Development of Breeder-Friendly KASP Markers for Low Concentration of Kunitz Trypsin Inhibitor in Soybean Seeds.

Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals' protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F3:5 lines derived from PI 547656 (low KTI) X Glenn (normal KTI) were genotyped using the BARCSoySNP6K_v2 Beadchip. F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.

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.

Proteomic analysis of anti-nutritional factors (ANF's) in soybean seeds as affected by environmental and genetic factors.

The genotype (G), environment (E), and the relationship between G and E on soybean seed anti-nutritional factors (ANF's) were examined under three different agro-climatic conditions. The field trials were conducted at Maryland, South Carolina and South Dakota using nine region specific genotypes. At each location, the nine genotypes were grown with two planting/sowing dates. Differentially expressed protein spots from the two-dimensional gel electrophoresis were analyzed using mass spectrometry. Seven ANF's corresponding to soybean agglutinin and Kunitz trypsin inhibitor were identified based on the statistical significance levels at p<0.005. The G and E conditions (planting/sowing season) influences the ANF's content. This initial study suggests that early sowing reduces the total ANF's content irrespective of genotypes and their growing locations.

The primary structure of Vipera ammodytes venom trypsin inhibitor I.

The primary structure of Vipera ammodytes venom trypsin inhibitor I consists of 61 amino acid residues [sequence in text]. The N-terminal group of the inhibitor is pyrrolidonecarboxylic acid. The sequential data were obtained by analysis of peptides isolated from tryptic and chymotryptic digests and by analysis of peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The primary structure of trypsin inhibitor I presented shows approximately 80% sequence homology with chymotrypsin inhibitor isolated from the venom of the same snake, and nearly 50% homology with bovine basic pancreatic trypsin inhibitor. It belongs to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Kunitz protease inhibitor-containing amyloid beta protein precursor immunoreactivity in Alzheimer's disease.

The amyloid beta protein (beta/A4) that is deposited in senile plaques and in cerebral vessels in Alzheimer's disease (AD) is derived from a larger membrane-associated glycoprotein, the amyloid beta protein precursor (APP). The gene encoding APP produces at least four major transcripts. Three of the four transcripts contain an alternatively-spliced exon encoding a Kunitz protease inhibitor domain (KPI). We now report the results of a series of experiments using novel immunohistochemical reagents to anatomically localize beta/A4, APP, and KPI-containing forms of APP (APP-KPI) in the hippocampal formation and temporal neocortex. A new monoclonal antibody against beta/A4 recognized senile plaques and vascular amyloid, but no cellular elements. Anti-APP and anti-KPI monoclonal antibodies stained neurons, including proximal axons and dendrites. The neuritic component of some plaques in patients with AD and in elderly control individuals were also immunoreactive for both APP and APP-KPI. Quantitative assessment of senile plaques in temporal neocortex showed that, on average, about one-third of beta/A4 immunoreactive plaques stained with either anti-APP or anti-KPI. Amyloid beta protein precursor and APP-KPI immunoreactivity were also found in the white and grey matter vessels of both AD patients and control individuals. These results suggest that KPI-containing forms of APP are present in dystrophic neurites of senile plaques, and normally in neurons, neuronal processes, and in the vascular compartment in the brain. Thus, APP-KPI is in a position to be intimately associated with beta/A4 deposition in the neuropil, in plaques and in amyloid angiopathy.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsin and monoclonal antibodies.

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea (Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor-enzyme and inhibitor-mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245,000 plates/m.

Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran. I: Synthesis and characterization.

The Kunitz-type soybean trypsin inhibitor (STI), a model protein, was conjugated with dextran (Mw, approximately 9900; STI-D), and its physicochemical and biochemical properties were studied to develop a novel delivery system for a protein drug. Conjugation was carried out using periodate oxidation, and cyanogen bromide, carbodiimide, cyanuric chloride, epichlorhydrin, and N-succiniimidyl-3-(2-pyridyldithio)propionate (SPDP) reagent methods. Dextran was conjugated to STI at a molar ratio of 1.5 to 4.6, but the degree of modification, as well as yield and contamination extent of unreacted STI and dextran, varied with the method of synthesis. Gel filtration and electrophoresis confirmed the covalent attachment of dextran to STI but also demonstrated the broad molecular weight distribution of the conjugates. The STI-D conjugate retained satisfactory activity, although the attachment partially reduced its inhibitory activity against trypsin. The periodate oxidation method seemed to be the best for the preparation of STI-D since it gave the conjugate with a high modification ratio (4.6 molecules per STI), high yield (95%), and satisfactory activity recovery (63%). Chemical modification of STI was also carried out with activated polyethylene glycol (PEG) for comparison. The STI-PEG conjugate was obtained in a satisfactory yield (96%) and modification degree (5.8 molecules per STI), but the remaining activity was considerably lower (34%). Thus, conjugation of protein with dextran by the periodate oxidation method is suggested to be preferable for preparing a protein-carrier system without significant diminution of its biological activity.

Assay for protease inhibitors: qualitative measurement and detection in polyacrylamide gel electrophoretograms.

A simple and fast method for qualitative measurement of protease inhibitors has been developed. The high sensitivity of the assay is based on a casein-precipitating reaction at pH 5.5. Cellulose paper strips, moistened with protease inhibitor-containing solutions, are applied to the surface of an agar-skim milk gel previously coated with a protease solution. After incubation for 2 h at 37 degrees C, inhibitory activity is indicated by a translucent zone (absence of casein degradation) on a white background of precipitated small casein fragments. In addition to being useful for rapid screening of a large number of complex samples, the method allows the localization of protease inhibitor activity in sodium dodecyl sulfate-polyacrylamide gel electrophoretograms.

Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel.

Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 microl of buffer by a modification of the procedure of Gombocz and Cortez. The electroeluate was freed of SDS, ultrafiltered and subjected to MALDI-TOF mass spectrometry. The masses of the five native proteins were found to be maintained after electrophoresis and electroelution in the presence of the potential contaminants SDS, barbituric acid and Cascade Blue. The procedure of protein transfer from SDS-PAGE into mass spectrometry, without excision of bands, gel maceration and protein recovery by diffusion, therefore is shown to be suitable for the identification by mass of intact proteins derived from gel electrophoretic bands.

Immunoassays of soy proteins.

Proteins of soybeans (Glycine max) are widely used in animal and human nutrition. In addition to the bulk of the seed storage proteins, which are classified as albumins and globulins, approximately 6% of soybean proteins are classified as inhibitors of trypsin and chymotrypsin and approximately 0.5% are sugar-binding lectins. The two major classes of inhibitors are the Kunitz trypsin inhibitor, which inhibits trypsin, and the Bowman-Birk inhibitor (BBI), which inhibits both trypsin and chymotrypsin. Unless removed or inactivated, these inhibitors and lectins can impair the nutritional quality and safety of soy-based diets. On the other hand, several studies suggest that BBI can also function as an anticarcinogen, possibly through interaction with a cellular serine protease. Good-quality soybean proteins contribute to the nutritional value of many specialty foods including infant soy formulas and milk replacers for calves, and provide texture to many processed foods. However, they may also induce occasional allergic responses in humans. This paper outlines immunoassays developed to analyze for soy proteins in different soybean lines, in processed foods, and in nonsoy foods fortified with soy proteins. An assessment of the current status of immunoassays, especially of enzyme-linked immunosorbent assays for soybean inhibitors of digestive enzymes, soy globulins, and soy lectins, demonstrates the usefulness of these methods in plant and food sciences and in medicine.

Relevance of multiple soybean trypsin inhibitor forms to nutritional quality.

Trypsin inhibitors contribute to the antinutritional component of raw soybean meal by inhibiting vertebrate pancreatic serine proteinases in the small intestine, resulting in a range of deleterious physiological effects in the animal. The variation in the nutritional quality of soybean cultivars stems partly from wide-ranging levels of trypsin inhibitor, and from varying proportions of trypsin inhibitors of two classes--the Kunitz and the Bowman-Birk inhibitor classes. The latter class is better able to survive heat processing and digestion in the stomach. Some variation in cultivars also arises from the array of isoinhibitors present in the seed. The three Kunitz isoinhibitors, Ti(a), Ti(b) and Ti(c) differ by as much as 1000-fold in their interaction with bovine trypsin. The Bowman-Birk isoinhibitors differ not only in their extent of interaction with trypsin, but in their spectrum of inhibition of the other pancreatic enzymes, chymotrypsin and elastase. In this chapter, we look at twenty-two Bowman-Birk inhibitors from ten soybean cultivars and find at least twelve which are different enough in amino acid composition and/or inhibitor activity to be distinct protein species. Of these, three pairs are related by proteolytic digestion. Quite ironically, the Bowman-Birk inhibitors, and to some extent the Kunitz inhibitors, contribute to the nutritional quality of soybeans by virtue of their high cystine content which supplements the low or negligible amounts of sulfur-containing amino acids in the storage proteins that comprise the bulk of the protein reserve in the seed.

ELISA analysis of soybean trypsin inhibitors in processed foods.

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Effect of heat on the nutritional quality and safety of soybean cultivars.

To evaluate whether soybean strains with reduced levels of trypsin inhibitors have enhanced nutritional and safety characteristics, we measured protease inhibitor content of a standard cultivar (Williams 82) and an isoline (L81-4590) lacking the Kunitz trypsin inhibitor, using enzyme inhibition assays and enzyme-linked immunosorbent assays (ELISA). Less heat was needed to inactivate the remaining trypsin inhibitory activity of the isoline than that of the standard soybean cultivar. In fact, autoclaving (steam heating at 121 degrees C) of the isoline for 20 min resulted in a near zero level of trypsin inhibitor activity, while 20% remained in the Williams 82 sample. Feeding studies with rats showed that the raw soy flour prepared from the isoline was nutritionally superior to the raw flour prepared from the standard variety, as measured by PER and pancreatic weights. Since the content of amino and fatty acids of the flours from both strains was identical and the hemagglutinating activities were within a factor of 2, the increased PER was likely due to the lower level of trypsin inhibitory activity in the isoline. Steam heating the flours for up to 30 min at 121 degrees C progressively increased the PER for both strains. Preliminary screening of several accessions from the USDA Soybean Germplasm Collection showed considerable variation in the content of trypsin inhibitors, sulfur amino acids, and lectins. The BBI content of these cultivars, determined by chymotrypsin inhibition assays, was identical to that found by ELISA. The results indicate that further screening studies could lead to the discovery of soybeans which yield flour that is safe and nutritious, with minimal need for heating.

Effect of soybean variety and processing on growth performance of young chicks and pigs.

The objective of this study was to determine whether soybeans without the Kunitz trypsin inhibitor and lectins could be fed effectively to young chicks and pigs. Specifically, we compared the growth performance of chicks and pigs fed diets containing modified soybeans: Kunitz trypsin inhibitor-free (KF), lectin-free (LF), lectin and Kunitz trypsin inhibitor-free (LFKF), conventional soybeans (CSB), and commercially obtained, dehulled, solvent-extracted soybean meal (SBM). A 7-d chick experiment was conducted to evaluate the nutritional value of CSB, KF, LF, LFKF, and SBM. The experiment was conducted as a completely randomized design, with four replicates, five treatments, and six male chicks per pen (n = 120). The five treatments consisted of 23% CP dextrose-soybean-based diets containing KF, LF, LFKF, CSB, or SBM as the source of dietary protein. A 28-d pig experiment was conducted to evaluate the nutritional value of CSB, LF, LFKF, and SBM. Pens of four pigs were assigned randomly to a control, corn-SBM, or one of six corn-soybean diets containing raw or extruded soybean varieties as a 2 x 3 factorial arrangement of treatments in a randomized complete block design with five blocks per treatment (n = 140). Chicks fed diets containing any of the raw soybean varieties gained less weight (P < 0.05) than chicks fed SBM (22.81 g/d for SBM vs. 14.17 g/d for the raw soybeans combined). Among the raw soybean treatments, there was a greater effect on growth performance (P < 0.05) by removing both lectins and Kunitz trypsin inhibitor (ADG of 16.56 g for LFKF) than by removing each antinutritional factor separately (ADG of 14.38 and 14.11 g for KF and LF, respectively). Pig growth performance was different (P < 0.001) for SBM (ADG of 409 g) and all the varieties when extruded (ADG of 450 g for CSB, 417 g for LF, and 408 g for LFKF) compared with the raw soybean treatments (ADG of 101 g for CSB, 165 g for LF, and 266 g for LFKF). Among the raw soybean treatments, growth performance improved (P = 0.003) as the antinutritional factor, lectin, was removed from the soybean and improved further (P = 0.045) when both lectins and Kunitz trypsin inhibitor were removed. The growth-inhibiting effect of feeding modified soybeans to young animals was more detrimental for pigs than for chicks in our experiments. Soybeans without the Kunitz trypsin inhibitor and lectins cannot be fed successfully to young chicks and pigs without heating.

Effect of heating on nutritional quality of conventional and Kunitz trypsin inhibitor-free soybeans.

Five 10-day chick growth experiments and an amino acid digestibility assay were conducted to assess the effect of steam heating on in vivo protein quality of raw, full-fat Kunitz trypsin inhibitor-free soybeans (KFSB) compared with raw, conventional full-fat soybeans (CSB). Protein solubility in .2% KOH was also evaluated as an in vitro test of in vivo protein quality for underprocessed CSB. The CSB and KFSB were autoclaved for 0, 3, 6, 9, 12, 15, 18, or 21 min at 121 C and 124 kPa. The soybeans were then fed to 8- or 9-day-old chicks as the sole source of protein in dextrose and soybean diets containing 23% protein. Growth performance of chicks fed raw KFSB was superior to that of chicks fed raw CSB. Growth performance of chicks fed autoclaved KFSB or CSB increased and pancreas weight (percentage of body weight) decreased as autoclaving time increased. Slightly less autoclaving time was consistently required to achieve maximum chick performance for KFSB compared with CSB. Less autoclaving time was also required to obtain maximum digestibility of amino acids in KFSB compared with CSB. Urease activity of the soybeans decreased as autoclaving time increased, whereas protein solubility in .2% KOH for CSB did not change consistently in response to heating time. The results of the present study indicate that raw KFSB must be heated to obtain maximum protein quality for chicks and that protein solubility in KOH is not a sensitive indicator of underprocessing of CSB soybeans.

Nutritional evaluation of lectin-free soybeans for poultry.

This study evaluated the nutritional value of raw lectin-free soybeans in comparison with raw Kunitz trypsin inhibitor-free soybeans, raw conventional soybeans, and commercial heat processed soybean meal (SBM). Analyzed lectin values (milligrams per kilogram) were 7.2, 7.1, and < 0.00015 for the Kunitz-free, conventional, and lectin-free soybeans, respectively. Three experiments were conducted using New Hampshire x Columbian male chicks fed 23% CP dextrose-soybean diets from 8 to 17 d of age. Growth performance of chicks fed lectin-free soybeans was greater (P < 0.05) than that of chicks fed raw conventional soybeans in all three experiments. However, performance of chicks fed lectin-free soybeans was lower than that of chicks fed Kunitz-free soybeans or SBM. The SBM yielded weight gains and feed efficiencies that were much higher than those observed from any of the raw soybeans. True amino acid digestibility and TMEn of the lectin-free and conventional soybeans were determined using the precision-fed cecectomized rooster assay. Seven roosters were crop-intubated with 30 g of soybeans and excreta were collected for 48 h. Digestibility coefficients of most amino acids for lectin-free soybeans were 5 to 8 percentage units higher than those for conventional soybeans, but the differences were not significant (P > 0.05). Likewise, the TMEn for lectin-free soybeans was 11% higher than that for raw conventional soybeans (3.577 vs 3.227 kcal/g DM) but the difference was not significant (P > 0.05). The results of this study indicate that the nutritional value of raw lectin-free soybeans is greater than raw conventional soybeans but is less than raw Kunitz-free soybeans and SBM, suggesting that trypsin inhibitor is a greater antinutritional factor than lectins.

[Thermodynamic and kinetic study of thermal denaturation of Kunitz soybean trypsin inhibitor by differential scanning microcalorimetry]. / Termodinamicheskoe i kineticheskoe issledovanie termicheskoi denaturatsii ingibitora tripsina Kunitsa iz bobov soi medodom differentsial'noi skaniruiushchei mikrokalorimetrii.

The thermal denaturation of soybean trypsin inhibitor (Kunitz inhibitor) has been studied in pH-region from 2.0 to 11.0 by differential scanning microcalorimetry. The thermodynamic characteristics have been determined. It has been established that the denaturation transition of protein may be described by a two-state model. It has been shown, that two side hydrogen bonds between carboxylate-ion and tyrosyl and carboxylate-ion and lysyl take part in the stabilization of the inhibitor's native structure. The activation of denaturation is accompanied by cleavage of one side hydrogen bond.

[Interpretation of thermograms of protein denaturation in non-equilibrium conditions]. / K voprosu ob interpretatsii termogramm denaturatsii belkov v neravnovesnykh usloviiakh.

Data are presented concerning the effect of heating rate on the denaturation parameters of small and oligomeric globular proteins: Kunitz trypsin inhibitor from soybeans and 1,5-Ribulose Bisphosphate Carboxylase from tobacco leaves. Substantional dependence of denaturation temperature on the heating rate reflects non-equilibrium pattern of denaturation of these proteins under experimental conditions. To interpret these data a kinetic approach is proposed, which permits determination of equilibrium value of the denaturation temperature and of the constant of de- and renaturation rate. The conformation transitions in the proteins studied are shown to be relatively slow processes. Their rate is comparable to the velocity of temperature change in a calorimeter, which is the cause of non-equilibrium effects in a calorimetric experiment.

[Biological evaluation of soybean line with low trypsin inhibitor activities]. / Avaliação biológica de soja com baixas atividades de inibidores de tripsina e ausência do inibidor Kunitz.

The soybean cultivar BR 36 with conventional levels of trypsin inhibitors activity and the soybean line BRM95-5262, which was genetically selected to contain low activity of trypsin inhibitors were used for biological assays with rats. BR 36 and BRM95-5262 contained 40 and 20, and 30 and 20% of relative residual activity of trypsin inhibitors, respectively. The mean values of PER and NPR showed that treatments with crude soybeans were minor than treatments with soybean thermically processed. However, the treatments of thermically processed soybean did not showed significative differences (p > or = 0.05). When the trypsin inhibitors activity were 8.61 and 8.44 UIT/mg of samples or 20 and 30% of relative residual activity of cultivar BR 36 and line BRM95-5262, respectively, it was observed that mean values of PER and NPR were not significatives. The mean values of CDA and CDV of treatments with crude soybeans were minor than treatment with casein and similar to the treatments with soybean thermically processed. So, it can be concluded that the biological evaluation obtained with soybean protein were dependent of initial trypsin inhibitors activities and of its respective thermical treatment. There was advantage in the use of BRM95-5262 soybean line with low trypsin inhibitors activity.

The heat-induced protein aggregate correlated with trypsin inhibitor inactivation in soymilk processing.

Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) have trypsin inhibitor activities (TIA), which could cause pancreatic disease if at a high level. It is not clear why some KTI and BBI lose TIA and some does not in the soymilk processing. This would be examined in this study. TIA assay showed residual TIA was decreased with elevated temperature and TIA was decreased quickly in the beginning and then slowly in boiling water bath. Interestingly, ultracentrifugation showed low residual TIA soymilk had more precipitate than high residual TIA soymilk and soymilk TIA loss had a high correlation coefficient (R(2) > 0.9) with precipitate amount. In addition, the TIAs of floating, supernatant, and precipitate obtained by ultracentrifugation were assayed and >80% residual TIA was concentrated in the supernatant. Tricine-SDS-PAGE showed KTI in supernatant was mainly a noncovalent bound form which might exist as itself and/or incorporated into a small protein aggregate, while KTI in precipitate was incorporated into a protein aggregate by disulfide and/or noncovalent bonds. Chymotrypsin inhibitor activity (CIA) assay showed about 89% of the original CIA remained after 100 °C for 15 min. Ultracentrifugation showed that >90% residual CIA was concentrated in supernatant. Tricine-SDS-PAGE showed soymilk (100 °C, 15 min) BBI mainly existed in supernatant but not in precipitate. It was considered that BBI tended to exist as itself with its natural conformation. Thus, it was suggested residual TIA was mainly from the free BBI and TIA inactivation was mainly from KTI incorporation into protein aggregate. This study is meaningful for a new strategy for low TIA soymilk manufacture based on the consideration of promoting protein aggregate formation.