Richard F. Wilson

Low-linolenic acid soybean oil—Alternatives to frying oils

Oil was hexane-extracted from soybeans that had been modified by hybridization breeding for low-linolenic acid (18∶3) content. Extracted crude oils were processed to finished edible oils by laboratory simulations of commercial oil processing procedures. Oils from three germplasm lines N83-375 (5.5% 18∶3), N89-2009 (2.9% 18∶3) and N85-2176 (1.9% 18∶3) were compared to commercial unhydrogenated soybean salad oil with 6.2% 18∶3 and two hydrogenated soybean frying oils, HSBOI (4.1% 18∶3) and HSBOII (<0.2% 18∶3). Low-18∶3 oils produced by hybridization showed significantly lower room odor intensity scores than the commercial soybean salad oil and the commercial frying oils. The N85-2176 oil with an 18∶3 content below 2.0% showed no fishy odor after 10 h at 190°C and lower burnt and acrid odors after 20 h of use when compared to the commercial oils. Flavor quality of potatoes fried with the N85-2176 oil at 190°C after 10 and 20 h was good, and significantly better at both time periods than that of potatoes fried in the unhydrogenated oil or in the hydrogenated oils. Flavor quality scores of potatoes fried in the N89-2009 oil (2.9% 18∶3) after 10 and 20 h was good and equal to that of potatoes fried in the HSBOI oil (4.1% 18∶3). Fishy flavors, perceived with potatoes fried in the low-18∶3 oils, were significantly lower than those reported for potatoes fried in the unhydrogenated control oil, and the potatoes lacked the hydrogenated flavors of potatoes fried in hydrogenated oils. These results indicate that oils with lowered linolenic acid content produced by hybridization breeding of soybeans are potential alternatives to hydrogenated frying oils.

Isolation and purification of diacylglycerol acyltransferase from germinating soybean cotyledons

Abstract A homogeneous preparation of the enzyme that catalyses triacylglycerol synthesis has been achieved for the first time from plant tissue. Diacylglycerol acyltransferase (EC 2.3.1.20) was solubilized from purified microsomes prepared from germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons with the non denaturing zwitterionic detergent, CHAPS. The enzyme was isolated from CHAPS solubilized microsomal proteins and purified by Sepharose CL-4B chromatography and agarose gel electrophoresis. Purified diacylglycerol acyltransferase exhibited a single Schlieren image during analytical ultracentrifugation. The homogeneity of the enzyme was confirmed by high-pressure liquid chromatography using gel filtration columns. The purified enzyme was devoid of lipase (EC 3.1.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15), lysolecithin acyltransferase (EC 2.3.1.23), acylglycerol acyltransferase (EC 2.3.1.22), and 1-acylglycerolphosphate acyltransferase (EC 2.3.1.51) activities. Purified diacylglycerol acyltransferase activity was stable for three months at −20°C. Hyperbolic enzyme kinetics were observed using sn-1,2-diolein and stearoyl-CoA or oleoyl-CoA as substrates. The apparent Km using sn-1,2-diolein was 60 μM with stearoyl-CoA, and 113 μM with oleoyl-CoA. This report has documented a method by which highly purified diacylglycerol acyltransferase may be obtained from germinating soybean cotyledons.

Phenotypic variation for saturated fatty acid content in soybean

SummaryConcern over high saturates in human diets has prompted the development of soybean [Glycine max (L.) Merr.] lines producing oil with reduced saturated fatty acid concentration. To better understand those factors that influence phenotypic expression for palmitic and stearic acid content in soybean, thirty soybean lines random for saturated fatty acid content were grown in eight field environments contrasting for mean temperature during seed-filling. Palmitic and stearic acid content varied significantly (P<0.01) both among genotypes and across environments, while genotype x environment interactions were reflected in changes in line variance and ranking for both traits. Therefore selection of a superior genotype for saturated fatty acid composition may not correlate well from one environment to another. In general, early-maturing lines were less sensitive than later-maturing lines in their response to changes in mean daily temperature for palmitic concentration. However, factors in addition to temperature appeared to influence genotype response for stearic acid content. It appears that genetic systems conditioning palmitic and stearic acids are independent, and that separate breeding strategies need be adopted to make simultaneous improvement for these two oil traits. In summary, development of soybean lines with low or high saturated fatty acid content may be accomplished through evaluation and selection in a few environments contrasting for temperature.

Triacylglycerol synthesis and metabolism in germinating soybean cotyledons

Abstract The objective of this study was to investigate the ability of germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons to synthesize triacylglycerol, for the purpose of identifying an alternative tissue source for the isolation and purification of the enzyme diacylglycerol acyltransferase (EC 2.3.1.20). Aspects of glycerolipid metabolism were examined in etiolated cotyledons and greening cotyledons with intact or detached epicotyls at various stages of germination. Regardless of tissue morphology or age, cotyledons from all treatments were found to be capable of triacylglycerol synthesis from [2-14C]acetate. In general, the rate of triacylglycerol synthesis in etiolated cotyledons was greater than in greening cotyledons with intact epicotyls. The greatest triacylglycerol synthetic activity (2.1 nmol 14C/h per g dry weight), however, was attained at 20 days after germination in greening cotyledons with detached epicotyls. That rate was approximately one-half of the maximal rate of triacylglycerol synthesis from [2-14C]acetate reported for developing Dare seed. Furthermore, it was shown that the de novo synthesized triacylglycerol in germinating soybean cotyledons exhibited zero-order metabolic kinetics. Hence these data indicated a subcellular compartmentation of the synthetic and hydrolytic mechanisms associated with triacylglycerol metabolism in germinating soybean cotyledons. The conclusion drawn from these findings was that germinating soybean cotyledons would be a suitable medium for the isolation, purification, and subcellular localization of diacylglycerol acyltransferase, the enzyme which catalyses triacylglycerol synthesis.

Temperature effects upon the expression of a high oleic acid trait in soybean

Soybeans (Glycine max L. Merr. cvs. N78-2245 and Dare) were grown to maturity under controlled environments to investigate temperature effects upon the fatty acid composition of developing seed. These genotypes exhibited genetic differences in oleic acid (18:1) content. Mature seed from N78-2245 germplasm normally contained ca. 43 mol% 18:1, and Dare seed contained ca. 18 mol% 18:1. When grown at 30/26 C or 22/18 C, the overall response of these genotypes to temperature resulted in lower 18:1 and higher linoleic (18:2) and linolenic (18:3) acid concentrations in mature seed. However, the genotypic response was much more pronounced in N78-2245 seed than in Dare seed. The basis for these genotypic differences appeared to be related to temperature effects upon the differentiation of the 18:1-synthetic and 18:1-desaturation mechanisms during seed development. Although the high-18:1 trait was expressed during N78-2245 seed development at both temperatures, high-18:1 glycerolipids accumulated during a shorter developmental period at 22/18 C than at 30/26 C. At 30/26 C, glycerolipids containing greater than 50% 18:1 were deposited between 20 and 45 days after flowering (DAF) and accounted for 84% (w/w) of the oil in mature seed. At 22/18 C, glycerolipids with similar fatty acid composition were formed between 30 and 45 DAF and accounted for only 40% (w/w) of the oil. Temperature effects upon 18:1-desaturation also appeared to mediate the overall differences in unsaturated fatty acid composition in these genotypes. The 18:1-desaturation mechanism in N78-2245 seed was more sensitive to temperature than that in Dare seed. These genotype-treatment combinations were ranked by degree of 18:1-desaturation in the order: Dare (22/18 C) = Dare (30/26 C) ≥ N78-2245 (22/18 C) > N78-2245 (30/26 C). It was proposed that the ranking of these genotype-treatment combinations may be attributed, in part, to the tissue levels of the 18:1-desaturase enzymes in soybean seed grown at different temperatures.

Effects of defoliation on seed protein concentration in normal and high protein lines of soybean

Two high (NC106, NC111) and two normal (NC103, NC107) seed protein concentration lines, derived from two different recurrent selection populations of soybean (Glycine max L. Merr.) were subjected to partial defoliation at beginning seed fill (R5) under outdoor pot culture and field conditions. The aim of this study was to test the hypothesis that capacity to store N in vegetative organs and/or to mobilize that N to reproductive organs is associated with the high seed protein concentration trait. Symbiotic N2 fixation was the sole source of N in the pot experiment and the major source of N (met > 50% of the N requirement) in the low N soil used in the field experiment. Seed protein concentration and seed yield at maturity in both experiments and N accumulation and mobilization between R5 and maturity in the pot experiment were measured. The four genotypes did not differ significantly with respect to the amount of N accumulated before beginning seed fill (R5). Removal of up to two leaflets per trifoliolate leaf at R5 significantly decreased the seed protein concentration of NC107/111 but had no effect on this trait in NC103/106. Defoliation treatments significantly decreased seed yield, whole plant N accumulation (N2-fixation) during reproductive growth and vegetative N mobilization of all genotypes. Differences in harvest indices between the high and low protein lines accounted for approximately 35% of the differences in protein concentration. The two normal protein lines mobilized more vegetative N to the seed (average. 5.26 g plant−1) than the two high protein lines (average. 4.28 g plant−1). The two high seed protein lines (NC106, NC111) exhibited significantly different relative dependencies of reproductive N accumulation on vegetative N mobilization, 45% vs. 29%, in the control treatment. Whereas, NC103 with normal and NC106 with high seed protein concentration exhibited similar relative dependencies of reproductive N accumulation on vegetative N mobilization, (47% vs. 45%). Collectively, these results indicate that N stored in shoot organs before R5 and greater absolute and relative contribution of vegetative N mobilization to the reproductive N requirement are not responsible for the high seed protein concentration trait.

Subunit and amino acid composition of diacylglycerol acyltransferase from germinating soybean cotyledons.

The subunit and amino acid composition of the enzyme that catalyses triacylglycerol synthesis was determined for the first time from plant tissues. Diacylglycerol acyltransferase (acyl-CoA:1,2-diacylglycerol O-acyltransferase, EC 2.3.1.20) purified from germinating soybean (Glycine max L. Merr. cv. Dare) cotyledons, dissociated into three nonidentical subunits having apparent molecular masses of 40.8, 28.7, and 24.5 kDa. The respective subunits occurred in a 1:2:2 molar ratio in the native enzyme. Five peptides in that molar ratio were assumed to constitute a monomer having a putative molecular mass of 153.1 kDa. Based upon the apparent molecular mass of purified diacylglycerol acyltransferase after delipidation (1539 kDa), there was a high probability that the complete structure of the native enzyme from soybean contained ten identical monomers. The polarity index of each subunit was less than 21%, far below the 40% boundary reported for membrane bound proteins. Hydrophobic amino acids accounted for greater than 48% of the composition in each subunit. It was predicted from these data that the native enzyme contained 12,525 amino acid residues, and that the two smaller subunits were more deeply embedded in the membrane than the 40.8 kDa subunit. Attempts to reactivate the denatured or delipidated protein were not successful.

Effect of fungal damage on seed composition and quality of soybeans

Fungal damage caused by pathogens such asFusarium, Cercospora, andPhomopsis can have a devastating impact on physical quality and farm price of soybeans. In some price-discount schedules, soybeans may be rejected with as low as 5% fungal damage. Although the severity of this problem varies throughout the United States, millions of bushels of fungus-damaged soybeans may be destroyed annually due to a lack of markets. The effect of fungal damage on seed composition was evaluated to assess potential utility of highly damaged soybeans. Graded samples of the cv. Centennial soybean were dried to 10% moisture and blended on a proportional weight basis to derive a series of treatments from 0 to 80% fungal damage. A positive correlation was found between fungal damage and both protein and oil concentrations. This condition was attributed to loss of residual seed mass. As a result, the protein concentration of defatted meal increased from ca. 54 to 66% over the range of 0 to 80% fungal damage. Mycotoxin contamination appeared to be insignificant in these high-protein meals. Fixed colors in bleached, alkali refined oils were intensified by heat treatment prior to extraction. No significant differences, however, were noted in total polar lipid content, phospholipid, or tocopherol composition among treatments of up to 20% fungal damage. Oils from treatments of more than 40% fungal damage were more severely oxidized and could not be degummed effectively. These data suggest that fungus-damaged soybeans may be blended with high-quality soybeans to alleviate the chemical symptoms associated with unacceptable product quality. Thus, through various blend ratios, processors may consider using fungus-damaged soybeans to gain economic advantage, especially when high-quality soybeans have lower protein concentration.

Lipid changes during pre-germination and germination ofstriga asiatica seeds

Witchweed (Striga asiatica L. Kuntze) seeds were incubated at 28 C in a moist environment for a 14-day period, after which seeds germinated only when exposed to specific natural or synthetic germination stimulants. Changes in lipid composition were determined during germination of witchweed seeds and during early seedling growth. Witchweed seeds contained 37.5% (w/w) oil. Increased levels of monogalactosyl-diacylglycerol and phosphatidylglycerol suggested the enlargement or multiplication of plastids after witchweed seeds had germinated. In contrast to the usual course of events in seeds with high oil reserves, witchweed seeds did not hydrolyze triacylglycerol rapidly during or after germination. These findings indicated that triacylglycerol in germinating witchweed seeds was conserved for subsequent use during haustorial formation and host invasion.

Role of Diacylglycerol Acyltransferase in Regulating Oil Content and Composition in Soybean

Accessions of the USDA Soybean ( Glycine max L. Merr.) Germplasm Collection exhibit genetic diversity for oil concentration ranging from 12 to 27 percent of dry weight. Although oil concentration is a highly heritable quantitative genetic trait, the genetic and biochemical basis for genotypic differences in the oil content of soybean seed is unknown. Recent knowledge on biological regulation of this trait has emerged from research on diacylglycerol acyltransferase (EC 2.3.1.20), the enzyme that catalyses triacylglycerol synthesis. Diacylglycerol acyltransferase purified from the cv. Dare has a native mass of about 1.5 MDa. Structural analysis suggests the protein consists of 10 monomers having three nonidentical subunits in a 1:2:2 molar ratio. Kinetics of the enzyme purified from soybeans exhibiting high or low oil content suggest that genotypic differences in oil content may be governed by gene dosage effects. However, subtle conformational changes in protein structure may influence oil composition, as evidenced by apparent substrate specificities observed in germplasm containing low-palmitic acid. Therefore, diacylglycerol acyltransferase may play a unique role in determining the content and composition of triacylglycerol in soybean.