proteins are hydrated, and the radicals suddenly become reactive. The findings suggest that a portion of the energy released from metastable radicals when powdered soy proteins are hydrated is involved in the generation of chemically-induced light.

Static headspace analysis of odorants in commercial rice proteins

Accurate identification of the odor-contributing compounds in aqueous slurries of rice proteins is necessary to improve their overall flavor characteristics. The objective of this study was to identify the primary odorants in rice protein slurries using static headspace analysis. Five commercial rice protein (RP) products, RP-G, RP-O, RP-RM, RP-RS1, and RP-RS2, were analyzed. RP-G contained the lowest levels of most of the odorants. Acetaldehyde was present in the highest amount in RP-O (0.434mg/m3). RP-RM had the highest levels of hexanal (5.907mg/m3), methanethiol (0.138mg/m3), pentanal (1.575mg/m3), and 2-pentylfuran (5.702mg/m3). Corresponding odor values were, 111, 86, 22 and 21, respectively. In RP-RS1 and RP-RS2, the predominant odorants were dimethyl disulfide, dimethyl trisulfide, and hexanal. The results showed the importance of the volatile compounds produced from amino acids, including the sulfur-containing compounds and acetaldehyde, as well as lipid oxidation derived odorants to the overall odor of rice proteins.

Effect of sequestering intrinsic iron on the electron paramagnetic resonance signals in powdered soy proteins.

This investigation examined iron in powdered soy protein products using electron paramagnetic resonance (EPR) spectroscopy, and the effect that selectively binding free iron in isolated soy protein (ISP) had on the occurrence of metastable radicals in powdered soy proteins. EPR analyses of soybean defatted flour, commercial ISP and laboratory ISP samples revealed a peak at g = 4.3 characteristic of high-spin ferric iron in a rhombic-coordinated environment. Commercial ISP samples examined contained higher levels of the rhombic ferric iron than laboratory-prepared ISP samples. During the first 6 wk of storage the primary singlet EPR signal at g = 2.0049 in the commercial ISP samples approximately doubled, and the laboratory prepared samples increased by about 9-fold. The EPR signal was initially about 4-times higher in the freshly prepared commercial samples compared to the corresponding laboratory ISP. Laboratory ISP samples prepared with added deferoxamine to sequester endogenous iron exhibited a large increase in the high-spin ferric iron EPR signal at g = 4.3. ISP treated with deferoxamine also exhibited a multiple-line EPR signal at about g = 2.007, instead of the typical singlet signal at g = 2.0049. The power at which the signal amplitude was half-saturated also changed from about 1 mW in the control ISP to about 20 mW in the deferoxamine treated ISP. The multiple-line EPR spectrum from the ISP treated with deferoxamine increased during storage over a 6-wk period by about 6-fold. The observed changes in EPR line-shape, g-value, and power saturation with the deferoxamine treatment indicate that the primary free-radical signal in powdered ISP samples may be from stabilized tyrosine radicals with spin densities distributed over the aromatic ring.

Redox cycling and generation of reactive oxygen species in commercial infant formulas

Three nationally prominent commercial powdered infant formulas generated hydrogen peroxide, ranging from 10.46 to 11.62 μM, when prepared according to the manufacturers instructions. Treating infant formulas with the chelating agent diethylene triamine pentaacetic acid (DTPA) significantly reduced H2O2 generation. In contrast, the addition of disodium ethylenediaminetetraacetic acid (EDTA) elevated the level of H2O2 generated in the same infant formulas by approximately 3- to 4-fold above the untreated infant formulas. The infant formulas contained ascorbate radicals ranging from about 138 nM to 40 nM. Treatment with catalase reduced the ascorbate radical contents by as much as 67%. Treatment with DTPA further reduced ascorbate radical signals to below quantifiable levels in most samples, further implicating the involvement of transition metal redox cycling in reactive oxygen species (ROS) formation. Supportive evidence of the generation of ROS is provided using luminol-enhanced luminescence (LEL) in both model mixtures of ascorbic acid and in commercial infant formulas.