Production, characterization and evaluation of anti-diabetes peptides from proteins of improved common bean (Phaseolus vulgaris L.)cultivars using bioinformatic tools, enzymatic systems, and in vitro and in vivo models

Black beans;peptides;diabetes;glucose absorption;Glucose transporter 2 (GLUT2);Sodium-dependent glucose cotransporter (SGLT1)

Mojica Contreras, Luis Alfonso

关键词: Black beans;    peptides;    diabetes;    glucose absorption;    Glucose transporter 2 (GLUT2);    Sodium-dependent glucose cotransporter (SGLT1);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/90816/MOJICACONTRERAS-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Diabetes is an important metabolic disease expected to increase worldwide 55% by 2035; with an estimated healthcare cost of $245 billion per year in the US alone. The use of alternative plant based treatments has the advantage of no side effects and could reduce treatment cost. Common beans health benefits are attributed to bioactive compounds; including resistant starch and oligosaccharides that contribute to dietary fiber; phenolic compounds and bioactive peptides released from proteins during enzymatic digestion. Biological potential of bean peptides has been related to antihypertensive, anticancer, anti-inflammatory, antioxidant and antifungal; however, the anti-diabetes potential of common bean protein fractions and pure peptides has not been studied. In that sense, the central hypothesis of this research was that common bean protein hydrolyzed fraction contains bioactive peptides that could modulate molecular targets of type 2 diabetes. To fill the gap in the knowledge, our long-term goal was to understand the mechanism by which common bean peptides contribute to the management of type 2 diabetes. To achieve this aim, we generated anti-diabetes peptides from common bean protein and determined their efficacy using bioinformatics tools, enzymatic systems, in vitro and in vivo models. Our research started with the selection and characterization of proteins, peptide profile, protein inhibitors, α-amylase inhibitors, phenolic compounds and anthocyanins of improved common bean cultivars from Mexico and Brazil (Aim 1). The peptides and phenolic compounds found presented outstanding anti-diabetes potential from the biochemical and in silico studies. Furthermore, we evaluated the peptides anti-diabetes potential after simulated gastrointestinal digestion and characterized their bioactive peptides, angiotensin converting enzyme, dipeptidyl peptidase IV, and α-glucosidase inhibition (Aim 2). Negro Otomi cultivar presented the lowest IC50 to inhibit DPP-IV (0.14±0.01 mg DW/ mL). The effect of processing was evaluated in Aim 3, where the goal was to determine the bioactive properties of the released peptides from commercially available precooked common beans (Phaseolus vulgaris). Bioactive properties and peptide profiles were evaluated in protein hydrolysates of raw and commercially precooked common beans. Hydrolysates from Navy beans were the most potent inhibitors of DPP-IV with no statistical differences between precooked and raw (IC50 = 0.093 and 0.095 mg protein/mL, respectively). In general, we observed that processing did not affect the bioactive properties of released peptides from precooked beans. With the objective to increase the anti-diabetes potential of common bean protein fractions, the fourth Aim was to optimize the production of anti-diabetes protein fractions from black bean (Phaseolus vulgaris L.) protein isolates. The results suggested that the best combination of conditions to generate anti-diabetes peptides were with alcalase for two h and E/S of 1:20, with high inhibitions values for dipeptidyl peptidase IV (DPP-IV, 96.7%), α-amylase (53.4%) and α-glucosidase (66.1%). The protein fractions were characterized using LC-ESI-MS/MS and 33 sequences were identified.Finally, we evaluated the anti-diabetes mechanism of action of common bean protein fractions and pure peptides using in silico, in vitro, and in vivo models. Sequenced peptides showed outstanding potential to bind and blocking intestinal glucose transporters SGLT1 and GLUT2. Besides, using Caco-2 cells monolayer insert we observed that protein fractions reduce glucose absorption by 6.5 and 24.5 after 30 min and 24 h, respectively.It could be due to protein fractions were also able to decrease protein expression and translocation to the membrane of GLUT2 and SGLT1.Furthermore, protein fractions were able to lower postprandial glucose by 24.5% in an oral glucose tolerance test in rats after receiving 50 mg/kg BW compared to untreated control.Also, a hyperglycemic rat model showed that protein fractions at 100 mg of PF/ Kg BW twice daily significantly reduced postprandial glucose levels by 48% compared to the non-treated diabetic group, without having consequences in body weight or other biochemical parameters. In conclusion pure peptides and protein fraction showed remarkable potential to decrease translocation of glucose transporters to the membrane and as a consequence reduction of glucose absorption.