The increase and prevalence of chronic degenerative pathologies derived from chronic cellular oxidation processes (associated with the increased consumption of meat products, processed flour, colorants and preservatives) has stimulated the search for new molecules with biological activity against cellular oxidation, where the proteins stand out as the most interesting molecules. There are several amino acid sequences (peptides) that exhibit bioactive properties of interest but are normally found in large and complex polypeptide sequences that
inhibit their biological activity
(Arnal et al., 2024). The biological active is correlated to the peptide amino acid composition, residues quantity, residues position in the carbon chain and the spatial structure
(Sun et al., 2024). In human health, therapeutic products enhanced with peptides are used mainly against the metabolic syndrome, antibacterial and antiviral therapies and especially for developing cell-targeting platforms and improving cell-penetrating properties (
Zhu et al., 2019). Peptides are base of dietetic formulas for infant and geriatric products and weight-control/high-energy products (
Kehinde and Sharma, 2020;
Valenzuela et al., 2022).
The use of animal sources, with the exception of some sub-products of the milk industry, has usually been discarded as a source of bioactive peptides, mainly because of high and sustained demand for these raw products by the food industry
(Shabir et al., 2023).
Protein from plant sources traditionally has not been considered as an important source of proteins due they often contain allergenic proteins and other anti-nutritional factors (
e.g. glycinin, trypsin inhibitors,
etc.); but when vegetal protein is hydrolyzed, antigenic and anti-nutritional behavior decreases significantly (
Clemente and Chambers, 2000). Bioactive peptides from legume have usually been described as a chains of 2 to 20 amino acid residues
(Acquah et al., 2022), where soybean and chickpea (
Castro and Sato, 2014) are the most widely used legumes.
Leucaena esculenta, a legume widely distributed in Mexico and Central America, has been utilized as a protein supplement in fodder feeds (
Parrota, 1992). The seeds are not commonly used in human nutrition due the presence of mimosine; however,
Leucaena esculenta seeds can be safely consumed when the seeds are soaking and cooking (
Balderas-León et al., 2004). Despite this limitation,
Leucaena esculenta seeds represent a promising nutritional resource due to their high protein content. Nevertheless, the utilization for human nutrition of
Leucaena esculenta seeds protein remains underexplored.
The aim of this study was to evaluate the antioxidant
in vitro activity (by DPPH*/ABTS*
+ radicals) and antidiabetic activity (α-amylase/α-glucosidase inhibitory activity) from the hydrolyzed protein from
Leucaena esculenta ripe seeds. Additionally, the study evaluate the correlations between the biological activity and hydrolyzed protein fractions sizes (≤100 kDa, ≤10 kDa and ≤3 kDa).
Collection of plant material
Ripe
Leucaena esculenta seeds, were collected in Oaxaca, Mexico (16°59’21’’N, 96°43’26’’W) during 2021. The identification of the species was carried on in the Herbarium Mtra. Graciela Calderón Díaz-Barriga and Dr. Jerzy Rzedowski.
Pre-treatment of plant material
Ripe seeds were cleaned in a commercial solution of 5% (v/v) of sodium hypochlorite. Seeds were reduced to particulate material (<0.149 mm).
Protein isolation
The protein fraction (native fraction) was obtained according to the methodology of
Ohara et al. (2020).
Protein quantification
The protein quantification was used according to the methodology of
Grimsley and Pace (2004).
Enzymatic hydrolysis
Enzymatic hydrolysis was performed according to
Hamada (2000). Three proteolytic enzymes were used: i) Alcalase (serine-type endoprotease.); ii) Flavourzyme (mixtures of different endo- and exoprotease) and iii) Neutrase (endoprotease random behavior).
The hydrolysis conditions were kept within the optimal ranges specified by the enzyme supplier (Novozymes). Alcalase: 3 ug
-1, pH 6.5, 60°C; Flavourzyme: 500 ug
-1, pH 6.0-50°C and Neutrase: 1.5 ug
-1, pH 6.0-50°C.
Determination of the degree of hydrolysis
It was determined by the OPA technique according to method of
Nielsen et al. (2021).
Recovery of protein fractions
Isolation of the protein fractions was performed using Centricon-Plus centrifugal filters of 100 kDa, 10 kDa and 3 kDa mesh sizes. Filters were preconditioned according the manufactures instructions. 2 mL of the protein hydrolysate solution was added to each filter. The filters loaded with the protein samples were centrifuged (3500 xg for 5 min at 25°C) to recover the filtrate. Protein fraction from 100-10 kDa was denominated as 100kDa fraction, the fraction from 10-3 kDa was denominated as 10 kDa fraction and the protein fraction ≤3kDa was denominated as 3 kDa.
Bioactive properties. Assessment of antioxidant activity; DPPH* assay
Antioxidant activity was determined according to the method of
Brand-Williams et al. (1995).
Bioactive properties. Assessment of antioxidant activity; ABTS+* assay
Antioxidant activity was determined according to the method of
Re et al. (1999).
Inhibition of α-amylase and α-glucosidase enzymes
The protein sample concentration was diluted with sodium phosphate solution (50 mM at pH 6.9) to obtain a protein content of 30 mg mL
-1.
Inhibition of α-amylase and α-glucosidase were evaluated according to the method of
Mojica and González (2015). The content of reducing sugars released by α-amylase is determined by the dinitrosalicylic acid method according to
Miller (1959).
The polysaccharide acarbose was used as a positive control (25 mg mL
-1; human standard therapeutic dose for glucose en blood control).
As negative control of enzymatic activity a solution of 10 mg mL
-1 of wheat starch was used.
Statistical analysis
Results of each protein sample were analyzed by an analysis of variance (ANOVA) with Tukey’s media test (significance level,
p<0.050). Correlation analysis was carried out by a multiple linear regression (significance level,
p<0.050). Analysis was performed using Statistica ver. 7.0.
Antioxidant capacity
In legumes, the use of proteases (
e.g., alcalase) is used as a strategy to obtain a higher concentration of peptides/increase the bioactive activity
(Kumar et al., 2022). Peptides of lower molecular size and/or enriched in hydrophobic amino acid have been associated with higher antioxidant potential
(Yust et al., 2012; González-Garza et al., 2017).
Our results show that hydrolysis by all the evaluated proteases increase the antioxidant activity (p<0.050). The Flavourzyme hydrolysates exhibit the highest antioxidant activity against the DPPH* and ABTS
+* radicals (Table 1). The fraction with a molecular size of 100 kDa, showed the highest ABTS
+* radical inhibitory capacity, 74.95% (Fig 1b). This could be attributed to the specific peptide sequences or sequences rich in hydrophobic amino acids.
The hydrolysate fraction from Neutrase with a molecular size of 10 kDa showed the highest DPPH* radical inhibitory capacity, 74.96% (Fig 1a).
However, both for the hydrolysates from Flavourzyme and Neutrase there is no significant correlation (
p=0.053) between the size and the antioxidant capacity.
Xu et al., (2024) suggests that the specific amino acid sequences (
e.g. histidine, methionine and cysteine - electron donators) may be more significant than peptide size.
It has been proposed that the antioxidant action of peptides is due their action on several signaling pathways: i) Kelch-like ECH-associated protein 1/NFE2-related factor 2/antioxidant response elements, Keap1-Nrf2/ARE (inactivation of the Keap1 Kelch active site); ii) growth factor-β/small mothers against decapentaplegic, TGF-β/SMAD (promoting the formation of collagen in fibroblasts by activating the TGF-β1/SMAD2); iii) AMP-activated protein kinase/Sirtuin/peroxisome proliferator-activated receptor γ coactivator-1α, AMPK/SIRT1/PGC-1α (inhibition mitochondria- mediated apoptosis, improving the mitochondrial function and avoiding the mitochondrial autophagy); iv) phosphatidylinositol 3-kinase/Akt, PI3K/Akt/mTOR (promoting the cytoprotective effects by activating the PI3K/Akt pathway) and v) nuclear factor-κB (inactivation of RelA (P65) and P50 of the NF-κB family)
(Feng et al., 2021; Li et al., 2022).
Enzymatic breakdown of polysaccharides (sugar molecules)
α-glucosidase and α-amylase are essential for metabolizing polysaccharides in food. The inhibition of those enzymes causing a lowering post-prandial glucose levels (
Elferink et al., 2020).
Inhibitory capacity of evaluated protein fractions against the α-glucosidase and α-amylase varied significantly (
p<0.050) (Table 1). Hydrolysate fraction form Alcalase with molecular size of 100 kDa, showed the highest α-glucosidase and α-amylase inhibitory capacity, 33.42% and 56.42% respectively (Fig 1c and 1d).
Lu et al., (2023) propose that peptides with a high concentration of hydrophobic residues effectively inhibit the α-amylase and α-glucosidase.
Protein fractions isolated from legumes exhibit a low inhibition capacity of α-glucosidase and α-amylase
(Acquah et al., 2022).
In α-amylase, three structural domains with catalytic activity over polysaccharides are observed, were the residues 1-99; 170-404; are described as the main active site for the glycosyl-enzyme intermediate complex
(Ramasubbu et al., 2004).
Our results show no significant correlation between the size of any of the hydrolysates evaluated and the inhibition of the enzymatic breakdown reaction of polysaccharides (
p>0.050), further suggesting that the inhibitory effect might be more related to the amino acid sequences. Previous studies have reported that α-amylase and α-glucosidase inactivation by peptides is correlated with the concentration of hydrophobic residues
(Wei et al., 2022; Fu et al., 2023).
Several authors report a significant increase in the inhibitory activity of α-glucosidase/α-amylase by protein fractions isolated from different legumes after hydrolysis (
Rivero-Pino et al., 2021;
Arnal et al., 2024).
