Chymotrypsin Inhibitory Activity Research Articles

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.

Chymotrypsin and Trypsin Inhibitory Activity of Some Medicinal Plants Collected from Rize (Türkiye).

In this research, the evaluation of in vitro chymotrypsin and trypsin inhibitory activities of ten plant species collected from Rize were aimed, and fractions that showed strong activity were analyzed through HPLC. Daphne pontica L. and Mentha longifolia (L.) L. were found to have the highest chymotrypsin inhibitory activities (87.75 and 84.24 % inhibition). Similarly, the highest trypsin inhibitory activity was observed in D. pontica (%99.93 inhibition), followed by Sambucus ebulus L. flowers (87.47 % inhibition). Extracts showing strong enzyme inhibition were fractioned and subjected to activity tests. The highest chymotrypsin inhibitory activity was observed in the n-hexane fraction of D. pontica (%80.70 inhibition), while the highest trypsin inhibitory activity was found in the n-butanol fraction of S. ebulus (%86.81 inhibition). HPLC studies determined that the 80 % ethanol extract of D. pontica and its dichloromethane and ethyl acetate fractions contained umbelliferone. It was found that chlorogenic acid was present in the 80 % ethanol extracts of S. ebulus flowers. M. longifolia was found to contain chlorogenic acid, caffeic acid, luteolin-7-glucoside, and rosmarinic acid. M. longifolia has been identified as the plant exhibiting the highest antioxidant activity in ABTS and CUPRAC tests, consistent with its high phenolic and flavonoid content.

Elemental sulfur concentration can be used as a rapid, reliable, and cost-effective predictor of sulfur amino acid content of soybean seeds

In this study, we have examined the feasibility of using elemental sulfur content of soybean seeds as a proxy for the overall sulfur amino acid content of soybean seeds. Earlier, we have identified by high throughput ionomic phenotyping several high and low sulfur containing soybean lines from the USDA Soybean Germplasm Collection. Here, we measured the cysteine and methionine content of select soybean lines by high-performance liquid chromatography. Our results demonstrate that those soybean lines which had high elemental sulfur content also had a higher cysteine and methionine content when compared to soybean lines with low elemental sulfur. SDS-PAGE and immunoblot analysis revealed that the accumulation of Bowman Birk protease inhibitor and lunasin in soybean seeds may only be marginally correlated with the elemental sulfur levels. However, we found a positive correlation between the levels of trypsin and chymotrypsin inhibitor activities and elemental sulfur and sulfur amino acid content of the seeds. Thus, elemental sulfur content and/or protease inhibitor activity measurement can be utilized as a rapid and cost-effective method to predict the overall sulfur amino acid content of soybean seeds. Our findings will benefit breeders in their endeavors to develop soybean cultivars with enhanced sulfur amino acid content.

Nutritional, metabolic and genetic profiling of ‘Cerato’ and ‘Curniciello’ bean landraces from Caserta, Southern Italy

Italy represents a territory rich in common bean landraces, most of which have not yet been characterized. Therefore, the proximal composition and metabolic profiles (fatty acid composition, total and free amino acids) as well as total polyphenols and antioxidant capacities of both ‘Cerato’ and ‘Curniciello’ dry beans, cultivated in Caserta’s rural areas (Southern Italy) were evaluated, in comparison with other local known dry beans. ‘Cerato’ dry beans have a lower content of crude proteins (21.18 vs 23.41 g/100 g), lipids (1.27 vs 2.08 g/100 g) and total amino acids (16.01 vs. 17.89 g/100 g) with respect to ‘Curniciello’ dry beans, considering the average values of two different harvest years (2020–2021), although slight statistical differences were found when the two harvest years were analysed separately. Two essential fatty acids (n-6 linoleic and n-3 alpha-linolenic) and oleic acid were the most abundant fatty acids in both dry beans, (∼90% of the total). Subsequently, the trypsin and chymotrypsin inhibitory activities in raw and boiled (2 h) dry beans, as well as the α-amylase and α-glucosidase inhibitory activities were investigated, considering their capability to reduce crude protein and carbohydrates intake and assimilation. In addition, AFLP analysis of two landraces shows different polymorphic patterns useful for their authentication and traceability. Overall, our data provide a starting point for promoting the cultivation and consumption of ‘Cerato’ and ‘Curniciello’ dry beans, thus contributing to the preservation of local culinary traditions and Italian biodiversity.

Temporal variability in foliar protein content and trypsin inhibitory levels in two host trees of tropical Tasar silkworm Antheraea mylitta, Drury

Aim: Variation in total soluble proteins and protease inhibitor levels were investigated to determine nutritional and plant defense status in different leaf types of Terminalia arjuna (Roxb.) Wight and Arn., and Schleichera oleosa (Lou.) Oken, two host tree species of the economically important, non-mulberry Tasar silkworm, A. mylitta. Methodology: Quantitative spectrophotometric assays based on diagnostic amidolytic substrates were conducted to assess serine protease inhibitor activities in young, semi-mature and mature leaf types. A microplate quantification assay for total protein estimations was used with leaf types sampled over a year. Efficacy of total trypsin inhibitor and chymotrypsin inhibitor activities detected in T. arjuna (a primary host plant) was further evaluated on gut extracts of fourth instar A. mylitta and Pieris brassicae L. (a Pierid pest of crucifers) to assess the physiological adaptation of larvae to dietary antifeedants. Molecular provenances with rbcL genes were obtained that are available as NCBI accessions #MN460810 for T. arjuna and #MT010554 for S. oleosa. Results: Intra-specific variations were evident in leaf phenology of two tree species. Generally, young leaf type of both tree species had high levels of total protein and trypsin inhibitory activities, while mature leaf type of T. arjuna had low total protein content and trypsin inhibitor levels. Mature leaf type of S. oleosa had low trypsin inhibitor levels during the months of July and August. Midgut proteases of A. mylitta and P. brassicae were significantly more susceptible (p≤0.05) to both trypsin and chymotrypsin inhibitors detected in young and semi-mature leaf types of T. arjuna than the mature leaf type. Interpretation: In this study, differential inhibition of digestive proteases in A. mylitta and P. brassicae by protease inhibitors from different leaf types of T. arjuna, suggested adaptation to dietary antifeedants. Such reports on nutritional quality, foliar antifeedants, phenology and host plant utilization are relevant for strategies to domesticate the tropical Tasar silkworm, A. mylitta. Key words: Antheraea mylitta, Protease Inhibitors, Terminalia arjuna, Total protein, Tree phenology

In vitro Antioxidant, Enzyme Inhibitory and Antimicrobial Evaluation of Sideritis caesarea H. Duman, Aytaç & Başer Extracts

Sideritis caesarea H. Duman, Aytaç & Başer of the family Lamiaceae is an endemic plant with different ethnobotanical uses in Anatolia. In this present study, the antioxidant and biological activity potential of different S caesarea extracts and purified compounds were evaluated in vitro using DPPH•, CUPRAC, and ABTS• methods. In addition, their urease and chymotrypsin enzyme inhibitory activities, antimicrobial potential, and total phenol and flavonoid contents were determined. The experimental results showed that the two isolated flavonoids {4′- O-methylhypolaetin-7- O-[6″′- O-acetyl- β-D-allopyranosyl-(1→2)]-6″- O-acetyl- β-D-glucopyranoside, and isoscutellarein-7- O-[6″′- O-acetyl- β-D-allopyranosyl-(1→2)]-6″- O-acetyl- β-D-glucopyranoside}, as well as their fractions, except the n-hexane and water residue, showed remarkable antioxidant activity. Interestingly, the ethyl acetate and water residue fractions inhibited the urease enzyme activity by 49.6% and 49.1%, respectively; while the n-hexane and dichloromethane fractions attracted attention with their relative high chymotrypsin inhibitory activity of 85.8%, and 82.4%, respectively. When evaluated for their in vitro antimicrobial potential, including Helicobacter pylori, only Bacillus subtilis was susceptible to the n-butanol fraction at 1 mg/mL, while all of the studied samples showed activity against the human pathogenic yeast Candida albicans with the range of 0.25 to 1 mg/mL minimum inhibitory concentrations. The initial antioxidant activity, enzyme inhibitions, and antimicrobial results of S caesarea extracts and compounds suggest the further potential for in vivo evaluations.

Chymotrypsin inhibitor assay: Expressing, calculating, and standardizing inhibitory activity in absolute amounts of chymotrypsin inhibited

AbstractThere is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in soybean and other legume products, although trypsin inhibitor activity has been primarily measured. Recently, for accurately measuring CIA, an optimized method was developed in our laboratory and published elsewhere. Like most reported methods, the new method expresses CIA as chymotrypsin units (CU) inhibited (CUI)/mg sample. This arbitrary unit makes comparison of results impossible among different methods. The present study solved this problem by expressing CIA in absolute amounts of chymotrypsin inhibited (CId) and standardizing against a reference chymotrypsin. With the new method, two experiments were conducted, using four chymotrypsin reagents having different specific activities (SA), respectively. Experiment 1 determined the relationship between absorbance at 400 nm (A400) and μg chymotrypsin. Experiment 2 measured raw and heated soybeans, expressed CIA as CUI/mg sample and μg CId/mg sample, respectively, and determined conversion factors between the two units. Conversion factors determined in both experiments matched each other but varied with chymotrypsin SA. Yet, after standardizing against the reference chymotrypsin having a fixed SA of 150 N‐benzoyl‐L‐tyrosine‐p‐nitroanilide (BTNA) (equivalent to 50 N‐benzoyl‐L‐tyrosine ethyl ester [BTEE]) units/mg protein, a standardized conversion factor of 1.5 CUI (= 0.015 ΔA400) = 1 μg CId was determined. It remained consistent regardless of chymotrypsin SA, sample heating history, and levels. The new method can now express results in μg CId/mg sample (or mg/g) by dividing CUI/mg sample by 1.5. When other methods follow the same strategies for determining standardized conversion factors, CIA results could be comparable.

Amino Acid Substitutions at P1 Position Change the Inhibitory Activity and Specificity of Protease Inhibitors BmSPI38 and BmSPI39 from Bombyx mori

It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid-base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.

Overexpression of soybean trypsin inhibitor genes decreases defoliation by corn earworm (Helicoverpa zea) in soybean (Glycine max) and Arabidopsis thaliana.

Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

The effect of radiofrequency heat treatment on trypsin inhibitor activity and in vitro digestibility of soybean varieties (Glycine max. (L.) Merr.)

Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved.

Method development and optimization for measuring chymotrypsin and chymotrypsin inhibitor activities.

Protease inhibitors of protein nature are rich in seeds of legume crops. There are two common types: Kunitz inhibitor, which mainly inhibits trypsin, and Bowman-Birk inhibitor, which inhibits both trypsin and chymotrypsin. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Reported methods for CIA assay vary greatly and are incompletely described. No standardized or official method is available. The present study focused on developing a robust method for accurately measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Since BTpNA is not water soluble, a water-miscible organic solvent must be present. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels (which affected % chymotrypsin inhibition), the sequence of adding reagents, extractant and extraction time, and so forth, an optimized method for CIA measurement was finally developed. It features dimethylformamide as the organic solvent, the enzyme-last sequence, 5ml total assay volume, and calculation of the inhibitor activity based on 40% chymotrypsin inhibition. The method can also be slightly modified for measuring chymotrypsin activity. The robust performance of the method was verified by measuring 11 assorted protein products, paving a way for standardization. PRACTICAL APPLICATION: With an increasing use of plant proteins, there is an urgent need to measure chymotrypsin inhibitor activity in various protein products with accuracy. After thoroughly investigating several factors, an optimized method for measuring chymotrypsin inhibitor activity in various protein products was developed. The proposed method is sensitive and robust, providing a basis for standardization. It can also be used for measuring chymotrypsin activity.

Determination of protease inhibitors, glycinin, and beta-conglycinin in soybeans and their relationships.

In order to search for suitable soybean varieties for different applications, the protein contents of Kunitz trypsin inhibitor (KTI), Bowman-Birk trypsin inhibitor (BBI), glycinin (11S), and β-conglycinin (7S) of 93 soybean samples from different sources and harvest years were quantified by sodium dodecyl sulfate polyacrylamidegelelectrophoresis. Meanwhile, the protease inhibitory activities against trypsin and chymotrypsin were determined. Results showed that the individual protein contents and trypsin inhibitor activities differed significantly (p<0.05) among soybean samples. KTI contents ranged from 5.25 to 14.60mg·g-1 ; BBI contents ranged from 1.81 to 5.74mg·g-1 ; 11S varied from 13.65% to 48.55% and 7S varied from 15.68% to 42.15% of total soluble protein; trypsin and chymotrypsin inhibitory activities were 8.93-20.95mg TI·g-1 and 4.18 -12.79mg CI·g-1 , respectively. Excellent linear relationships existed between trypsin inhibitor contents and their activities. The regression equations offer a rapid method for estimating the activity of KTI or BBI in raw soybeans. PRACTICAL APPLICATION: The regression equations established based on a large number of soybean varieties offered a rapid method to estimate the activity of trypsin inhibitors. The data presented here provided useful information for the food industry or breeders to select soybean varieties with different inhibitory activities or protein contents for different food processing applications.

Trypsin inhibitor activity, phenolic content and antioxidant capacity of soymilk as affected by grinding temperatures, heating methods and soybean varieties

Two soybean varieties (yellow and black) were ground under three temperature regimes (cold, ambient and hot) and then the raw soymilk underwent three thermal treatments (traditional stove cooking, one-phase ultra-high temperature (UHT) processing, and two-phase UHT processing). Trypsin inhibitor activity (TIA), chymotrypsin inhibitor activity (CIA), total phenolic content (TPC), total flavonoid content (TFC), condensed tannin content (CTC), DPPH free radical scavenging activity, and oxygen radical absorbing capacity (ORAC) were significantly affected by the above factors and their interactions. For raw yellow soymilk, hot grinding generated 64 and 69% lower TIA than cold and ambient grinding, respectively. For raw black soymilk, hot grinding generated 40 and 43% lower TIA than cold and ambient grinding, respectively. After heating process, the advantage of hot grinding was reduced. By suppression of the development of lipoxygenase-induced hydroperoxides, hot grinding preserved the most sulfhydryl and disulfide groups. Stove cooking following ambient grinding resulted in the lowest CIA. Except TFC, hot grinding recovered the highest phenolic content and antioxidant capacity. CTC from hot grinding was about twice that from ambient grinding. Black soymilk contained significantly higher antioxidant capacity than yellow soymilk and UHT treatments showed advantage over traditional stove cooking in the preservation of phenolics.

Bowman-Birk inhibitor in soybean: Genetic variability in relation to total trypsin inhibitor activity and elimination of Kunitz trypsin inhibitor

Bowman-Birk inhibitor (BBI) is a soybean seed serine protease inhibitor whose nutraceutical properties far exceed its anti-nutritional property arising from its trypsin and chymotrypsin inhibitor activity. High BBI soybean genotypes are sought for the commercial preparation of this nutraceutical biomolecule while low BBI content in Kunitz trypsin inhibitor (KTI)-free genetic background is desirable in soymeal manufacturing. In the present investigation, monoclonal antibody assay of 95 soybean genotypes revealed 11-fold genetic variation for BBI concentration. The study led to the identification of 3 very high (&gt;20 mg/g defatted soy flour) and 5 very low BBI (&lt; 4 mg/g defatted soy flour) soybean genotypes. Contribution of BBI to total trypsin inhibition activity ranged from 2.2 to 53.5% with average value of 11.6%. Genotypes with varying level of BBI raised consecutively for two years exhibited non-significant (p&gt;0.05) effect of growing year on the trait. BBI content in BC3F2 derived KTI-free lines was at par with the recurrent parent. Low and high BBI content genotypes with diverse genetic background identified in the study may be exploited to develop mapping population to identify genomic regions underlying BBI in soybean.

The impact of enzymatic hydrolysis using three enzymes on the nutritional properties of a chickpea protein isolate

AbstractBackground and objectivesEnzymatic hydrolysis has shown potential for improving the functional properties of pulse protein products; however, this process may also result in modified nutritional properties. Several studies concerning this topic have already been performed; however, in many cases these studies focus either on a single enzyme, or a single level of hydrolytic treatment. The goal of this study was instead to analyze a chickpea protein isolate (CPI) hydrolyzed under a variety of conditions, in an attempt to better understand any emergent relationships present. To achieve this, three different proteases (trypsin, pepsin, and papain), and three levels of hydrolysis (DH 5%, 10%, and 15%), that is, nine enzymatically treated CPI preparations, were used to generate protein hydrolysates. They are then tested for levels of four selected bioactive compounds (trypsin and chymotrypsin inhibitory activity, total phenolics, and tannins), as well as limiting amino acid score (LAS), in vitro protein digestibility (IV‐PD), and in vitro protein digestibility‐corrected amino acid scores (IV‐PDCAAS).FindingsIncreasing degree of pepsin and papain hydrolysis led to progressive decreases in trypsin inhibitory activity. Hydrolysis with all enzymes led to decreases in chymotrypsin inhibitory activity. Total phenolic contents exhibited little change following hydrolysis. Tannins could not be detected in any of the protein isolates. IV‐PD did not change following hydrolysis. Tryptophan was the most limiting amino acid in the untreated isolate, following hydrolysis cysteine + methionine also became increasingly limited. IV‐PDCAAS scores generally declined following enzymatic treatments.ConclusionsHydrolysis of &lt;5% DH resulted in minimal negative effects to the protein quality of a CPI. CPI products hydrolyzed to more than 5% DH should be fortified with sulfur containing amino acids for optimal protein nutrition.Significance and noveltyA total of nine distinct enzyme treatments were compared with respect to the nutritional properties of a chickpea protein material, showing low hydrolysis can improve the nutritional properties of CPI.

Effect of high-temperature, short-time cooking conditions on in vitro protein digestibility, enzyme inhibitor activity and amino acid profile of selected legume grains

African yam beans (Sphenostylis stenocarpa), Bambara groundnut (Vigna subterranean) and Pigeon pea (Cajanus cajan) flours were extruded in a single screw extruder at two extrusion temperatures; 100 °C and 140 °C, and the effect of extrusion cooking temperature on the chemical composition; crude protein, crude fibre, ether extract and nitrogen-free extracts, protein digestibility, enzyme inhibitor activity and amino acid profiles was investigated. The crude protein, amino acid profile and ether extract of the grain legumes were negatively affected (p < 0.05) by the extrusion cooking process, with a significant increase in nitrogen-free extracts for all grain legumes, and increased crude fibre of Bambara groundnut and Pigeon pea extrudates. Extrusion cooking of African yam beans and Pigeon pea produced extrudates with significantly lower trypsin, chymotrypsin and amylase inhibitor activity as well as improved protein digestibility. However, extrusion cooking did not modify the chymotrypsin and amylase inhibitor activity of Bambara groundnut extrudates. Extrusion cooking at 140 °C compared to 100 °C significantly reduced the protein quality of extrudates resulting in 22.94–51.27%, 5.11–25.18%, and 7.78–38.42% reduction in amino acid concentration of African yam beans, Bambara groundnut and Pigeon pea, respectively.

In vitro screening of peptidase inhibitory activity in some plants of North India

In the present study, trypsin and chymotrypsin inhibitory activity of some plants of different families was evaluated. A total of 55 plants were screened, out of which six showed the maximum trypsin inhibitory activity namely Acacia concinna, Caesalpinia bonducella, Lathyrus sativus, Mucuna pruriens, Psoralea corylifolia and Sapindus mukorossi. Results suggested that the plants showing trypsin inhibitory activity (TIA) also have chymotrypsin inhibitory activity (CIA). Both trypsin and chymotrypsin inhibitory activities were high in seeds compared to leaves followed by flowers. It was also observed that TIA was maximally present in Sapindaceae family whereas CIA was maximum in fabaceae family followed by others.

Effect of interaction between tea polyphenols with soymilk protein on inactivation of soybean trypsin inhibitor

Soymilk is a popular health beverage worldwide, but its nutritional value is limited by soybean trypsin inhibitors (STIs). The interaction of tea polyphenols (TPs) with soymilk protein in the complex system was investigated in this work. TPs interacted with soymilk protein via static quenching process and hydrophobic interaction, with binding constant (Ka) of 5.22 × 103 L mol−1 at 298 K. Synchronous fluorescence suggested that the binding site of TPs to soymilk protein was mainly at Trp residues compared with Tyr residues. FTIR analysis revealed that hydrogen bonds were also observed in TPs-soymilk system. CD spectroscopy suggested that the protein conformation became more stable with addition of TPs by reducing β-sheet and random coil, and increasing α-helix and β-turn. The trypsin and chymotrypsin inhibitory activities (TIA and CIA) were reduced at 0.6 mg/g TPs in cooked soymilk from 788.3 ± 10.4 U/mL and 918.7 ± 18.0 U/mL to 388.3 ± 35.5 U/mL and 633.3 ± 52.8 U/mL, respectively.

Comparing Nutritional Quality, Antioxidant, and Antiulcer Activity of Two Amaranthaceae Plants: Achyranthes aspera and Amaranthus spinosus

Achyranthes aspera and Amaranthus spinosus are species of medicinal plants from the Amaranthaceae family, used to treat wounds, asthma, malaria, gonorrhea, burns, and diabetes. In this study, the nutritional value and phytochemical constituents in A. aspera and A. spinosus were determined using inductively coupled plasma-optical emission spectrometry and column chromatography, respectively. The antiulcer activity was determined using the α-chymotrypsin assay while the antioxidant activity was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl radical scavenging assay, ferric reducing antioxidant power, and phosphomolybdate assay. The results showed leaves of A. spinosus to contribute between 43.4% and 97.8% toward the recommended dietary allowance for iron; therefore, it is recommended for consumption by patients suffering from chronic anemia. Arsenic, cadmium, and lead were not detected in the leaves of the two species giving credence to their use as nutraceuticals in South Africa while the concentration of cobalt, nickel, and selenium were below the detection limit of the instrument. The phytochemical analysis resulted in the isolation of oleanolic acid, lutein, pheophytin a, and chondrillasterol glucoside. The antioxidant activity was high for lutein, ethyl acetate extract from A. aspera, chondrillasterol glucoside, and the methanol extract from A. spinosus, relative to the control. The antiulcer activity using the α-chymotrypsin inhibition assay showed lutein to have maximum chymotrypsin inhibitory activity. Findings from this study show lutein, pheophytin a, chondrillasterol, and its derivatives to serve as potential taxonomic markers for species in the Amaranthaceae family.

Phytochemical analysis, antimicrobial, antioxidant and enzyme inhibitory activities of ethanolic extract ofCentaureasolstitialis L. and its different fractions

Resistance to conventional antimicrobial regimes is one of the issues of concern in healthcare and it drives the need for development of new antimicrobial agents. Medicinal plants, as rich source of biochemical and bioactive compounds, serve as potential source for new drugs. Here, we evaluated the ethanolic extract of Centaureasolstitialis L.and its different fractions (n-hexane, choloroform and n-butanol soluble fraction) for antimicrobial, antioxidant and enzymes inhibitory activities. Antibacterial activity against two gram-positive and three gram-negative bacteria species were determined by using agar well diffusion and 96-wells microplate methods. Similarly, antifungal activity against two fungal strains was also evaluated by agar well diffusion method. Antioxidant activity analyzed by measuring the scavenging activity of DPPH radicals and acetylcholinesterase, butrylcholinesterase and chymotrypsin inhibitory activity was determined at 10 µg/mL and 1.0 mg/mL concentrations. Results revealed that the ethanolic extract of C. solstitialis and its different fractions possesses significant ( p &lt;0.05) antibacterial activity and effective against fungi Aspergillus niger and Macrophominaphaseolina . Significant ( p &lt;0.05) DPPH scavenging activity (88.52±0.23%) among all fractions was noted. n-butanol fraction showed significant acetyl-cholinesterase (78.55±0.76%) and butrylcholinesterase inhibitory activity (78.1±0.41%) with IC 50 values of 54.6±0.39 µg/mL and 211.9±0.15 µg/mL, respectively. Maximum chymotrypsin inhibition activity was shown by crude ethanolic extract (87.76±1.17) with IC 50 valueof 38.23±0.75 µg/mL. It is concluded that C. solstitialis extract and its fractions possess significant antimicrobial, antioxidant and enzyme inhibitory activity