Optimization, identification and comparison of peptides from germinated chickpea (Cicer arietinum) protein hydrolysates and their effects on markers of type 2 diabetes and bitterness using biochemical, in silico and in vitro techniques

Chandrasekaran, Subhiksha

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https://hdl.handle.net/2142/116131 Copy

Description

Title

Optimization, identification and comparison of peptides from germinated chickpea (Cicer arietinum) protein hydrolysates and their effects on markers of type 2 diabetes and bitterness using biochemical, in silico and in vitro techniques

Author(s)

Chandrasekaran, Subhiksha

Issue Date

2022-07-22

Director of Research (if dissertation) or Advisor (if thesis)

de Mejia, Elvira

Committee Member(s)

• Pepino de Gruev, Marta Yanina
• Chen, Hong

Department of Study

Food Science & Human Nutrition

Discipline

Food Science & Human Nutrition

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

• chickpea
• bitterness, type 2 diabetes, glucose, DPP-IV, adipocytes, ficin

Abstract

Chickpea (Cicer arietinum) is currently the second most produced legume worldwide. There is a steady growth in alternative protein consumption, allowing for commercial increased use of plant-based proteins as functional ingredients. The objective was to optimize and compare the production of antidiabetic peptides from germinated chickpea isolated protein using either papain or ficin. Additionally, this study’s objective was also to evaluate the optimum germinated chickpea protein hydrolysate (GCPH) and its peptides in vitro for their effect on markers of type 2 diabetes (T2D) and bitter taste receptor expression in intestinal epithelial cells and adipocytes. Kabuli chickpeas were germinated for 2, 4 and 6 days. Proteins were isolated, and hydrolysates were produced based on a central composite design selecting human dipeptidyl peptidase (DPP-IV) inhibition as a response. Peptide sequencing was performed to identify and evaluate the physiochemical, biochemical and bitterness properties. DPP-IV inhibition using papain was 84.66 ± 8.72%, with ficin being 72.05 ± 1.20%. The optimum hydrolysate conditions were 6 days germination, 1:10 E/S, and 30 min ficin hydrolysis; SPGAGKG, GLAR, and STSA were identified. Pure SPGAGKG had relatively high affinity for DPP-IV (−7.2 kcal/mol) and α-glucosidase inhibition (−5.9 kcal/mol), with an IC50 of 0.27 mg/mL for DPP-IV inhibition. Caco-2 cells were used to determine glucose uptake and extra-oral bitter receptor activation. Three peptides, VVFW, GEAGR, and FDLPAL, were identified in legumin in GCPH made with a new batch of the same cultivar of chickpeas. FDLPAL was the most potent peptide in molecular docking studies with a DPP-IV energy of affinity of -9.8 kcal/mol. GCPH significantly inhibited DPP-IV production by Caco-2 cells (IC50 = 2.1 mM). Glucose uptake was inhibited in a dose-dependent manner (IC25 2.0 mM). Among pure peptides tested, peptide VVFW was the most potent in glucose uptake inhibition, with 38.7% inhibition at 50 µM. Pure peptide FDLPAL was the most potent in docking with sucrase-isomaltase (-7.8 kcal/mol) and according showed the highest inhibition of sucrase (44.1%), maltase (64.2%) and isomaltase activity (51.2%) at 100 µM among peptides tested, A negative correlation was found between glucose uptake and PLCβ2 expression in Caco-2 cells (R value, -0.62) and additionally, a positive correlation was found between GLUT2 expression and PLCβ2 expressio\n in Caco-2 cells (R value 0.77). In adipocytes (3T3-L1 MBX cells), peptide GEAGR was the most potent inhibitor of lipid accumulation (38.2%) at 250 µM. Peptide GEAGR was the most potent inhibitor of FAS activity, with 68.6% inhibition at 100 µM. GCPH also reduced PPARγ expression at 75 µM and 100 µM, and pure peptides FDLPAL and GEAGR at all concentrations. The activation of TAS2R4 by GCPH showed a positive correlation with PPARγ expression in adipocytes, as GCPH at 250 µM activated both PPARγ and TAS2R4 expression (R value, 0.63). Overall, in Caco-2 cells, blocking the bitter signaling pathway appeared to inhibit glucose uptake. In adipocytes, increased expression of the bitter signaling pathway appeared to promote browning. GCPH showed potential to be commercialized as a functional ingredient.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Subhiksha Chandrasekaran

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