Bowman-Birk and Kunitz Protease Inhibitors among Antinutrients and Bioactives Modified by Germination and Hydrolysis in Brazilian Soybean Cultivar BRS 133

Soybean contains constituents that have antinutritional and bioactive properties. Enzymatic hydrolysis and germination can enhance the biological activity of these compounds in soybean. The objective of this study was to investigate the effect of germination, Alcalase (protease) hydrolysis, and their combination on the concentrations of antinutritional and bioactive compounds in Brazilian soybean cultivar BRS 133. A combination of germination and Alcalase hydrolysis resulted in the degradation of Bowman-Birk inhibitor (BBI), Kunitz trypsin inhibitor (KTI), and lunasin by 96.9, 97.8, and 38.4%. Lectin was not affected by any of the processing treatments when compared to nongerminated and nonhydrolyzed soy protein extract. Total isoflavones (ISF) and total saponins (SAP) increased by 16.2 and 28.7%, respectively, after 18 h of germination, while Alcalase hydrolysis led to the reduction of these compounds. A significant correlation was found between concentrations of BBI and KTI, BBI and lunasin, BBI and ISF, KTI and lunasin, KTI and ISF, KTI and SAP, lunasin and ISF, and ISF and SAP. Germination and Alcalase hydrolysis interacted in reducing BBI, ISF, and SAP. This study presents a process of preparing soy flour ingredients with lower concentrations of antinutritional factors and with biologically active constituents, important for the promotion of health associated with soybean consumption. In conclusion, 18 h of germination and 3 h of Alcalase hydrolysis is recommended for elimination of protease inhibitors, while bioactives are maintained by at least 50% of their original concentrations.     

beta-Conglycinins among sources of bioactives in hydrolysates of different soybean varieties that inhibit leukemia cells in vitro

Soybean is a complex matrix containing several potentially bioactive components. The objective was to develop a statistical model to predict the in vitro anticancer potential of soybean varieties based on the correlation between protein composition and bioactive components after simulated gastrointestinal enzyme digestion with their effect on leukemia mouse cells. The IC 50 values of the hydrolysates of soy genotypes (NB1-NB7) on L1210 leukemia cells ranged from 3.5 to 6.2 mg/mL. Depending on genotype, each gram of soy hydrolysates contained 2.7-6.6 micromol of total daidzein, 3.0-4.7 micromol of total genistein, 0.5-1.3 micromol of glycitein, 2.1-2.8 micromol of total saponins, 0.1-0.2 micromol of lunasin, and 0.1-0.6 micromol of Bowman-Birk inhibitor (BBI). The IC 50 values calculated from a partial least-squares (PLS) analysis model correlated well with experimental data ( R (2) = 0.99). Isoflavones and beta-conglycinin positively contributed to the cytotoxicity of soy on L1210 leukemia cells. Lunasin and BBI were potent L1210 cell inhibitors (IC 50 = 13.9 and 22.5 microM, respectively), but made modest contributions to the activity of defatted soy flour hydrolysates due to their relatively low concentrations. In conclusion, the data demonstrated that beta-conglycinins are among the major protein components that inhibit leukemia cell growth in vitro. Furthermore, it was feasible to differentiate soybean varieties on the basis of the biological effect of their components using a statistical model and a cell-based assay.     

Contents and bioactivities of lunasin, bowman-birk inhibitor, and isoflavones in soybean seed

It has been previously demonstrated that lunasin is a novel and promising cancer preventive peptide from soybean. The Bowman-Birk protease inhibitor (BBI) and isoflavones are well-studied substances from soy. This study evaluated the levels and bioactivities of these three compounds as affected by stages of seed development and sprouting under light and dark conditions. BBI and lunasin appear at 7 and 6 weeks, respectively, after flowering and increase as the seed matures. Daidzein and genistein both decrease during seed maturation. During sprouting under light, BBI increases up to the 6th day and decreases thereafter, disappearing at the 9th day after soaking. Under dark conditions, BBI increases up to the 7th day after soaking and decreases thereafter, disappearing at the 10th day. Lunasin starts to decrease at 2 days after soaking and disappears completely at 7 days under light and dark conditions. Daidzein and genistein increase continuously during the 10 days of soaking, and both increase more in the dark than under light conditions. Protein extracts from early seed development (2-5 weeks after flowering) suppress cell viability to a greater degree than those from later stages (6-9 weeks). Inhibition of foci formation by protein extracts from later stages is greater than those from earlier stages. Lunasin and BBI suppress foci formation more than the isoflavones. Sprouting decreases lunasin and BBI contents but increases isoflavones. Protein extracts from early soaking times inhibit foci formation more and suppress cell viability less than those from later soaking times. Light and dark conditions have no influence on the bioactivities of protein extracts. These data are useful in the preparation of soy fractions enriched in lunasin, BBI, and isoflavones and in making dietary recommendations.     

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.     

Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb)

Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention.     

Inhibition of core histone acetylation by the cancer preventive peptide lunasin

Lunasin is a unique 43 amino acid soy peptide that has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model in this work against oncogenes and chemical carcinogens. The observation that lunasin inhibits core histone acetylation led to the proposal of an epigenetic mechanism by which lunasin selectively kills cells that are being transformed by disrupting the dynamics of cellular histone acetylation-deacetylation when the transformation event is triggered by the inactivation of tumor suppressors that function via histone deacetylation. Here is reported for the first time the core histone H3- and H4-acetylation inhibitory properties of lunasin from different Korean soybean varieties used for various food purposes and from tissues of rats fed with lunasin-enriched soy (LES) to measure bioavailability. Lunasin was analyzed by immunostaining and inhibition of core histone acetylation by a non-radioactive histone acetyl transferase assay. Various amounts of lunasin are found in the soybean varieties, which correlated with the extent of inhibition of core histone acetylation. Both soy lunasin and synthetic lunasin inhibit core histone acetylation in a dose-dependent manner. Lunasin in LES is protected from in vitro digestion by pepsin. Lunasin extracted from blood and liver of rats fed with LES is intact and inhibits core histone acetylation.     

Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin

Food-derived bioactive peptides with ACE-inhibitory properties are receiving special attention due to their beneficial effects in the treatment of hypertension. In this work we evaluate the impact of a simulated gastrointestinal digestion on the stability and activity of two bioactive peptides that derive from ovalbumin by enzymatic hydrolysis, YAEERYPIL and RADHPFL. These peptides possess in vitro ACE-inhibitory activity and antihypertensive activity in spontaneously hypertensive rats (SHR). The results showed that YAEERYPIL and RADHPFL were susceptible to proteolytic degradation after incubation with pepsin and a pancreatic extract. In addition, their ACE-inhibitory activity in vitro decreased after the simulated digestion. The antihypertensive activity on SHR of the end products of the gastrointestinal hydrolysis, YAEER, YPI, and RADHP, was evaluated. The fragments YPI and RADHP significantly decreased blood pressure, 2 h after administration, at doses of 2 mg/kg, but they probably did not exert their antihypertensive effect through an ACE-inhibitory mechanism. It is likely that RADHP is also the active end product of the gastrointestinal digestion of the antihypertensive peptides FRADHPFL (ovokinin) and RADHPF (ovokinin 2-7).     

Lunasin concentration in different soybean genotypes, commercial soy protein, and isoflavone products

Lunasin is a unique and novel cancer preventive peptide originally isolated from soy. Information on lunasin concentration of soybean cultivars and commercial soy proteins would be useful in developing lunasin-enriched cultivars and soy products. We report the development of an enzyme-linked immunosorbent assay (ELISA) method to identify lunasin and quantify the variations in concentration in 144 selected, diverse soybean accessions from the U.S. Department of Agriculture Soybean Germplasm Collection, several commercially available soy protein fractions and isoflavone-enriched products. With synthetic lunasin and monoclonal antibody, ELISA shows a linear concentration range of 24-72 ng/mL, good reproducibility, a detection limit of 8 ng/mL, and a recovery of 90% on spiked soy samples. Lunasin concentrations in the tested materials range from 0.10 to 1.33 g/100 g flour. Differences that exceeded 100% have been observed among accessions of similar maturity that were grown in the same environment, indicating that genetic differences in soybeans exist for lunasin. The mean of 23 major ancestral lines of U.S. cultivars is similar to the mean of 16 modern cultivars selected to represent the current diversity of the crop, but the highest values were found within the ancestral and exotic accessions. Soy protein concentrate, isolate, and hydrolyzate contain 2.81 +/- 0.30, 3.75 +/- 0.43, and 4.43 +/- 0.59 g lunasin/100 g flour, respectively, while soy flour and soy flakes contain 1.24 +/- 0.22 g lunasin/100 g flour. Isoflavone-enriched products contain very little or no lunasin. The relative mass (M(r)) of lunasin in the samples is 5.45 +/- 0.25 kDa. The wide range of lunasin concentrations within the Glycine max species indicates that the levels of this important bioactive peptide can be genetically manipulated. Furthermore, soy isolates and hydrolyzed soy proteins contain the highest concentrations of lunasin.     

Bioactive peptides in amaranth (Amaranthus hypochondriacus) seed

Amaranth seeds are rich in protein with a high nutritional value, but little is known about their bioactive compounds that could benefit health. The objectives of this research were to investigate the presence, characterization, and the anticarcinogenic properties of the peptide lunasin in amaranth seeds. Furthermore, to predict and identify other peptides in amaranth seed with potential biological activities. ELISA showed an average concentration of 11.1 microg lunasin equivalent/g total extracted protein in four genotypes of mature amaranth seeds. Glutelin fraction had the highest lunasin concentration (3.0 microg/g). Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDI-TOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence. Glutelin extracts digested with trypsin, showed the induction of apoptosis against HeLa cells. Prediction of other bioactive peptides in amaranth globulins and glutelins were mainly antihypertensive. This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions.     

Identification and characterization of topoisomerase II inhibitory peptides from soy protein hydrolysates

Topoisomerases are targets of several anticancer agents because their inhibition impedes the processes of cell proliferation and differentiation in carcinogenesis. With very limited information available on the inhibitory activities of peptides derived from dietary proteins, the objectives of this study were to employ co-immunoprecipitation to identify inhibitory peptides in soy protein hydrolysates in a single step and to investigate their molecular interactions with topoisomerase II. For this, soy protein isolates were subjected to simulated gastrointestinal digestion with pepsin and pancreatin, and the human topoisomerase II inhibitory peptides were co-immunoprecipitated and identified on a CapLC- Micromass Q-TOF Ultima API system. The inhibitory activity of these peptides from soy isolates toward topoisomerase II was confirmed using three synthetic peptides, FEITPEKNPQ, IETWNPNNKP,and VFDGEL, which have IC 50 values of 2.4, 4.0, and 7.9 mM, respectively. The molecular interactions of these peptides evaluated by molecular docking revealed interaction energies with the topoisomerase II C-terminal domain (CTD) (-186 to -398 kcal/mol) that were smaller than for the ATPase domain (-169 to -357 kcal/mol) and that correlated well with our experimental IC 50 values ( R (2) = 0.99). In conclusion, three peptides released from in vitro gastrointestinal enzyme digestion of soy proteins inhibited human topoisomerase II activity through binding to the active site of the CTD domain.     

Angiotensin I converting enzyme inhibitory peptides from in vitro pepsin-pancreatin digestion of soy protein

Angiotensin I converting enzyme (ACE) inhibitory activity was determined in the soy protein isolate (SPI) digest produced by in vitro pepsin-pancreatin sequential digestion. The inhibitory activity was highest within the first 20 min of pepsin digestion and decreased upon subsequent digestion with pancreatin. An IC(50) value of 0.28 +/- 0.04 mg/mL was determined after 180 min of digestion, while no ACE inhibitory activity was measured for the undigested SPI at 0.73 mg/mL. Chromatographic fractionation of the SPI digest resulted in IC(50) values of active fractions ranging from 0.13 +/- 0.03 to 0.93 +/- 0.08 mg/mL. Although many of the fractions showed ACE inhibition, peptides with lower molecular masses and higher hydrophobicities were most active. The findings show that many different peptides with ACE inhibitory activities were produced after in vitro pepsin-pancreatin digestion of SPI and lead to the speculation that physiological gastrointestinal digestion could also yield ACE inhibitory peptides from SPI.     

Chitosan-alginate microcapsules for oral delivery of egg yolk immunoglobulin (IgY)

Chitosan-alginate microcapsules were evaluated as a method of oral delivery of IgY antibodies. Physical characteristics, encapsulation efficiency (EE%), the loading capacity for IgY (IgY loading percentage, %, w/w of microcapsules), gastro-resistance, and release characteristics of these microcapsules in vitro under varying pH were investigated. Optimum physical factors were established for preparation of homogeneous, spherical, and smooth microcapsules. IgY loading% was not significantly altered by pH of the encapsulation medium. Encapsulation efficiency was highest (73.93%) at a pH of 3.5, above which EE% decreased significantly (p < 0.05). IgY was released from microcapsules upon exposure to simulated intestinal fluid (SIF, pH 6.8), and decreasing pH increased significantly IgY release (p < 0.05). The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and the residual activity was not affected by pH of the encapsulation medium. Moreover, microencapsulated IgY was significantly resistant to pepsin hydrolysis. This approach may enable intact IgY to reach target microorganisms within the lower digestive tract.     

Beta-conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro

Obesity is a worldwide health concern because it is a well-recognized predictor of premature mortality. The objective was to identify soybean varieties that have improved potential to inhibit fat accumulation in adipocytes by testing the effects of soy hydrolysates having a range of protein subunit compositions on lipid accumulation and adiponectin expression in 3T3-L1 adipocytes. The results showed that differences in the protein distribution of 15 soy genotypes led to different potentials for the reduction of fat accumulation. The inhibition of lipid accumulation of soy alcalase hydrolysates in 3T3-L1 adipocytes ranged from 29 to 46%. Soy hydrolysates made from genotypes with 45.3 +/- 3.3% of total protein as beta-conglycinin, on average, showed significantly higher inhibition of lipid accumulation compared to those with 24.7 +/- 1.5% of extracted total protein as beta-conglycinin. Moreover, after in vitro simulated digestion with pepsin-pancreatin of the soy alcalase hydrolysates, 86% of the original activity remained. Adiponectin expression was induced in 3T3-L1 adipocytes treated with 15 soy hydrolysates up to 2.49- and 2.63-fold for high and low molecular weight adiponectin, respectively. The inhibition of lipid accumulation calculated from a partial least squares (PLS) analysis model correlated well with experimental data (R(2) = 0.91). In conclusion, it was feasible to differentiate soy varieties on the basis of the potential of their proteins to reduce fat accumulation using a statistical model and a cell-based assay in vitro. Furthermore, beta-conglycinin embeds more peptides than glycinin subunits that inhibit lipid accumulation and induce adiponectin in 3T3-L1 adipocytes. Therefore, soy ingredients containing beta-conglycinin may be important food components for the control of lipid accumulation in adipose tissue.     

Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.     

Stability and bioaccessibility of isoflavones from soy bread during in vitro digestion

The impact of simulated digestion on the stability and bioaccessibility of isoflavonoids from soy bread was examined using simulated oral, gastric, and small intestinal digestion. The aqueous (bioaccessible) fraction was isolated from digesta by centrifugation, and samples were analyzed by high-performance liquid chromatography (HPLC). Isoflavonoids were stable during simulated digestion. Partitioning of aglycones, acetylgenistin, and malonylgenistin into the aqueous fraction was significantly (P < 0.01) affected by the concentration of bile present during small intestinal digestion. Omission of bile resulted in nondetectable genistein and <40% of total daidzein, glycitein, and acetylgenistin in the aqueous fraction of digesta. Partitioning of these compounds into the aqueous fraction was increased by physiological concentrations of bile extract. These results suggest that micellarization is required for optimal bioaccessibility of isoflavonoid aglycones. We propose that the bioavailability of isoflavones from foods containing fat and protein may exceed that of supplements due to enhanced bile secretion.     

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.     

Preparation and characterization of nanoparticles based on hydrophobic alginate derivative as carriers for sustained release of vitamin D3

Hydrophobic alginate derivative was prepared by modification of alginate by acid chloride reaction using oleoyl chloride without organic solvents. The conjugate of oleoyl alginate ester (OAE) was confirmed by FT-IR and (1)H NMR. The degree of substitution (DS) of OAE was determined by (1)H NMR, and it ranged from 0.84 to 3.85. In distilled water, OAE formed self-assembled nanoparticles at low concentrations in aqueous medium, and nanoparticles retained their structural integrity both in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The loading and release characteristics of nanoparticles based on OAE were investigated using vitamin D(3) as a model nutraceutical. As the concentration of vitamin D(3) increased, the loading capacity (LC) increased, whereas the loading efficiency (LE) decreased. Nanoparticles could release vitamin D(3) at a sustained rate in gastrointestinal fluid. These results revealed the potential of OAE nanoparticles as oral carriers for sustained release of vitamin D(3).     

Enhanced survival of probiotic Lactobacillus acidophilus by encapsulation with nanostructured polyelectrolyte layers through layer-by-layer approach

The encapsulation of probiotic Lactobacillus acidophilus through layer-by-layer self-assembly of polyelectrolytes (PE) chitosan (CHI) and carboxymethyl cellulose (CMC) has been investigated to enhance its survival in adverse conditions encountered in the GI tract. The survival of encapsulated cells in simulated gastric (SGF) and intestinal fluids (SIF) is significant when compared to nonencapsulated cells. On sequential exposure to SGF and SIF for 120 min, almost complete death of free cells is observed. However, for cells coated with three nanolayers of PEs (CHI/CMC/CHI), about 33 log?% of the cells (6?log?cfu/500?mg) survived under the same conditions. The enhanced survival rate of encapsulated L. acidophilus can be attributed to the impermeability of polyelectrolyte nanolayers to large enzyme molecules like pepsin and pancreatin that cause proteolysis and to the stability of the polyelectrolyte nanolayers in gastric and intestinal pH. The PE coating also serves to reduce viability losses during freezing and freeze-drying. About 73 and 92 log?% of uncoated and coated cells survived after freeze-drying, and the losses occurring between freezing and freeze-drying were found to be lower for the coated cells.     

Pea (Pisum sativum L.) protease inhibitors from the Bowman-Birk class influence the growth of human colorectal adenocarcinoma HT29 cells in vitro

The Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybean has been described as a potential cancer chemopreventive agent. We have compared the effects of BBI with those of two variant recombinant pea (Pisum sativum L.) seed protease inhibitors, rTI1B and rTI2B, homologous to BBI but differing in inhibitory activity, on the growth of human colorectal adenocarcinoma HT29 cells in vitro. A significant and dose-dependent decrease in the growth of HT29 cells was observed using all protease inhibitors, with rTI1B showing the largest decrease (IC50 = 46 microM). Inclusion of the pan-caspase inhibitor, Boc-D-FMK, did not negate the effects of rTI1B or rTI2B in the cell assays. The relative effectiveness of rTI1B and rTI2B may correlate with a variant amino acid sequence within their respective chymotrypsin inhibitory domain, in agreement with a chymotrypsin-like protease as a potential target.