Antioxidant and Chelating Activity of Nontoxic <i>Jatropha curcas</i> L. Protein Hydrolysates Produced by <i>In Vitro</i> Digestion Using Pepsin and Pancreatin

Gallegos Tintoré, Santiago; Torres Fuentes, Cristina; Solorza Feria, Javier; Alaiz, Manuel; Girón Calle, Julio; Martínez Ayala, Alma Leticia; Chel Guerrero, Luis; Vioque, Javier

doi:https://doi.org/10.1155/2015/190129

Journal of Chemistry

On this page

Abstract Introduction Materials and Methods Results and Discussion Conclusions Abbreviations Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2015 | Article ID 190129 | https://doi.org/10.1155/2015/190129

Antioxidant and Chelating Activity of Nontoxic Jatropha curcas L. Protein Hydrolysates Produced by In Vitro Digestion Using Pepsin and Pancreatin

Santiago Gallegos Tintoré,^1,2Cristina Torres Fuentes,³Javier Solorza Feria,¹Manuel Alaiz,³Julio Girón Calle,³Alma Leticia Martínez Ayala,¹Luis Chel Guerrero,²and Javier Vioque³

Academic Editor: Henryk Kozlowski

Received14 Dec 2014

Accepted24 Feb 2015

Published11 Mar 2015

Abstract

The antioxidant and metal chelating activities in J. curcas protein hydrolysates have been determined. The hydrolysates were produced by treatment of a nontoxic genotype with the digestive enzymes pepsin and pancreatin and then were characterized by fast protein liquid chromatography and reverse phase chromatography. Peptidic fractions with higher radical scavenging activity were analysed by matrix-assisted laser desorption/ionization mass spectrometry. The antioxidant activity was determined by measuring inhibition of the oxidative degradation of β-carotene and by measuring the reactive oxygen species (ROS) in Caco-2 cell cultures. Cu²⁺ and Fe²⁺ chelating activities were also determined. The hydrolysates inhibited the degradation of β-carotene and the formation of ROS in Caco-2 cells. The lower molecular weight peptidic fractions from FPLC had stronger antioxidant activity in cell cultures compared with the hydrolysates, which correlated with a higher content in antioxidant and chelating amino acids. These fractions were characterized by a large presence of peptides with different molecular masses. The hydrolysates exhibited both Cu²⁺ and Fe²⁺ chelating activity. It was concluded that J. curcas is a good source of antioxidant and metal chelating peptides, which may have a positive impact on the economic value of this crop, as a potential source of food functional components.

1. Introduction

Jatropha curcas L., a member of the Euphorbiaceae family, is a drought resistant small tree of significant economic importance, because of its various industrial and medicinal uses [1]. Its seeds are a good source of oil that can be used as a diesel substitute. Its oil is also used for manufacturing pharmaceuticals and cosmetics in tropical countries. Although Jatropha cake byproduct obtained after oil extraction is rich in proteins, it is not particularly used for feeding animals because of its toxicity, which is attributed to the presence of phorbol esters [2]. However, nontoxic J. curcas genotypes that do not contain phorbol esters have been reported in Mexico [3], where traditional dishes are elaborated using the roasted fruits of these nontoxic genotypes.

The production of protein isolates has been proposed as a method to reduce antinutritional and toxic factors [4]. Protein isolates can be further processed in order to produce protein hydrolysates that have better functional and nutritional properties [5]. In addition, protein hydrolysates are also a source of bioactive peptides that may have beneficial biological effects [6]. Thus, bioactive peptides with antihypertensive, immunomodulatory, opioid, antioxidant, hypocholesterolemic, and metal chelating activity have been described [7].

The chelating properties of bioactive peptides have been related with their antioxidant activity [8]. Various metabolic pathways produce free radical species in living tissues. When these free radicals are generated in excess, or when cellular defences are deficient, biomolecules may be damaged by oxidative stress, a phenomenon that seems to be involved in cellular aging, cardiovascular diseases, cancer, and neurological degenerative diseases [9]. Oxidative reactions may also be responsible for alterations in foods. Thus, unsaturated fatty acids are prone to suffer peroxidative degradation, resulting in adverse effects on nutritional and functional properties. Lipid peroxidation products may even have toxic effects on mammalian cells [10].

Although copper and iron are essential cofactors for a variety of enzymes, their excess may also have deleterious prooxidant effects in vivo, by promoting the formation of reactive oxygen species. In addition, they also promote oxidative reactions in foods that have a negative impact on the flavour, texture, nutritive value, and shelf life of food products. Chelating peptides may constitute a powerful tool for preventing these oxidative processes, both in foods and in vivo [11]. Among chelating peptides, phosphopeptides derived from milk proteins have been the best characterized. The positive effect of these peptides on the in vivo and in vitro absorption of minerals such as calcium, zinc, or iron has been reported [12, 13].

The aim of this work was to investigate the antioxidant and chelating properties of J. curcas protein hydrolysates obtained from a nontoxic genotype in order to determine their possible use for producing bioactive peptides that could be used for manufacturing of functional foods. In addition, because pepsin and pancreatin are involved in gastrointestinal digestion in human, the results might provide valuable information on the production of bioactive peptides with antioxidant and metal chelating properties, during physiological digestion of J. curcas proteins.

2. Materials and Methods

2.1. Materials

J. curcas seeds from the August-September 2009 harvest in Huitzilán, Puebla (Mexico), were used to prepare protein hydrolysates. Porcine pepsin and pancreatin, butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP), 2,4,6-trinitrobenzenesulfonic (TNBS) acid, β-carotene, linoleic acid, and Tween 20 were from Sigma (St. Louis, MO); Hanks’ balanced salt solution (HBSS), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum, penicillin-streptomycin, and nonessential amino acids solution were purchased from Gibco products (Grand Island, NY). All other reagents were of analytical grade.

2.2. Preparation of Dehulled Defatted Meal and Proximate Composition

J. curcas seeds were dehulled by hand using pliers, and the resulting kernels were ground using a domestic blender. Flour was sifted through a 20-mesh screen and then defatted by Soxhlet hexane extraction for 11 hours.

Proximate composition of kernels was determined by AOAC methods [14]: moisture (934.01), fat (920.39), ash (923.03), and crude fibre (962.09). Protein was calculated as nitrogen × 6.25 and carbohydrate content was estimated as nitrogen-free extract (NFE).

2.3. Preparation of Protein Isolate

The protein isolate was obtained according to Hoover et al. [15] with modifications. A suspension of dehulled defatted meal in water pH 10.5, 1 : 6 (w/v) was stirred for 1 h at room temperature. The suspension was then filtered twice through an 80-mesh screen, and then the solids on the screen were washed using distilled water. The filtrate was strained through a 150-mesh wet screen in order to get rid of the finest particles that still remained in suspension. Starch was eliminated by centrifugation at 487 ×g for 15 minutes, and proteins in the supernatant were precipitated by acidifying the alkaline extract to pH 4.5 using 1 M HCl. Proteins were recovered by centrifugation at 7796 ×g for 15 minutes at 5°C and then freeze-dried.

2.4. Preparation of J. curcas Protein Hydrolysates

J. curcas protein hydrolysates were prepared according to Megías et al. [12] using a hydrolysis reactor vessel equipped with stirrer, thermometer, and pH electrode. Freeze-dried protein isolate/water (5% w/v) were digested at 37°C using pepsin at pH 2.5 for 60 minutes and then with pancreatin at pH 7.5 for 120 minutes. A 1/20 (w/w) enzyme to substrate ratio was used for both enzymes. Aliquots were taken at different times and hydrolysis was stopped by heat inactivation at 80°C for 20 minutes. The resulting hydrolysates were clarified by centrifugation at 11227 ×g for 30 minutes at 5°C, frozen at −20°C, and stored at the same temperature until further use.

2.5. Protein Determination

Protein content (g kg⁻¹) in protein isolate and hydrolysates was determined by elemental analysis as % nitrogen content × 6.25 using a LECO CHNS-932 analyzer (St. Joseph, MI).

2.6. Degree of Hydrolysis

The degree of hydrolysis was calculated by measuring the free amino groups () by reaction with TNBS [16]. The total number of amino groups () was determined in samples 100% hydrolyzed by treatment with 6 N HCl at 110°C for 24 h.

The degree of hydrolysis was calculated using the formula % DH = (h/h_t) × 100.

2.7. Amino Acid Analysis

Amino acid analysis was carried out by acid hydrolysis and HPLC, after derivatization with diethyl ethoxymethylenemalonate, according to the method described by Alaiz et al. [17] using D,L-α-aminobutyric acid as internal standard.

2.8. Antioxidant Activity: β-Carotene Bleaching Method

Antioxidant activity was determined by using the β-carotene bleaching method, with modifications as described [18]. A mixture of β-carotene (1 mL, 10 mg/mL in chloroform), linoleic acid (20 mg), and Tween 20 (200 mg) was vigorously stirred by vortexing. After removal of chloroform under a stream of nitrogen, a clear solution was obtained by mixing in oxygen-sparged distilled water (50 mL). Protein hydrolysates (500 μg) and β-carotene/linoleic acid/Tween 20 solution (200 μL) were added to wells in a 96-well plate and incubated at 50°C in the dark. Negative controls consisted of incubations containing the β-carotene/linoleic acid/Tween 20 solution, in the absence of hydrolysates. Positive controls consisted of incubations containing the β-carotene/linoleic acid/Tween 20 solution and 10 μg of BHT. The peroxidative degradation of β-carotene was monitored by recording absorbance at 450 nm for 135 min using a microplate reader.

2.9. Copper and Iron Chelating Activity

Cu²⁺ chelating activity was determined using the pyrocatechol violet reagent according to Saiga et al. [19]. Hydrolysates (500 μg protein) were added to 96-well plates containing 250 μL of 50 mM sodium acetate pH 6.0, 6.25 μL of 4 mM pyrocatechol violet, and Cu (1 μg, CuSO₄). EDTA (50 μg) was used as a positive control. Absorbance at 632 nm was determined after incubation for 1 minute at room temperature. Copper chelating activity was calculated as follows:

Fe²⁺ chelating activity was determined by measuring the formation of the Fe²⁺-ferrozine complex according to Carter [20]. Hydrolysates (500 μg protein) were added to 96-well plates containing 250 μL 100 mM sodium acetate buffer pH 4.9 and 25 μL FeCl₂ solution (0.1 mg/mL water). EDTA (50 μg) was used as a positive control. Absorbance at 562 nm was determined after addition of a ferrozine solution (12.5 μL, 40 mM in water) and incubation for 30 minutes at room temperature. Iron chelating activity was calculated as follows:

2.10. Fast Protein Liquid Chromatography (FPLC) and Reverse Phase-HPLC

FPLC was carried out in an AKTA-purifier system equipped with a Superdex Peptide column (GE Healthcare); bed dimensions were 10 × 300–310 mm. Injection volume was 200 μL (10 mg protein hydrolysate/mL) and elution was carried out using 0.75 M ammonium bicarbonate at 1 mL/min. Elution was monitored at 214 nm and molecular mass was estimated using molecular weight standards from Pharmacia: blue dextran (2000000 Da), cytochrome c (12500 Da), aprotinin (6512 Da), bacitracin (1450 kDa), cytidine (246 Da), and glycine (75 Da).

RP-HPLC was carried out by injecting hydrolysates (20 μL, 2 μg/μL) in a Discovery BIO Wide Pore C₁₈ column (25 cm × 4.6 mm, 5 μm) (Supelco), using a Beckman-Coulter HPLC system. A linear gradient acetonitrile 0.1% trifluoroacetic acid : water 0.1% trifluoroacetic acid, 0 : 100 to 30 : 70 (v/v) in 60 minutes, at a flow of 1 mL/min and 25°C, was used for elution. The eluent was monitored for absorbance at 215 nm.

2.11. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Analysis

RP-HPLC fractions that exhibited strong radical scavenging activity were subjected to MALDI-TOF analysis using a QSTAR XL quadruple time-of-flight mass spectrometer. The samples were dissolved in 600 μL of acetonitrile at 20% (v/v). To enhance ionization, the matrix α-cyano-4-hydroxycinnamic acid (a-cyano, CHCA) was used in a 1 : 1 matrix : sample relation. Samples were analyzed in positive ion reflector mode in a range of 500–3000 Da.

2.12. Antioxidant Activity in Caco-2 Cells

Antioxidant activity in Caco-2 cells was determined according to Wolfe and Riu [21] with modifications. Caco-2 cells from the European Collection of Cell Cultures were grown in DMEM, supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO₂. Cells were seeded on 96-well microplates (2 × 10⁴ cells/well). Twenty-four hours after seeding, growth medium was removed and cells were washed with PBS pH 7.4 before incubation for 1 hour in HBSS (100 μL/well), containing protein hydrolysates (100, 200, or 500 μg protein) and DCFH-DA, 25 μM. After incubation with the hydrolysates and DCFH-DA, wells were washed using PBS (100 μL), and HBSS (100 μL) containing 600 μM of ABAP was added. Fluorescence (excitation at 485 nm and emission at 555 nm) was measured after 120 min. Each plate included positive control wells, which consisted of cells treated with DCFH-DA and free radical generator ABAP in the absence of hydrolysates, and negative control wells, which consisted of cells treated with DCFH-DA in the absence of hydrolysates and ABAP. The decrease in cellular fluorescence (fluorescence units), when compared to the control cells, indicated the antioxidant capacity of the hydrolysates.

2.13. Statistical Analysis

A one-way analysis of variance and Fisher’s LSD test were used to determine statistically significant differences (), using Statgraphics plus version 5.1.

3. Results and Discussion

3.1. Kernels Proximate Composition and Production of J. curcas Protein Hydrolysates

Protein content of Jatropha kernels ( g kg⁻¹) was similar to that reported for other J. curcas genotypes from Mexico (311 g kg⁻¹, for flour from Papantla, Veracruz; 321 g kg⁻¹, for flour from Yautepec, Morelos). Devappa and Swamylingappa [4] reported 248 g kg⁻¹ for flour from Karnataka, India. Ash content was g kg⁻¹, similar to that reported for different genotypes from Mexico (47, 51 g kg⁻¹) and India (41 g kg⁻¹); crude fat was 618.7 g kg⁻¹, slightly higher than other Mexican genotypes (577 g kg⁻¹ Papantla, Veracruz; 553 g kg⁻¹ Yautepec, Morelos) [2]; however, Devappa and Swamylingappa [4] reported that Jatropha kernel contained 400–600 g oil kg⁻¹. Crude fiber ( g kg⁻¹) was also similar to the content (41.6 g kg⁻¹) reported by these authors.

Preparation of J. curcas protein isolate yielded a protein concentration of g kg⁻¹, which is similar to that reported by Makkar et al. [1]. Treatment with pepsin for 40 minutes yielded a 6.8% degree of hydrolysis, which did not increase any further during treatment for up to 60 minutes (Figure 1). This result is consistent with previous reports on the use of pepsin to hydrolyze seed proteins [22]. Thus, Torres-Fuentes et al. [23] reported a 9% degree of hydrolysis after treating chickpea protein with pepsin for 60 minutes. Quist et al. [24] obtained a roasted peanut hydrolysate with 9.6% degree of hydrolysis after 30 minutes of pepsin treatment. The final J. curcas protein hydrolysate that was produced by treatment with pepsin for 60 minutes, followed by pancreatin for 120 minutes, had 31.5% degree of hydrolysis (Figure 1), which is similar to that reported by Megías et al. [22] for sunflower protein isolates (37%).

Pepsin is the main enzyme generated in the human stomach during food digestion; it has endopeptidase activity and is most efficient in cleaving peptide bonds between hydrophobic and preferably aromatic amino acids, such as Phe, Tyr, and Trp. Pancreatin includes proteases, such as trypsin or chymotrypsin, released by the pancreas in the small bowel. Trypsin is an endopeptidase that is more efficient in cleaving peptide bonds between basic amino acids, such as Arg and Lys; chymotrypsin is an endopeptidase which is more efficient in cleaving peptide bonds between aromatic amino acids, such as Tyr, Phe, and Trp [12, 25–27].

3.2. Fast Protein Liquid Chromatography and Reversed Phase Chromatography

The most prominent feature in the J. curcas protein isolate as determined by FPLC was a major protein fraction with an apparent molecular weight of kDa, corresponding to nonhydrolysed protein, that eluted with the void volume; there were also two species detected with negligible presence and lower molecular weight (Figure 2), probably related to the degree of hydrolysis in the protein isolate (2.7%). Lower molecular weight fractions appeared in the hydrolysates obtained by treatment with pepsin and pancreatin. Thus, in addition to a common fraction of ~ kDa, hydrolysis for 20, 60, 80, and 180 minutes yielded hydrolysates with major peptidic fractions, with apparent molecular weight of , , , and kDa, respectively. Chromatographic analysis using RP-HPLC revealed that the hydrolysates were made up of a complex mixture of peptides (Figure 3(a)). Hydrolysis with pepsin for 20 and 40 minutes resulted in similar peptidic profiles. Additional hydrolysis using pancreatin resulted in increasing amounts of more hydrophilic peptides that elute between 10 and 50 minutes and disappearance of hydrophobic peptides with elution times higher than 50 minutes (Figure 3(a)).

(a)

(b)

The chromatogram corresponding to 180 min hydrolysate, as shown in Figure 2, was divided into five fractions (A to E), corresponding to peptides eluted between 7.9 and 11.2 mL (fraction A), 11.2 and 14.5 mL (fraction B), 14.5 and 18.6 mL (fraction C), 18.6 and 20.5 mL (fraction D), and 20.8 and 23.5 mL (fraction E). Antioxidant activity was determined using DPPH radical, detecting the highest radical scavenging activity in C, D, and E fractions (data not shown). Antioxidant activity in Caco-2 cells was determined in the lower molecular weight fractions C, D, and E, testing 50 μg of protein.

3.3. MALDI-TOF-MS Analysis

RP-HPLC fractions that exhibited strong radical scavenging activity (Figure 3(a) marked with circles) were subjected to MALDI-TOF analysis. The peptidic fraction obtained at 5–10 min of elution time in RP-HPLC analysis (Figure 3(b)) contains several peptides with different molecular masses: a peptide with high presence and molecular mass of 1134 Da was detected, followed by two more peptides with moderate presence and molecular masses of 1025, 1083 Da. The peptidic fraction obtained at 20–25 min of elution time showed a high quantity of peptides with different molecular masses (669 to 2313 Da). These peptides are present in fractions D and E from FPLC (Figure 3(a)). The results were in concert with the general finding that short peptides with 2–10 amino acids exert greater antioxidant potential and other bioactive properties than their parent native proteins or large polypeptides [28].

3.4. Antioxidant Activity: β-Carotene Bleaching Method

Antioxidant activity in the J. curcas protein hydrolysates increased with degree of hydrolysis (Figure 4); this was probably because the enzyme hydrolysis may have unfolded the native protein and exposed amino acid residues, which were electron donors and could react with free radicals, convert them to more stable products, and terminate the free radical chain reaction [29]. The highest antioxidant activity was observed in samples produced by hydrolysis from 60 to 180 minutes (treatment with pepsin for 60 minutes and 0 to 120 minutes additional treatment with pancreatin). The inhibition of the oxidative decomposition of β-carotene by these samples was higher than the inhibition caused by the negative control and by the samples that were generated by hydrolysis for 5 to 40 minutes; further treatment with pancreatin did not result in higher antioxidant activity. The activity of these hydrolysates at a concentration of 500 μg/well as shown in Figure 4 was similar to the activity of 10 μg BHT/well. These results are comparable to those reported for sunflower protein hydrolysates produced using pepsin and pancreatin [12].

3.5. Metal Chelating Activity

Hydrolysis of the J. curcas protein isolates results in an increasing copper chelating activity as shown in Figure 5(a). This activity roughly correlated with the degree of hydrolysis and reached a maximum in the hydrolysates produced by treatment with pepsin for 60 minutes, followed by treatment with pancreatin for 100 to 120 minutes, which chelated 78% of the copper ion in the assay. Thus, protein hydrolysis led to an enhanced Cu²⁺ chelating ability, which may be due to an increased concentration of carboxylic acid groups. These groups promote the conversion of proteins into an anionic form, which facilitates chelation of Cu²⁺ also, an increased exposure of metal chelating amino acid residues, such as histidine, which is a well-known metal chelating amino acid [29, 30]. It is worth mentioning that J. curcas protein isolate is a good source of histidine (29–35 g kg⁻¹) [1, 31]. Another reason could be a combination of high His content and small peptide size in the hydrolysates [23].

(a)

(b)

Treatment with pepsin and pancreatin also led to an increase in iron chelating activity although the maximum iron chelating activity, which corresponded to the hydrolysates produced by treatment with pepsin for 60 minutes, followed by treatment with pancreatin for at least 20 minutes, was about three times lower than the maximum copper chelating activity (Figures 5(a) and 5(b)). Peng et al. [29] have also noted that whey protein hydrolysates have a far weaker Fe²⁺ chelating capacity than Cu²⁺ chelating capacity. Kong and Xiong [32] mentioned that this behavior may be related to the higher coordination number of Fe²⁺ (thus, more chelators required) than in Cu²⁺.

3.6. Antioxidant Activity in Caco-2 Cell Cultures

In order to assess whether the hydrolysates may have antioxidant activity “in vivo,” they were assayed using Caco-2 cells treated with ABAP as a free radical generator and DCFH-DA as an indicator of intracellular reactive oxygen species. Treatment with ABAP causes oxidative stress, resulting in the generation of reactive oxygen species. DCFH-DA is taken up by cells and cleaved by nonspecific esterases to form DCFH, which can be oxidized to the highly fluorescent derivative DCF by reactive oxygen species. The hydrolysates produced by treatment solely with pepsin for 60 minutes, or by treatment with pepsin for 60 minutes followed by pancreatin for 120 minutes, were used for these experiments. The former showed antioxidant effect, that was not dependent on protein concentration, while the latter showed a statistically significant () antioxidant effect that was similar at 100 and 200 μg/well and higher at 500 μg/well (Figures 6(a) and 6(b)).

(a)

(b)

(c)

Figure 6

Antioxidant activity of increasing concentrations of J. curcas hydrolysates in Caco-2 cell cultures treated with the free radical generator ABAP. Formation of reactive oxygen species was determined by oxidation of the fluorescent dye DCFH. Antioxidant effect of hydrolysate produced by treatment solely with pepsin for 60 minutes (a) or treatment with pepsin for 60 minutes followed by treatment with pancreatin for 120 minutes (b). (c) FPLC fractions C, D, and E (50 μg protein) from 180 min hydrolysate. Values are the mean of data from five wells ± standard deviation. Experiments were conducted in duplicate on two different days. Different letters indicate significant differences ().

Factors other than intrinsic antioxidant activity may have a great impact in the intracellular antioxidant effect of the hydrolysates, as compared to simpler chemical assays. Thus, cell permeability and susceptibility to hydrolysis by cellular exo- and endoproteases could limit or completely prevent antioxidant peptides from getting within cells.

The highest antioxidant activity was detected in fractions D and E (Figure 6(c)); these were especially rich in tyrosine, phenylalanine, arginine, and leucine (Table 1). It has been reported that histidine, tyrosine, and leucine residues are abundant in antioxidant peptides [33]; arginine has also been related to antioxidant activity [12, 34]. Recent studies have shown that tripeptides consisting of Trp or Tyr residues at the C-terminal and Phe at their N-terminal had strong free radical scavenging activity and inhibition of peroxidation of linoleic acid [35]. Moreover, antioxidant peptides often include hydrophobic amino acid residues such as Val or Leu at the N-terminus of the peptides and Pro, His, Tyr, Trp, Met, and Cys in their sequences [36].

Apart from the amino acid composition, the molecular weight of peptides may also determine their antioxidant activity [33]. The majority of the antioxidant peptides derived from food sources have molecular weights ranging from 500 to 1800 Da [36]. For example, in corn gluten meal hydrolysates, the antioxidant activity of peptides with a MW between 500 and 1500 Da was higher than that of peptides above 1500 Da and below 500 Da [33]. This could explain why fraction D had a stronger antioxidant activity when compared with fraction E.

4. Conclusions

This work has proven the presence of naturally occurring antioxidant and chelating peptides in nontoxic J. curcas seeds. These peptides were released from the native proteins by in vitro treatment, with the digestive enzymes pepsin and pancreatin, which mimics physiological digestion of proteins. Treatment with pepsin for one hour followed by treatment with pancreatin for two hours yielded the highest antioxidant and metal chelating properties. The antioxidant activity was determined using a cell culture system, supporting the view that these bioactive peptides might have activity in vivo. J. curcas protein digestion with gastrointestinal enzymes generates a broad spectrum of antioxidant peptides of different molecular sizes. This information might increase the economic value of J. curcas defatted meal from nontoxic J. curcas genotypes, which could be used for producing functional food components.

Abbreviations

ABAP:	2, 2′-Azobis(2-amidinopropane) dihydrochloride
AOAC:	Association of Official Analytical Chemists
BHT:	Butylated hydroxytoluene
DCFH-DA:	2′, 7′-Dichlorofluorescin diacetate
DCFH:	Intracellular 2′, 7′-dichlorofluorescin
DCF:	Fluorescent dichlorofluorescin
DMEM:	Dulbecco’s modified Eagle medium
DPPH:	2,2-Diphenyl-1-picrylhydrazyl
DH:	Degree of hydrolysis
EDTA:	Ethylenediaminetetraacetic acid
FPLC:	Fast protein liquid chromatography
HBSS:	Hanks’ balanced salt solution
HPLC:	High performance liquid chromatography
kDa:	Kilodaltons
MALDI-TOF:	Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
MW:	Molecular weight
PBS:	Phosphate buffer saline
RP-HPLC:	Reverse phase-high performance liquid chromatography
ROS:	Reactive oxygen species
TNBS:	2, 4, 6-Trinitrobenzenesulfonic acid.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by the Mexican National Council of Science and Technology (CONACYT), the National Polytechnic Institute (IPN), the Spanish Ministry of Innovation and Science, through Research Project AGL2007-63580, and the European Union through FEDER funds. Thanks are due to María Dolores García-Contreras and Alicia Chagolla for technical assistance.

References

H. P. S. Makkar, G. Francis, and K. Becker, “Protein concentrate from Jatropha curcas screw-pressed seed cake and toxic and antinutritional factors in protein concentrate,” Journal of the Science of Food and Agriculture, vol. 88, no. 9, pp. 1542–1548, 2008.
View at: Publisher Site | Google Scholar
J. Martínez-Herrera, P. Siddhuraju, G. Francis, G. Dávila-Ortíz, and K. Becker, “Chemical composition, toxic/antimetabolic constituents, and effects of different treatments on their levels, in four provenances of Jatropha curcas L. from Mexico,” Food Chemistry, vol. 96, no. 1, pp. 80–89, 2006.
View at: Publisher Site | Google Scholar
H. P. S. Makkar, J. Martínez-Herrera, and K. Becker, “Variations in seed number per fruit, seed physical parameters and contents of oil, protein and phorbol ester in toxic and non-toxic genotypes of Jatropha curcas,” Journal of Plant Sciences, vol. 3, no. 4, pp. 260–265, 2008.
View at: Publisher Site | Google Scholar
R. K. Devappa and B. Swamylingappa, “Biochemical and nutritional evaluation of Jatropha protein isolate prepared by steam injection heating for reduction of toxic and antinutritional factors,” Journal of the Science of Food and Agriculture, vol. 88, no. 5, pp. 911–919, 2008.
View at: Publisher Site | Google Scholar
A. Villanueva, J. Vioque, R. Sánchez et al., “Peptide characteristics of sunflower protein hydrolysates,” Journal of the American Oil Chemists' Society, vol. 76, no. 12, pp. 1455–1460, 1999.
View at: Publisher Site | Google Scholar
J. Vioque, R. Sánchez-Vioque, A. Clemente, J. Pedroche, M. M. Yust, and F. Millán, “Péptidos bioactivos en proteínas de reserva,” Grasas y Aceites, vol. 51, no. 5, pp. 361–365, 2000.
View at: Google Scholar
M. Gobbetti, L. Stepaniak, M. De Angelis, A. Corsetti, and R. Di Cagno, “Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing,” Critical Reviews in Food Science and Nutrition, vol. 42, no. 3, pp. 223–239, 2002.
View at: Publisher Site | Google Scholar
D. D. Kitts, “Antioxidant properties of caseinphosphopeptides,” Trends in Food Science and Technology, vol. 16, no. 12, pp. 549–554, 2005.
View at: Publisher Site | Google Scholar
V. A. Tironi and M. C. Añón, “Amaranth proteins as a source of antioxidant peptides: effect of proteolysis,” Food Research International, vol. 43, no. 1, pp. 315–322, 2010.
View at: Publisher Site | Google Scholar
Y. Li, B. Jiang, T. Zhang, W. Mu, and J. Liu, “Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH),” Food Chemistry, vol. 106, no. 2, pp. 444–450, 2008.
View at: Publisher Site | Google Scholar
C. Megías, J. Pedroche, M. Yust et al., “Affinity purification of copper chelating peptides from chickpea protein hydrolysates,” Journal of Agricultural and Food Chemistry, vol. 55, no. 10, pp. 3949–3954, 2007.
View at: Publisher Site | Google Scholar
C. Megías, J. Pedroche, M. M. Yust et al., “Production of copper-chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin,” LWT: Food Science and Technology, vol. 41, no. 10, pp. 1973–1977, 2008.
View at: Publisher Site | Google Scholar
H. Korhonen and A. Pihlanto, “Bioactive peptides: production and functionality,” International Dairy Journal, vol. 16, no. 9, pp. 945–960, 2006.
View at: Publisher Site | Google Scholar
Association of Official Analytical Chemists (AOAC), Official Methods of Analysis, Association of Official Analytical Chemists, Washington, DC, USA, 15th edition, 1997.
R. Hoover, S. Rorke, and A. Martin, “Isolation and characterization of Lima bean (Phaseolus lunatus) starch,” Journal of Food Biochemistry, vol. 15, no. 2, pp. 117–136, 1991.
View at: Publisher Site | Google Scholar
J. Adler-Nissen, “Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid,” Journal of Agricultural and Food Chemistry, vol. 27, no. 6, pp. 1256–1262, 1979.
View at: Publisher Site | Google Scholar
M. Alaiz, J. L. Navarro, J. Giron, and E. Vioque, “Amino acid analysis of high-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate,” Journal of Chromatography, vol. 591, no. 1-2, pp. 181–186, 1992.
View at: Publisher Site | Google Scholar
G. J. Marco, “A rapid method for evaluation of antioxidants,” Journal of the American Oil Chemists' Society, vol. 45, no. 9, pp. 594–598, 1968.
View at: Publisher Site | Google Scholar
A. Saiga, S. Tanabe, and T. Nishimura, “Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment,” Journal of Agricultural and Food Chemistry, vol. 51, no. 12, pp. 3661–3667, 2003.
View at: Publisher Site | Google Scholar
P. Carter, “Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine),” Analytical Biochemistry, vol. 40, no. 2, pp. 450–458, 1971.
View at: Publisher Site | Google Scholar
K. L. Wolfe and H. L. Rui, “Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements,” Journal of Agricultural and Food Chemistry, vol. 55, no. 22, pp. 8896–8907, 2007.
View at: Publisher Site | Google Scholar
C. Megías, M. Yust, J. Pedroche et al., “Purification of an ACE Inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates,” Journal of Agricultural and Food Chemistry, vol. 52, no. 7, pp. 1928–1932, 2004.
View at: Publisher Site | Google Scholar
C. Torres-Fuentes, M. Alaiz, and J. Vioque, “Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates,” Food Chemistry, vol. 129, no. 2, pp. 485–490, 2011.
View at: Publisher Site | Google Scholar
E. E. Quist, R. D. Phillips, and F. K. Saalia, “The effect of enzyme systems and processing on the hydrolysis of peanut (Arachis hypogaea L.) protein,” LWT—Food Science and Technology, vol. 42, no. 10, pp. 1717–1721, 2009.
View at: Publisher Site | Google Scholar
R. A. da Silva Gomes, R. Pedutti Batista, A. C. de Almeida, D. N. da Fonseca, L. Juliano, and V. Hial, “A fluorimetric method for the determination of pepsin activity,” Analytical Biochemistry, vol. 316, no. 1, pp. 11–14, 2003.
View at: Publisher Site | Google Scholar
J. E. Folk and P. W. Cole, “Chymotrypsin C. II. Enzymatic specificity toward several polypeptides,” The Journal of biological chemistry, vol. 240, pp. 193–197, 1965.
View at: Google Scholar
F. Sanger and H. Tuppy, “The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates,” The Biochemical Journal, vol. 49, no. 4, pp. 481–490, 1951.
View at: Google Scholar
Y. Ma, Y. L. Xiong, J. Zhai, H. Zhu, and T. Dziubla, “Fractionation and evaluation of radical scavenging peptides from in vitro digests of buckwheat protein,” Food Chemistry, vol. 118, no. 3, pp. 582–588, 2010.
View at: Publisher Site | Google Scholar
X. Peng, B. Kong, X. Xia, and Q. Liu, “Reducing and radical-scavenging activities of whey protein hydrolysates prepared with Alcalase,” International Dairy Journal, vol. 20, no. 5, pp. 360–365, 2010.
View at: Publisher Site | Google Scholar
A. Pihlanto, “Antioxidative peptides derived from milk proteins,” International Dairy Journal, vol. 16, no. 11, pp. 1306–1314, 2006.
View at: Publisher Site | Google Scholar
S. Gallegos-Tintoré, C. Torres-Fuentes, A. L. Martínez-Ayala et al., “Antioxidant and chelating activity of Jatropha curcas L. protein hydrolysates,” Journal of the Science of Food and Agriculture, vol. 91, no. 9, pp. 1618–1624, 2011.
View at: Publisher Site | Google Scholar
B. Kong and Y. L. Xiong, “Antioxidant activity of zein hydrolysates in a liposome system and the possible mode of action,” Journal of Agricultural and Food Chemistry, vol. 54, no. 16, pp. 6059–6068, 2006.
View at: Publisher Site | Google Scholar
B. H. Sarmadi and A. Ismail, “Antioxidative peptides from food proteins: a review,” Peptides, vol. 31, no. 10, pp. 1949–1956, 2010.
View at: Publisher Site | Google Scholar
M. N. Maillard, C. Billaud, Y. N. Chow, C. Ordonaud, and J. Nicolas, “Free radical scavenging, inhibition of polyphenoloxidase activity and copper chelating properties of model Maillard systems,” LWT—Food Science and Technology, vol. 40, no. 8, pp. 1434–1444, 2007.
View at: Publisher Site | Google Scholar
J. M. Broncano, J. Otte, M. J. Petrón, V. Parra, and M. L. Timón, “Isolation and identification of low molecular weight antioxidant compounds from fermented ‘chorizo’ sausages,” Meat Science, vol. 90, no. 2, pp. 494–501, 2012.
View at: Publisher Site | Google Scholar
A. G. P. Samaranayaka and E. C. Y. Li-Chan, “Food-derived peptidic antioxidants: a review of their production, assessment, and potential applications,” Journal of Functional Foods, vol. 3, no. 4, pp. 229–254, 2011.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2015 Santiago Gallegos Tintoré et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2090

Downloads

1136

Citations