Soybean trypsin inhibitor and urease activities and their correlations as affected by heating method,duration, sample matrix,and prior soaking

Urease activity (UA) in soybeans has historically been measured to indicate heating inadequacy. Yet, over the years, controversy has emerged regarding the reliability of UA as a heating index and surrogate for trypsin inhibitor activity (TIA). In Experiments 1–4, raw soybean materials with different matrices (whole beans, flakes, full-fat and defatted flours) were selectively subjected to steaming, boiling, or dry oven toasting for various durations. For steaming or boiling soybeans, with or without prior soaking was another factor. Reduction rates of TIA and UA with heating time were compared, their correlation coefficients were determined and statistically treated. Experiment 5 entailed collecting 30 commercial soybean meals and measuring TIA and UA without further treatments. By combining the five experiments into a single study, the most comprehensive spectra regarding relative decreasing rates of TIA and UA with heating time and their correlations were obtained. Results show that the reduction rate of UA could be slower than, close to, or faster than that of TIA, depending on combinations of four factors (sample matrix, with or without prior soaking, heating method and interval). UA reached zero within shorter heating durations, while TIA maintained residual values at the longest durations. Consequently, positive correlations between TIA and UA varied from insignificant to very strong. UA was a reliable index for heating inadequacy and surrogate for TIA in soybean products heated by several single combinations of the four factors, but for those heated by other single or mixed combinations, it was unreliable, and TIA should be measured directly.     

Heat inactivation of trypsin inhibitor,lipoxygenase and urease in soybeans: Effect of acid and base additives

Effects of chemical additives on the heat inactivation of trypsin inhibitor (TI), lipoxygenase and urease in soybeans were investigated. The nutritional value of soybeans increases when antigrowth factors, such as TI, are inactivated. Inactivation of lipoxygenase enhances palatability and storage stability. Heat inactivation of antinutritional factors during immersion cooking of dry soymeats was studied without additives. Processing time was varied from 15 min to 2 hr over a temperature range of 120–212 F. The experiments were repeated, with the addition of NaOH or HCl to the cooking water. Without additives, lipoxygenase proved to be the most heat labile and TI, the least. With either acid or base additives, the initial inactivation of urease and lipoxygenase was accelerated significantly; however, while TI inactivation was accelerated by base, it was retarded by acid addition. Presented at the AOCS Meeting, Ottawa, September 1972. ARS, USDA.     

Evaluation of ammonium thiosulfate as a soil urease inhibitor

Interest in use of ammonium thiosulfate (ATS) in conjunction with urea as a fertilizer has been stimulated by recent reports that this compound retards hydrolysis of urea by soil urease and thereby reduces volatilization of urea N as ammonia from soils fertilized with urea. We evaluated ATS as a soil urease inhibitor by studying its effects on urea hydrolysis, seed germination, and early seedling growth in soil. We found that ATS significantly retarded urea hydrolysis only when applied at rates as high as 2,500 or 5,000µg g^–1 soil, whereasN-(n-butyl) thiophosphoric triamide (NBPT) (a patented inhibitor of urea hydrolysis in soil) caused substantial retardation of urea hydrolysis when applied at rates as low as 1µg g^–1 soil. We also found that ATS had an adverse effect on germination of corn or wheat seeds in soil when applied at the rate of 2,500 or 5,000µg g^–1 soil and caused a dramatic reduction of early seedling growth of corn or wheat when applied at the rate of 1,000, 2,500, or 5,000µg g^–1 soil. These findings indicate that ATS has little, if any, potential value for retarding hydrolysis of urea fertilizer in soil.     

Toasted soybean flour components with trypsin inhibitor activity

Compounds in toasted soybean flour having trypsin inhibitor (TI) activity were isolated and characterized. Sodium hydroxide (0.01N) extracts of toasted soybean flour had an average of 2.59 mg TI/g sample. These extracts, after trichloroacetic acid (TCA) precipitation and dialysis, yielded supernatant and precipitate fractions. Addition of polyvinylpyrrolidone to eliminate free tannins and phenolics in the extracts, which may lead to overestimation of TI activity, was unnecessary. Material balance studies revealed 91% protein recovery and 92% recovery of TI activity in the TCA supernatant (1.1% protein, 2.0% TI) and precipitate (89.8% protein, 90.0% TI) fractions. Column chromatography and electrophoresis showed the TCA supernatant and precipitate fractions to contain proteins, including those having TI activity. Kunitz type TI and Bowman-Birk type protease inhibitors accounted for most residual TI activity of toasted soybean flour, as verified by column chromatography, isoelectric focusing, sodium dodecyl sulfate polyacrylamide electrophoresis, and size-exclusion high performance liquid chromatography, using the two similarly treated protease inhibitors as standards. Immunoblotting was also used to detect and identify Kunitz type TI’s in toasted soybean meal extracts. This study established the proteinaceous nature of residual trypsin inhibitor activity in toasted soybean flour and the presence of both Kunitz and Bowman-Birk inhibitors. Presented in part at the AOCS meeting in Dallas, TX, in 1984.     

土壤脲酶及脲酶抑制剂

本文介绍了影响尿素利用率的主要因素和一种新型脲酶抑制剂的研究方法。     

Effect of feeding soy products with varying trypsin inhibitor activities on growth of shrimp

Six isonitrogenous, isocaloric diets containing commercially defatted, toasted and lightly toasted soy flours (SF) (diets 1 and 2) and four soy protein concentrates (SPC) (diets 3–6) as replacements for 40% of animal protein were fed to satiation to juvenile shrimp (Penaeus vannamei) for 10 weeks. The SPCs used in diets 3 and 5 were chemically modified products with reduced trypsin inhibitor (TI) content. The chemical modification of SF in diet 2, which resulted in an SPC for diet 3, and of SPC in diet 4 consisted of heating at 70°C for 1 hr with 50 mM Na₂S₂O₅, followed by dialysis to remove salt residues. To keep all diets isocaloric, cornstarch was added to replace the oli-gosaccharides lost during processing to an SPC. The TI contents, in mg TI/g diet, were 0.77, 6.14, 0.64, 1.40, 0.92 and 1.72 for diets 1–6, respectively. Shrimp fed lightly toasted SF had the highest weight gain, which was significantly higher than shrimp fed SPC diets 4, 5 and 6, but not significantly higher than shrimp fed diets 1 and 3. No significant difference was observed in survival rates. Shrimp fed diet 3 (with lowest TI) had the highest body percentages of crude protein, while toasted soy flour diet 1, also with low TI, had the lowest content of this constituent. In general, a high body protein reflects good health of the animal and excellent utilization of the feed. At the replacement levels of soy evaluated, TI content did not affect overall weight gain. Presented at the 1991 AOCS Meeting, May 12–15, 1991, Chicago, IL.     

Mercapto-pyridines and mercapto-pyrimidines as urease inhibitors

Several mechanisms of urease hydrolysis of urea have been proposed. Major advances in this area were made when it was recognized that nickel played an important role in the hydrolysis of urea. Two recent literature reports indicate that dipyridyls can be used to complex with nickel. In both of these reports, the dipyridyl was used as a ligand for stabilizing an intermediate nickel(II) complex. In addition, it is known that heterocyclic mercapto compounds inhibit urease through the formation of disulfide bonds. Because of the stability of these complexes, compounds with similar structures were examined as potential urease inhibitors. From our initial tests it was not clear which functional group of these multifunctional compounds was responsible for the inhibition or whether it was necessary to retain the disulfide linkage for other potential inhibitors in this class. Therefore, a series of mercapto-pyridines, a series of mercapto-pyrimidines, and pyridine-N-oxide itself were tested for their urease inhibitory characteristics. It appears that the key functional group responsible for the inhibition is the mercapto group itself, with the N-oxide function adding little to the urease inhibitory power in the monomeric compounds. It is suspected that the presence of the N-oxide moiety in the dimeric compounds results in increases in solubility and a weakening of the strength of the disulfide bond, thus producing a more active inhibitor.     

Influence of urea fertiliser formulation, urease inhibitor and season on ammonia loss from ryegrass

This paper reports the results of experiments to determine whether ammonia (NH₃) loss can be reduced and nitrogen (N) use efficiency improved by using two relatively new commercial urea formulations rather than granular urea and urea ammonium nitrate. Four nitrogen treatments were applied at a rate of 40 kg N ha^?1: granular urea, ‘Green Urea? 14’ [containing 45.8 % N as urea and ‘Agrotain^®’ (N-(n-butyl) thiophosphoric triamide) @ 5 L t^?1 of urea as a urease inhibitor], ‘Nhance’, a fine particle spray [containing 46 % N as urea, ‘Agrotain’ @ 1 L t^?1 of urea and gibberellic acid (applied at a rate of 10 g ha^?1)] and urea ammonium nitrate in solution (UAN) surface applied. Ammonia loss was determined in autumn and spring using a micrometeorological method. In autumn, use of the Green Urea and Nhance reduced NH₃ loss from the 30 % of applied N lost from the granular urea to 9 and 23 % respectively. Loss from all treatments in spring was very small (<2 % of applied N), because 4 mm of rain fell within 24 h of application onto an already wet site. The use of the Nhance and Green Urea instead of granular urea did not result in increased agronomic efficiency or recovery efficiency of the applied N, and this is most likely due to the presence of sufficient available N from both fertiliser application and the soil. A ¹⁵N study recovered 72.8 % of the applied N in the plants and soil, and showed that 30 % of the total N taken up by the plant was derived from the fertiliser, and 70 % from the soil.     

Colorimetric determination of urease activity in soybean meals

Summary A quantitative method for the determination of urease activity in soybean meals is described. The procedure is based on the incubation of the meal sample with a known amount of urea, followed by colorimetric determination of the urea with p-dimethylaminobenz-aldehyde after incubation. Urease activity is expressed as the amount of urea decomposed under the specified conditions. The test is both accurate and specific for urease. The method is very reproducible and sensitive, especially at low urease activities. Presented at the spring meeting, American Oil Chemists' Society, Memphis, Tenn., April 21 to 23, 1958.     

Rice bran oil. IV. Storage of the bran as it affects hydrolysis of the oil

Summary and Conclusions The oil, contained in bran from regularly milled rice, when stored at prevailing atmospheric temperature, humidity, and natural moisture content is subject to rapid hydrolysis which increases the free fatty acid content of the oil to a point where it cannot be economically refined. Data have been presented showing the effects of a) temperature, b) drying at temperatures of 70°, 85°, 100°, and 110°C. for various periods of time up to 5 hours, c) different relative humidities before and after drying, and d) added moisture on the rate of formation of free fatty acids during storage in bran from both regular and “Converted” rice. Decreasing the storage temperature tends to retard the formation of free fatty acids. In the case of regular rice bran deterioration during storage occurred at a fairly rapid rate even at 3°C. whereas bran from “Converted” rice was fairly stable when stored at this temperature. The investigation of the effect of heating or drying and the effect of different relative humidities on the storage of rice bran have shown that bran from both regular and “Converted” rice can be stored for periods of at least four months without excessive increase in the content of free fatty acids, provided the bran is dried sufficiently and is maintained at a low moisture content. An increase in the moisture content of predried bran causes a rapid increase in the free fatty acid content of the oil in the bran. Investigations of the effect of chemical inhibitors and of inert atmosphere on the rate of free fatty acid formation of regular rice bran indicated that these were ineffective in preventing deterioration. Presented at the 40th Annual Meeting of the American Oil Chemists’ Society, New Orleans, La., May 10–12, 1949; Report of a Study Made Under the Research and Marketing Act of 1946. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Retention of activity of urease immobilized on grafted polymer films

Expanded poly(tetrafluoroethylene) (ePTFE) films grafted with 2‐hydroxyethyl methacrylate (HEMA) and 2‐hydroxyethyl acrylate (HEA) were applied to a polymer support for urease immobilization. The HEMA‐ and HEA‐grafted ePTFE (ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA) films prepared by the combined use of the plasma treatment and photografting possessed high water‐absorptivities. Imidazole groups were introduced to grafted PHEMA and PHEA chains with 1,1′‐carbonyldiimidazole (CDI) in acetonitrile. The activity of urease covalently immobilized to the ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA films in a pH 7.0 buffer at 4°C had the maximum value at the optimum pH value of 7.5 for native urease. Urease immobilized on the ePTFE‐g‐PHEMA films with the extent of CDI bonding of about 20% had the maximum activity, and the repeatedly measured activity was kept almost constant. The relative activity of immobilized urease stayed almost constant in the range of the immobilized amounts between 10 and 30 mg/g for both grafted ePTFE films, and decreased at higher immobilized amounts because of the crowding of immobilized urease molecules in the grafted layers. The relative activity of immobilized urease had the maximum values at the grafted amounts of 1.2 and 1.7 mmol/g for the ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA films, respectively, and the further increase in the grafted amount resulted in the decrease in the relative activity. The optimum temperature of the activity for immobilized urease was shifted from 30 to 50°C for native urease by the covalent immobilization on both grafted ePTFE films and immobilized urease was repeatedly usable without a considerable decrease in the activity in the regions of the pH 6.0–9.0 and 10–60°C. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4886–4896, 2006     

Comparison of ISO 14902:2001 with AOCS Ba 12a-2020 for determining trypsin inhibitor activity in protein products

For measuring trypsin inhibitor activity (TIA), there are two major official methods: American Oil Chemists Society (AOCS) method Ba 12a-2020 and International Organization for Standardization (ISO) 14902:2001. The former was recently approved. The two methods differ in sample preparation, extraction, colorimetric assay systems and TIA calculations. In this study, the two methods were symmetrically compared using three unique sets of samples: assorted protein products of soybeans, pulses, and grains; soybeans boiled for varied durations; and soy white flakes toasted for varied durations. For given samples, significant differences existed in TIA measured by the two methods, resulting from effects related to the assay systems and TIA calculations, not from the difference in sample preparation and extraction. When the same trypsin was used, TIA (in mg trypsin inhibited/g sample) measured by the two methods were highly correlated (r = 0.9973, n = 27), giving an equation of y = 0.5464x − 0.4887, where y represents ISO values and x for AOCS values. The line connecting ratios of ISO/AOCS in TIA and AOCS values remained relatively flat around 0.53 but started to curve down when TIA approached the lowest. Furthermore, for the same samples, TIA values measured by the ISO method decreased with increasing specific activity of trypsin used, while AOCS values remained consistent, leading to decreasing ratios of ISO/AOCS. Therefore, accurate and direct comparison of the two methods was impossible. It could not be resolved by simply changing ISO method's calculations as hypothesized earlier. Regardless, for most samples, ISO values were roughly about 55% of AOCS values.     

Thermal bowing and how it affects the design of fire separating construction

This paper provides new data, both experimental and theoretical, on the thermal bowing of building elements such as walls and floors, when exposed to fire on one side, and suggests how the data can be used in design to tolerate unwanted thermal bowing in fire walls.     

Synthesis,crystal structures and urease inhibitory activity of copper(II) complexes with Schiff bases

Two thiocyanate-coordinated Schiff base copper(II) complexes, [CuL¹(NCS)] (1) and [Cu₂(L¹)₂(μ_1,3-NCS)₂]_n (2), where L¹ and L² are the deprotonated forms of 4-bromo-2-[(2-propylaminoethylimino)methyl]phenol (HL¹) and 2-ethoxy-6-[(2-propylaminoethylimino)methyl]phenol (HL²), respectively, have been prepared and structurally characterized. Single crystal X-ray diffraction indicates that 1 is a mononuclear copper(II) complex, while in 2, the adjacent two complex molecules are linked through two phenolate O atoms, forming a dimer. The dimers in 2 are further weakly linked through end-to-end thiocyanate bridges to form a two-dimensional sheet. The Cu atom in 1 is four-coordinate in a square planar geometry, and that in 2 is six-coordinate in an octahedral geometry. Complex 1 shows strong urease inhibitory activity, while complex 2 shows no activity.     

Genetics of the kunitz trypsin inhibitor: An antinutritional factor in soybeans

The major trypsin inhibitor present in soybean seed [Glycine max (L.) Merrill] is the Kunitz trypsin inhibitor or soybean trypsin inhibitor A₂ (SBTI-A₂). Four types of SBTI-A₂9 have been identified in the U.S. soybean germplasm collection. Three of the types designated Ti^a, Ti^b, and Ti^c are electrophoretically distinguishable from one another by their different Rf values of 0.79, 0.75, and 0.83, respectively. The three types are inherited as codominant alleles in a multiple allelic system at a single locus. The fourth type which is the absence of SBTI-A₂ is found in P.I. 157440 and P.I. 196168. The gene for the lack of SBTI-A₂ is designated ti and is inherited as a recessive allele to the other three SBTI-A₂ 9 types. Ti^a is the most common SBTI-A₂ type in the germplasm collection. Linkage studies revealed that the SBTI-A₂ protein is inherited independently of certain morphological characters and chemical components of seed. Potential applications of the SBTI-A₂ types are discussed.     

Phospholipase activity of rat tissues and its modification by trypsin1,2

Treatment with proteolytic enzymes before the addition of the phospholipid substrate increases the activity of the phospholipases of the spleen, thymys, bone marrow, lung, and liver of the rat. In contrast, the phospholipase activity of the intestine, which is higher than that of all other normal tissues, is not increased when incubated with proteases. The results of fractionation studies by high-speed centrifugation and gel filtration and differences in enzyme kinetics support the conclusion that the intestinal phospholipases differ substantially from phospholipases found in the other tissues. Supported in part by Grant No. CA04605-06, US Public Health Service; Contract No. AT-(40-1)-3329, US Atomic Energy Commission. Presented in part at the AOCS Meeting, Houston, April 1965.     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

Effect of PbII on the secondary structure and biological activity of trypsin

The effects of Pb(II) on the secondary structure and biological activity of trypsin have been examined by monitoring changes in its conductivity and IR and circular dichroism (CD) spectra. The results show that Pb(II) reacts with trypsin, and that the binding sites might be -OH and -NH groups in pepsin. The CD spectra indicate that interaction with Pb(II) significantly affects the secondary structure of trypsin, the beta-sheet-structure content being increased by about 42%, whilst those of alpha-helix and beta-turn structures are decreased by 13% and 21%, respectively. The results clearly demonstrate that Pb(II) affects the biological activity of trypsin by modifying its secondary structure. Most interesting is that Pb(II) up-regulates the activity of trypsin at low concentrations while down-regulating it at high concentrations.