An International Collaborative Study on Trypsin Inhibitor Assay for Legumes,Cereals, and Related Products

For determining trypsin inhibitor activity (TIA) in soy products, the American Oil Chemists' Society (AOCS) Method Ba 12-75 has been used. It measures differences in absorbance at 410 nm of bovine trypsin activity toward a synthetic substrate (Nα-benzoyl-DL-arginine-p-nitroanilide) in the absence and presence of an inhibitor. Recently, a significantly improved method was developed (JAOCS, 2019, 96:635–645), featuring 5 mL of total assay volume, enzyme-last sequence, and single inhibitor level in duplicate. It is proposed as the AOCS Method Ba 12a-2020. As a part of the AOCS method approval process, a collaborative study involving 12 international laboratories was conducted to evaluate the performance of the proposed method. The study involved measuring TIA in 10 selected test samples plus a blind duplicate. They included soybeans, pulses, cereals, and their processed products (flours, concentrates, and isolates). After rigorous statistical treatment of the data, only three outliers were removed from the data of two samples. Repeatability relative standard deviations (RSDr) for the 11 samples ranged from 0.99% to 5.52%. Reproducibility RSD (RSD_R) ranged from 7.07% to 22.92%, with seven samples having RSD_R around 10% or less. The remaining four samples had very low TIA, and their RSD_R values ranged from 13.34% to 22.92%. The study has demonstrated reliable performance of the proposed AOCS method. Several collaborators carried out additional experiments addressing some aspects of the method, leading to further refinements. The proposed method is undergoing evaluation by the AOCS Uniform Methods Committee for adoption as an Official Method for measuring TIA in various legume and grain products.     

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.     

Soybean Trypsin Inhibitor Assay: Further Improvement of the Standard Method Approved and Reapproved by American Oil Chemists’ Society and American Association of Cereal Chemists International

For measuring trypsin inhibitor (TI) activities in soybean products, the current standard method, approved and reapproved by American Oil Chemists Society (Method Ba 12-75) and American Association of Cereal Chemists International (Method 22-40.01), features mixing trypsin with a series of inhibitor levels and then adding a substrate to start the colorimetric reaction. Yet, previous studies have shown flaws with the method, particularly with using several inhibitor levels and the sequence of adding the substrate last. The present study showed that with varying levels of dilution and volumes of a dilute sample extract, the pH of the premix (the mixture of a dilute sample extract and trypsin solution) ranged 3.30–3.60 for raw soy flour, and 3.20–6.70 for toasted soy. Within these premix pH ranges, the standard method of adding substrate last would give TI values equal to or less than those measured by the same method except for adding the enzyme last. The standard method was subsequently improved by using a single sample extract level and the enzyme-last sequence. Other modifications included making stock solutions for reagents, adding Ca²⁺ to the trypsin solution, diluting sample extracts to a level that causes 30–70% of inhibition, and running both reference and sample blanks for better controls. Alternatively, the full volume assay (10 mL total, as in the standard method) was further modified by using half the volume of each reagent with the same concentration. Compared to the standard method, the improved methods gave more consistent results when assaying 11 selected soy products. The half volume (5 mL) and full volume methods gave the same results, but the former could increase assay sensitivity and reduce amounts of reagents used.     

Localization of phenolic acids and antioxidant activity in sorghum kernels

White and red of sorghum grains were sequentially pearled into 11 pearling fines and the corresponding 11 pearled kernels to study the localization of phenolic acids within sorghum grains. All fractions were analyzed for phenolic acids content by HPLC and antioxidant capacity by FRAP assays. Four phenolic acids identified in all fractions were caffeic, p-coumaric, ferulic, and sinapic acids. Data showed that the distribution of phenolic acids and antioxidant capacity in two sorghum genotypes was heterogeneous. Around 60% of the phenolic acids and the antioxidant capacity were recovered in the initial three pearling fine fractions that constituted about 20% surface removal. The concentrations of the phenolic acids decreased significantly with sequential pearling. Significant correlation between total phenolic acids and antioxidant capacity was observed with all fractions except for the first pearling fines from the red sorghum.     

Rheological behaviors,structural properties and freeze–thaw stability of normal and waxy genotypes of barley starch: a comparative study with mung bean,potato, and corn starches

Food Science and Biotechnology - The rheological behaviors, structural properties and freeze-thaw stability of starch isolated from Tetonia barley (Normal genotype, Reg. No. CV-334, PI 646199) and...     

Trypsin Inhibitor Assay: Expressing,Calculating, and Standardizing Inhibitor Activity in Absolute Amounts of Trypsin Inhibited or Trypsin Inhibitors

For expressing trypsin inhibitor activity (TIA), trypsin units inhibited (TUI), trypsin inhibited, and trypsin inhibitors have been used. Although the last two units are preferred, their calculations in current practices require refinement. With the proposed AOCS method Ba 12a-2020, four experiments were conducted, using four trypsin preparations having specific activity of 11,625, 12,602, 13,728, and 14,926 Nα-benzoyl-L-arginine ethyl ester (BAEE) units/mg protein, respectively. Experiment 1 determined the relationship between absorbance at 410 nm (A410) and trypsin concentration. Experiment 2 involved assaying raw and heated soybeans, expressing TIA as TUI/mg sample and μg trypsin inhibited/mg sample, and determining conversion factors between the two units. Experiment 3 resembled Experiment 2 except for using purified soybean Kunitz inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Conversion factors determined correlated highly with trypsin-specific activity (R² = 0.9789). After standardizing against a reference trypsin having 15,000 BAEE units/mg protein, a standardized conversion factor of 0.03 A410 (1.5 TUI) = 1 μg trypsin inhibited was determined. It remained consistent regardless of trypsin specific activity, with or without inhibitors, and type of inhibitor samples. By using purified inhibitors (Experiment 3), conversion values between TUI and μg trypsin inhibitor and between μg trypsin inhibited and μg trypsin inhibitor could also be calculated, enabling expression of TIA in amounts of pure KTI, BBI or their equivalents. Furthermore, when the AOCS method was modified with half substrate concentration, half trypsin concentration or half both (Experiment 4), TIA values in TUI could change with modifications but values in mg trypsin inhibited (standardized) or trypsin inhibitor remained consistent.     

Changes in Lipid Composition During Dry Grind Ethanol Processing of Corn

During the dry grind ethanol process, ground corn is fermented and the major co-product is a feed called distillers dried grains with solubles (DDGS). This study investigated the changes that occur in the composition of corn oil that can be extracted from various process fractions during the dry grind ethanol process. In the first part of this study, samples of distillers dried grains, thin stillage, condensed distillers solubles (also known as syrup), and DDGS were obtained from 7 dry grind ethanol plants. The levels of deleterious free fatty acids were high (>7%) and those of valuable total phytosterols were also high in all fractions (>2%). In the second part of this study, changes in the content and composition of the fatty acids, phytosterols, tocopherols and tocotrienols were quantitatively analyzed in crude oil samples extracted from nine dry grind process fractions from three commercial ethanol plants. Fatty acid and phytosterol composition remained nearly constant in all nine fractions, although some significant variations in phytosterol composition existed among the fractions. Examination of the tocopherols and tocotrienols revealed that γ-tocopherol was the most abundant tocol in ground corn but an unknown tocol became the predominant tocol after fermentation and persisted in the remaining processing fractions and in the final DDGS product. Overall, the remaining majority of tocols remained relatively unchanged.     

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.     

Fractionation of Condensed Distillers Solubles and Compositional Characterization of Its Co-Products

Condensed distillers solubles (CDS) was fractionated into a protein-mineral fraction and a glycerol fraction by a chemical method; protein and glycerol-mineral fractions by a physical method; and protein, mineral, and glycerol fractions by a physicochemical method. The co-products from each method, along with CDS, were characterized for concentrations of key constituents (protein, oil, ash, glycerol and other carbohydrates), mineral profile, and amino acid composition. Recovery of mass and main constituents was also investigated. With the chemical method, about two-third of the mass went to the protein-mineral fraction, while by the physical method the equal amounts of mass went to the protein and glycerol-mineral fractions. Protein, minerals, and glycerol were mostly recovered into their respective fractions. CDS and its fractions contained six major minerals (Ca, Mg, P, K, Na, and S) and four trace ones (Cu, Fe, Mn, and Zn). Both chemical and physical treatments caused significant reduction of mineral contents (dry matter basis) in the protein or glycerol fraction. Several amino acids differed significantly in percent relative to total amino acids among fractions but the extent was not substantial for protein-rich fractions. These changes resulting from CDS fractionation are favorable for value added utilization of the new co-products.