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Stability of sunflower protein hydrolysates in simulated gastric and intestinal fluids and Caco-2 cell extracts
LWT - Food Science and Technology 42 (2009) 1496–1500
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Stability of sunflower protein hydrolysates in simulated gastric and intestinal fluids and Caco-2 cell extracts Cristina Megı´as, Justo Pedroche, Marı´a del Mar Yust, Manuel Alaiz, Julio Giro´n-Calle, Francisco Milla´n, Javier Vioque* Instituto de la Grasa (CSIC), Padre Garcı´a Tejero 4, 41012-Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2008 Received in revised form 6 March 2009 Accepted 30 April 2009
The in vitro stability of bioactive properties in sunflower protein hydrolysates and purified peptides was studied by using simulated gastric fluid, simulated intestinal fluid, and Caco-2 cells extracts. Protein hydrolysates that inhibit the angiotensin converting enzyme and copper chelating peptides produced by hydrolysis with the microbial protease alcalase were partially resistant to incubation with simulated gastric and intestinal fluids. These hydrolysates and others produced by hydrolysis using pepsin plus pancreatin were also partially resistant to incubation with Caco-2 cells extracts. In addition, the ACE inhibitory peptide FVNPQAGS that is generated by extensive hydrolysis using pepsin and pancreatin, was found to be resistant to hydrolysis by Caco-2 cells extracts. These results suggest that stability in the digestive tract should not be a problem for the bioavailability of these bioactive peptides. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Sunflower Protein hydrolysate Bioactive peptides Gastrointestinal fluids Caco-2
1. Introduction Food proteins are increasingly been recognized as a source of beneficial bioactive peptides that are released during physiological digestion. A variety of bioactive peptides derived from plant and animal food products with positive effects on the immune, nervous, digestive and vascular systems have been described (Vioque et al., 2000). In addition to be absorbed by the digestive tract, these peptides have to be resistant to digestive proteases and peptidases in order to have a physiological effect. A variety of sunflower bioactive hydrolysates and peptides have been described in our laboratory, including angiotensin converting enzyme (ACE) inhibitory peptides (Megı´as et al., 2006, 2004), and metal chelating peptides (Megı´as et al., 2007, 2008). These peptides have been produced by hydrolysis of sunflower seed protein isolates using microbial (alcalase and flavourzyme) or digestive (pepsin and pancreatin) enzymes, and their bioactivity has been determined using in vitro methods. However, the bioavailability of these peptides remains to be determined whether these bioactive peptides are stable enough to hydrolysis by digestive proteases and peptidases in order to be susceptible to
* Corresponding author. Tel.: þ34 954 611 550; fax: þ34 954 616 790. E-mail addresses: [email protected], [email protected] (J. Vioque). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.04.008
be absorbed and reach the cardiovascular system in active form. In addition to loss of activity, gastrointestinal digestion may actually lead to generation of more potent bioactive peptides. Thus, an increase in the ACE inhibitory activity of fermented casein solutions was observed after incubation with digestive enzymes (Vermeirssen, Van Camp, Decroos, Van Wijmelbeke, & Verstraete, 2003). In the present work, the stability the ACE inhibitory activity and the copper chelating activity in sunflower protein hydrolysates and purified peptides has been studied using simulated gastric fluid, simulated intestinal fluid, and Caco-2 cells extracts as in vitro models of physiological digestion.
2. Material and methods 2.1. Materials Deffated sunflower meal was supplied by MIGASA (Sevilla, Spain). Alcalase 2.4L was from NovoNordisk (Bagsvaerd, Denmark). Bradford reagent, hippuryl-histidyl-L-leucine (HHL), 2,4,6-trichloros-triazine (TT, dioxane, pepsin, pancreatin and b-carotene were purchased from Sigma) (Tres Cantos, Madrid, Spain). Dulbeco´s Modified Eagle´s Medium, fetal bovine serum and antibiotics for cell culture were from GIBCO (Barcelona, Spain).
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
80 70 60 50 40 30 20 10 0
SGF 1 h + SIF 3 h
2.2.5. Incubation with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and Caco-2 cells extract SGF and SFI were prepared according to the United States Pharmacopeial Convention Council of Experts (2004), with modifications. The composition was as follows: SFG: 0.00064% pepsin (w/v), 30 mM NaCl, 0.7% (v/v) HCl, The enzyme/substrate ratio was 1/20 (w/w). The incubation was continued for 1 h under shacking at 37 C and pH 1.2. SFI: 0.0005% (w/v) pancreatin, 0.05 M KH2PO4, 19% (v/v) NaOH 0.2 N. The enzyme/substrate ratio was 1/20 (w/w). The incubation was continued for 3 h under shacking at 37 C and pH 7.5.
Treatment of a sunflower protein isolate with the microbial protease alcalase for 5 min yielded a high ACE inhibitory activity in the resulting hydrolysate (Megı´as et al., 2006) This hydrolysate has now been incubated with SGF and SIF in order to determine the stability of the ACE inhibitory activity to digestive proteases and peptidases. As shown in Fig. 1, incubation with SGF did not affect ACE inhibitory activity. Further incubation in SIF did not suppress activity, although it caused a time-dependent decrease in ACE inhibitory activity, resulting in an increase of the IC50 from 20,4 1,2 mg/mL after incubation with SGF, to 68,2 0,95 mg/mL
SGF 1 h + SIF 1 h
2.2.4. Determination of ACE inhibitory activity ACE activity was determined as described by Hayakari, Koudo, and Izomi (1978) with modifications. This method uses HHL as substrate for ACE, and relies on the reaction of the resulting hippuric acid with 2,4,6-trichloro-s-triazina (TT). The assay mixture (0.5 ml) consisted of potassium phosphate buffer pH 8.3 (40 mmol), sodium chloride (300 mmol), HHL (1.5 mmol), and 1 mg of pig lung ACE extract prepared as previously described (Megı´as et al., 2004). Incubation was carried out at 37 C for 15 min and was terminated by addition of TT in dioxane (1.5 ml 3% w/v), followed by 0.2 M phosphate buffer pH 8.3 (3 ml). Absorbance at 382 nm was determined in the supernatant obtained after centrifugation at 10,000 g for 10 min. The IC50 value, defined as the concentration of peptide in mg protein/mL required to produce 50% inhibition of ACE, was determined by regression analysis of ACE inhibition (%) versus peptide concentration. Experiments were ran in triplicate.
3.1. Incubation of sunflower bioactive hydrolysates and purified peptides with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)
SGF 1 h
2.2.3. Purification of chelating peptides and determination of antioxidant activity Sunflower protein hydrolysates with the highest antioxidant activity in the presence of copper were selected for purification of chelating peptides as previously described (Megı´as et al., 2007) using Vivapure Metal Chelate Maxi spin columns (Vivascience, Sartorius, Madrid, Spain) charged with copper. Antioxidant activity was assayed using the b-carotene oxidation method as previously described with modifications (Pedroche et al., 2006). Final assay mixtures contained different concentrations of hydrolysates, b-carotene (119 mM), and Cuþþ (0.1 mM) in a final volume of 200 mL. The degradation of b-carotene was monitored by recording the decrease in absorbance at 470 nm using a microplate reader. Experiments were ran in triplicate.
3. Results and discussion
SGF 0.5 h
2.2.2. Determination of peptides concentration Peptide concentrations of the hydrolysates were determined after amino acid analysis of derivatives according to the method of Alaiz, Navarro, Giro´n, and Vioque (1992). Samples were treated with 4 ml 6 mol/L HCl in tubes sealed under nitrogen for 24 h at 110 C for hydrolysis. Amino acids were determined by highperformance liquid chromatography of the derivatives obtained by reaction with diethyl ethoxymethylenemalonate.
SGF 0 h
2.2.1. Production of sunflower protein hydrolysates Sunflower protein isolates (Sa´nchez-Vioque, Clemente, Vioque, Bautista, & Milla´n, 1999) were used for production of protein hydrolysates using alcalase or pepsin plus pancreatin as previously described (Megı´as et al., 2004; Megı´as et al., 2007). Hydrolysis was stopped by heating at 80 C for 20 min. Hydrolysates were clarified by ultrafiltration through 0.45 mm filters (Millipore, Bedford, MA) and lyophilized for storage at 20 C.
Samples were collected at different times and were inactivated by heating at 80 C for 15 min and them centrifuged. Results are the average SD of three determinations. Caco-2 cells homogenates were prepared according to Augustijns, Annaert, Heylen, Van den Mooter, and Kinget (1998) with modifications. Briefly, Caco-2 cells monolayers were homogenized in 5 mL DMEM by sonication for 10 s. After sonication, samples were centrifuged at 14,000 g for 5 min and the supernatant was recovered. Protein content in the supernatant was determined according to Bradford (1976). Cells were obtained from the European Collection of Cell Cultures and kept at 5% CO2 in Dulbecco´s Modified Eagle´s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. Stability assays in Caco-2 cells extracts were done according to Vermeirssen et al. (2005) by incubation at 37 C for 2 h under shacking using a cell protein to sample protein ratio of 1–6 (w/w). Results are the average SD of three determinations.
IC50
2.2. Methods
1497
Fig. 1. Incubation of an ACE inhibitory sunflower hydrolysate with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 5 min. After incubation in SGF for 1 h, pH was adjusted and pancreatin added in order to carry out incubation in SIF for 3 h. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
0,00008
0,0004
0,00007 0,0003
0,00006 0,0002
Slope (PH)
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SGF 1 h + SIF 3 h
SGF 1 h + SIF 1.5 h
SGF 1 h
0,00004 SGF 0.5 h
0,0001
SGF 0 h
after incubation for 3 more hours in SIF. Direct incubation in SIF did not cause any major change in ACE inhibitory activity (Fig. 2). Similar results concerning stability of ACE inhibitory peptides have been described by other groups. Thus, Wu and Ding (2002) observed that soy derived ACE inhibitory peptides generated with alcalase where resistant to hydrolysis by pepsin and were only partially hydrolyzed by pancreatin. In other studies, the ACE inhibitory activity of peptides of known sequence increased, decreased, or remained unchanged after incubation in SGF and SIF depending on the presence in the peptidic sequence of the specific targets for the digestive proteases (Go´mez, Ramos, & Recio, 2004; Parrot, Degraeve, Curia, & Martial-Gros, 2003). The stability of the copper chelating activity in sunflower hydrolysates generated by treatment with alcalase (Megı´as et al., 2007) during incubation in SGF and SIF has also been studied. The chelating activity of a hydrolysate produced by incubation with alcalase for 30 min increased during incubation in SGF, suggesting that new chelating peptides were being generated. Although subsequent incubation in SIF for 3 h lead to a decrease in chelating activity, activity was not completed suppressed (Fig. 3). The copper chelating peptides in this hydrolysate that were purified by affinity chromatography using immobilized Cuþþ were also found to be fairly stable in incubations with SGF and SIF (Fig. 3). Direct incubation in SIF showed an increase in chelating activity after incubation for 1.5 h in both the alcalase hydrolysate and the corresponding affinity purified fraction (Fig. 4). These results suggest that degradation by physiological proteases and peptidases during digestion should not prevent the sunflower copper chelating peptides from exerting antioxidant effects in vivo. Thus, these peptides could have an antioxidant effect in the intestinal lumen, and if absorbed through the intestinal barrier they could reach the bloodstream and inhibit LDL oxidation (Burkitt, 2001), as well as the progression of certain diseases that have been linked to oxidative processes such as Alzheimer´s disease and Parkinson´s disease (Lin, Xue, Wang, Zhao, & Chen, 2006). It is interesting to note that direct incubation in SIF caused a much lower decrease in ACE-inhibitory activity and copper chelating activity than incubation in SIF following incubation in SGF. Thus, protecting the bioactive peptides by encapsulation in microgels in conditions that facilitate their release in the intestine could greatly increase bioavailability of these peptides.
Slope (CP)
1498
Fig. 3. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min. After incubation in SGF for 1 h, pH was adjusted and pancreatin added in order to carry out incubation in SIF for 3 h. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
3.2. Stability of sunflower bioactive peptides to Caco-2 cells extracts Confluent monolayers of Caco-2 cells, derived originally from a colorectal carcinoma, constitute a model of the normal, healthy intestinal epithelium when they are allowed to grow for a certain time (Engle, Goetz, & Alpers, 1998). Thus, this type of confluent cultures is a very popular in vitro model of the intestinal barrier for studies of absorption of nutrients, bioactive diet components, and drugs (Artursson, Palm, & Lothman, 2001). Caco-2 cells extracts have now been used as an in vitro model to test the stability of the ACE inhibitory activity and the copper chelating activity in sunflower bioactive hydrolysates previously described by us. These studies complement those that were carried out using SGF and SIF. A high ACE inhibitory activity in a sunflower hydrolysate that was produced by incubation of a protein isolate with pepsin for 3 h and pancreatin for another 3 h was previously described. The peptide of sequence FVNPQAGS, corresponding to a fragment of
0,000064
0,0004 100
0,00006
90
Slope (CP)
IC 50
70 60 50
0,000056 0,0002
Slope (PH)
80
0,0003
0,000052
40 30 0,0001
20 10 0
SIF 0 h
SIF 1 h
SIF 2 h
SIF 3 h
Fig. 2. Incubation of an ACE inhibitory sunflower hydrolysate with simulated intestinal fluid (SIF). The hydrolysate, produced by treatment of a sunflower protein isolate with alcalase for 5 min was incubated with SIF for 3 h. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
SIF 0 h
SIF 1.5 h
SIF 3 h
0,000048
Fig. 4. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min, and a peptididc fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
0,0004
100
0,00006
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60
Slope (CP)
120
80
IC 50
1499
40 0
0h
1h
2h
0
20
0
0h
1h
2h
Fig. 5. Incubation of two ACE inhibitory hydrolysates obtained by hydrolysis with alcalase and the ACE inhibitory peptide FVNPQAGS with Caco-2 extracts. The hydrolysates were produced by treatment with alcalase for 5 min (–C– A5), or pepsin for 180 min plus pancreatin for another 180 min (–,– PP360). The FVNPQAGS (–6–) was previously purified from the PP360 hydrolysate. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
0,0001
0,0005
0,00008
0,0004
0,00006
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0,00004
0,0002
0,00002
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incubation with Caco-2 extracts (Fig. 5), indicating that the bioavailability of this peptide is not limited at all by the peptidases produced by the Caco-2 cells. Incubation of the copper chelating hydrolysate produced by treatment with alcalase for 30 min and the corresponding affinity purified fraction (Megı´as et al., 2007) with Caco-2 extracts lead to a reduction of chelating activity of about 50% after 1 h (Fig. 6). The chelating activity was not further reduced, or even was increased, during the second hour of incubation. Similarly, incubation of the hydrolysate produced by treatment with pepsin plus pancreatin and the fraction purified by affinity chromatography from this hydrolysate (Megı´as et al., 2008), showed that the copper chelating activity did not change during the first hour of incubation, but decreased by about 50% by the end of the 2 h incubation (Fig. 7). In conclusion, the bioactive properties of sunflower protein hydrolysates and related fractions that are described in this article have been found to be partially resistant to hydrolysis by SGF, SIF, and Caco-2 extracts, which represent in vitro models of physiological digestion. It is particularly interesting the case of the FVNPQAGS peptide, which is produced by treatment with pepsin and pancreatin, and is resistant to hydrolysis by Caco-2 extracts. This peptide has been shown to be a true inhibitor and not a substrate of ACE (Megı´as et al., 2004).
Acknowledgements
Slope (CP)
Slope (PH)
helianthin, the main storage protein in sunflower seeds, was found in this hydrolysate (Megı´as et al., 2004). Incubation of this hydrolysate and the FVNPQAGS peptide with Caco-2 extracts was carried out in order to determine stability to the peptidases that are produced by these cells. The sunflower hydrolysate produced by incubation with alcalse for 5 min as described in the previous section was incubated with Caco-2 extracts as well. As shown in Fig. 5, the ACE inhibitory activity in both hydrolysates decreased by about 60% after incubation for 2 h. Similar results were obtained by Vermeirssen et al. (2005) when pea and whey peptides were incubated with Caco-2 extracts. Thus, the ACE inhibitory activity in the sunflower hydrolysates is reduced not only by incubation in SGF and SIF, but also by the peptidases produced by Caco-2 cells. Nevertheless, a significant portion of the inhibitory activity survives exposure to all the proteases and peptidases contained in these in vitro systems that mimic in vivo digestion. In contrast, the activity of the FVNPQAGS peptide was not affected by
Fig. 7. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with Caco-2 extracts. The hydrolysate was produced by treatment of a sunflower protein isolate with pepsin and pancreatin, and a peptidic fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented).
This work was supported by research grants AGL 2004-03930 (F.M.) and AGL 2005-01120 (J.G.-C.) from the Spanish Ministry of Education and Science partially supported by FEDER funds from the EU.
References
0
0h
1h
2h
0
Fig. 6. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with Caco-2 extracts. The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min, and a peptidic fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
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Burkitt, M. J. (2001). A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, a-tocopherol, thiols, and ceruloplasmin. Archives of Biochemistry and Biophysics, 394, 117–135. Engle, M. J., Goetz, G. S., & Alpers, D. H. (1998). Caco-2 cells express a combination of colonocyte and enterocyte phenotypes. Journal of Cellular Physiology, 174, 362–369, [0021–9541]. Go´mez, R. J. A., Ramos, M., & Recio, I. (2004). Angiotensin converting enzymeinhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. International Dairy Journal, 14, 1075–1080. Hayakari, M., Koudo, Y., & Izomi, H. (1978). A rapid and simple spectrophotometric assay of angiotensin-converting enzyme. Analytical Biochemistry, 84, 361–369. Lin, X., Xue, L.-Y., Wang, R., Zhao, Q.-Y., & Chen, Q. (2006). Protective effects on endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation. FEBS Journal, 273, 1275–1284. Megı´as, C., Pedroche, J., Yust, M. M., Alaiz, M., Giron-Calle, J., Milla´n, F., et al. (2006). Affinity purification of angiotensin converting enzyme inhibitory peptides using immobilized ACE. Journal of Agriculture and Food Chemistry, 54, 7120–7124. Megı´as, C., Pedroche, J., Yust, M. M., Giro´n-Calle, J., Alaiz, M., Milla´n, F., et al. (2007). Affinity purification of copper chelating peptides from sunflower protein hydrolysates. Journal of Agriculture and Food Chemistry, 55, 6509–6514. Megı´as, C., Pedroche, J., Yust, M. M., Giro´n-Calle, J., Alaiz, M., Milla´n, F., et al. (2008). Production of copper chelating peptides after hydrolysis of sunflower proteins with pepsin and pancreatin. LWT – Food Science and Technology, 41, 1973–1977. Megı´as, C., Yust, M. M., Pedroche, J., Lquari, H., Giro´n-Calle, J., Alaiz, M., et al. (2004). Purification of an ACE inhibitory peptide after hydrolysis of sunflower (Helianthus annuus L.) protein isolates. Journal of Agriculture and Food Chemistry, 52, 1928–1932.
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Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Stability of sunflower protein hydrolysates in simulated gastric and intestinal fluids and Caco-2 cell extracts Cristina Megı´as, Justo Pedroche, Marı´a del Mar Yust, Manuel Alaiz, Julio Giro´n-Calle, Francisco Milla´n, Javier Vioque* Instituto de la Grasa (CSIC), Padre Garcı´a Tejero 4, 41012-Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2008 Received in revised form 6 March 2009 Accepted 30 April 2009
The in vitro stability of bioactive properties in sunflower protein hydrolysates and purified peptides was studied by using simulated gastric fluid, simulated intestinal fluid, and Caco-2 cells extracts. Protein hydrolysates that inhibit the angiotensin converting enzyme and copper chelating peptides produced by hydrolysis with the microbial protease alcalase were partially resistant to incubation with simulated gastric and intestinal fluids. These hydrolysates and others produced by hydrolysis using pepsin plus pancreatin were also partially resistant to incubation with Caco-2 cells extracts. In addition, the ACE inhibitory peptide FVNPQAGS that is generated by extensive hydrolysis using pepsin and pancreatin, was found to be resistant to hydrolysis by Caco-2 cells extracts. These results suggest that stability in the digestive tract should not be a problem for the bioavailability of these bioactive peptides. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Sunflower Protein hydrolysate Bioactive peptides Gastrointestinal fluids Caco-2
1. Introduction Food proteins are increasingly been recognized as a source of beneficial bioactive peptides that are released during physiological digestion. A variety of bioactive peptides derived from plant and animal food products with positive effects on the immune, nervous, digestive and vascular systems have been described (Vioque et al., 2000). In addition to be absorbed by the digestive tract, these peptides have to be resistant to digestive proteases and peptidases in order to have a physiological effect. A variety of sunflower bioactive hydrolysates and peptides have been described in our laboratory, including angiotensin converting enzyme (ACE) inhibitory peptides (Megı´as et al., 2006, 2004), and metal chelating peptides (Megı´as et al., 2007, 2008). These peptides have been produced by hydrolysis of sunflower seed protein isolates using microbial (alcalase and flavourzyme) or digestive (pepsin and pancreatin) enzymes, and their bioactivity has been determined using in vitro methods. However, the bioavailability of these peptides remains to be determined whether these bioactive peptides are stable enough to hydrolysis by digestive proteases and peptidases in order to be susceptible to
* Corresponding author. Tel.: þ34 954 611 550; fax: þ34 954 616 790. E-mail addresses: [email protected], [email protected] (J. Vioque). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.04.008
be absorbed and reach the cardiovascular system in active form. In addition to loss of activity, gastrointestinal digestion may actually lead to generation of more potent bioactive peptides. Thus, an increase in the ACE inhibitory activity of fermented casein solutions was observed after incubation with digestive enzymes (Vermeirssen, Van Camp, Decroos, Van Wijmelbeke, & Verstraete, 2003). In the present work, the stability the ACE inhibitory activity and the copper chelating activity in sunflower protein hydrolysates and purified peptides has been studied using simulated gastric fluid, simulated intestinal fluid, and Caco-2 cells extracts as in vitro models of physiological digestion.
2. Material and methods 2.1. Materials Deffated sunflower meal was supplied by MIGASA (Sevilla, Spain). Alcalase 2.4L was from NovoNordisk (Bagsvaerd, Denmark). Bradford reagent, hippuryl-histidyl-L-leucine (HHL), 2,4,6-trichloros-triazine (TT, dioxane, pepsin, pancreatin and b-carotene were purchased from Sigma) (Tres Cantos, Madrid, Spain). Dulbeco´s Modified Eagle´s Medium, fetal bovine serum and antibiotics for cell culture were from GIBCO (Barcelona, Spain).
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
80 70 60 50 40 30 20 10 0
SGF 1 h + SIF 3 h
2.2.5. Incubation with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and Caco-2 cells extract SGF and SFI were prepared according to the United States Pharmacopeial Convention Council of Experts (2004), with modifications. The composition was as follows: SFG: 0.00064% pepsin (w/v), 30 mM NaCl, 0.7% (v/v) HCl, The enzyme/substrate ratio was 1/20 (w/w). The incubation was continued for 1 h under shacking at 37 C and pH 1.2. SFI: 0.0005% (w/v) pancreatin, 0.05 M KH2PO4, 19% (v/v) NaOH 0.2 N. The enzyme/substrate ratio was 1/20 (w/w). The incubation was continued for 3 h under shacking at 37 C and pH 7.5.
Treatment of a sunflower protein isolate with the microbial protease alcalase for 5 min yielded a high ACE inhibitory activity in the resulting hydrolysate (Megı´as et al., 2006) This hydrolysate has now been incubated with SGF and SIF in order to determine the stability of the ACE inhibitory activity to digestive proteases and peptidases. As shown in Fig. 1, incubation with SGF did not affect ACE inhibitory activity. Further incubation in SIF did not suppress activity, although it caused a time-dependent decrease in ACE inhibitory activity, resulting in an increase of the IC50 from 20,4 1,2 mg/mL after incubation with SGF, to 68,2 0,95 mg/mL
SGF 1 h + SIF 1 h
2.2.4. Determination of ACE inhibitory activity ACE activity was determined as described by Hayakari, Koudo, and Izomi (1978) with modifications. This method uses HHL as substrate for ACE, and relies on the reaction of the resulting hippuric acid with 2,4,6-trichloro-s-triazina (TT). The assay mixture (0.5 ml) consisted of potassium phosphate buffer pH 8.3 (40 mmol), sodium chloride (300 mmol), HHL (1.5 mmol), and 1 mg of pig lung ACE extract prepared as previously described (Megı´as et al., 2004). Incubation was carried out at 37 C for 15 min and was terminated by addition of TT in dioxane (1.5 ml 3% w/v), followed by 0.2 M phosphate buffer pH 8.3 (3 ml). Absorbance at 382 nm was determined in the supernatant obtained after centrifugation at 10,000 g for 10 min. The IC50 value, defined as the concentration of peptide in mg protein/mL required to produce 50% inhibition of ACE, was determined by regression analysis of ACE inhibition (%) versus peptide concentration. Experiments were ran in triplicate.
3.1. Incubation of sunflower bioactive hydrolysates and purified peptides with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)
SGF 1 h
2.2.3. Purification of chelating peptides and determination of antioxidant activity Sunflower protein hydrolysates with the highest antioxidant activity in the presence of copper were selected for purification of chelating peptides as previously described (Megı´as et al., 2007) using Vivapure Metal Chelate Maxi spin columns (Vivascience, Sartorius, Madrid, Spain) charged with copper. Antioxidant activity was assayed using the b-carotene oxidation method as previously described with modifications (Pedroche et al., 2006). Final assay mixtures contained different concentrations of hydrolysates, b-carotene (119 mM), and Cuþþ (0.1 mM) in a final volume of 200 mL. The degradation of b-carotene was monitored by recording the decrease in absorbance at 470 nm using a microplate reader. Experiments were ran in triplicate.
3. Results and discussion
SGF 0.5 h
2.2.2. Determination of peptides concentration Peptide concentrations of the hydrolysates were determined after amino acid analysis of derivatives according to the method of Alaiz, Navarro, Giro´n, and Vioque (1992). Samples were treated with 4 ml 6 mol/L HCl in tubes sealed under nitrogen for 24 h at 110 C for hydrolysis. Amino acids were determined by highperformance liquid chromatography of the derivatives obtained by reaction with diethyl ethoxymethylenemalonate.
SGF 0 h
2.2.1. Production of sunflower protein hydrolysates Sunflower protein isolates (Sa´nchez-Vioque, Clemente, Vioque, Bautista, & Milla´n, 1999) were used for production of protein hydrolysates using alcalase or pepsin plus pancreatin as previously described (Megı´as et al., 2004; Megı´as et al., 2007). Hydrolysis was stopped by heating at 80 C for 20 min. Hydrolysates were clarified by ultrafiltration through 0.45 mm filters (Millipore, Bedford, MA) and lyophilized for storage at 20 C.
Samples were collected at different times and were inactivated by heating at 80 C for 15 min and them centrifuged. Results are the average SD of three determinations. Caco-2 cells homogenates were prepared according to Augustijns, Annaert, Heylen, Van den Mooter, and Kinget (1998) with modifications. Briefly, Caco-2 cells monolayers were homogenized in 5 mL DMEM by sonication for 10 s. After sonication, samples were centrifuged at 14,000 g for 5 min and the supernatant was recovered. Protein content in the supernatant was determined according to Bradford (1976). Cells were obtained from the European Collection of Cell Cultures and kept at 5% CO2 in Dulbecco´s Modified Eagle´s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. Stability assays in Caco-2 cells extracts were done according to Vermeirssen et al. (2005) by incubation at 37 C for 2 h under shacking using a cell protein to sample protein ratio of 1–6 (w/w). Results are the average SD of three determinations.
IC50
2.2. Methods
1497
Fig. 1. Incubation of an ACE inhibitory sunflower hydrolysate with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 5 min. After incubation in SGF for 1 h, pH was adjusted and pancreatin added in order to carry out incubation in SIF for 3 h. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
0,00008
0,0004
0,00007 0,0003
0,00006 0,0002
Slope (PH)
0,00005
SGF 1 h + SIF 3 h
SGF 1 h + SIF 1.5 h
SGF 1 h
0,00004 SGF 0.5 h
0,0001
SGF 0 h
after incubation for 3 more hours in SIF. Direct incubation in SIF did not cause any major change in ACE inhibitory activity (Fig. 2). Similar results concerning stability of ACE inhibitory peptides have been described by other groups. Thus, Wu and Ding (2002) observed that soy derived ACE inhibitory peptides generated with alcalase where resistant to hydrolysis by pepsin and were only partially hydrolyzed by pancreatin. In other studies, the ACE inhibitory activity of peptides of known sequence increased, decreased, or remained unchanged after incubation in SGF and SIF depending on the presence in the peptidic sequence of the specific targets for the digestive proteases (Go´mez, Ramos, & Recio, 2004; Parrot, Degraeve, Curia, & Martial-Gros, 2003). The stability of the copper chelating activity in sunflower hydrolysates generated by treatment with alcalase (Megı´as et al., 2007) during incubation in SGF and SIF has also been studied. The chelating activity of a hydrolysate produced by incubation with alcalase for 30 min increased during incubation in SGF, suggesting that new chelating peptides were being generated. Although subsequent incubation in SIF for 3 h lead to a decrease in chelating activity, activity was not completed suppressed (Fig. 3). The copper chelating peptides in this hydrolysate that were purified by affinity chromatography using immobilized Cuþþ were also found to be fairly stable in incubations with SGF and SIF (Fig. 3). Direct incubation in SIF showed an increase in chelating activity after incubation for 1.5 h in both the alcalase hydrolysate and the corresponding affinity purified fraction (Fig. 4). These results suggest that degradation by physiological proteases and peptidases during digestion should not prevent the sunflower copper chelating peptides from exerting antioxidant effects in vivo. Thus, these peptides could have an antioxidant effect in the intestinal lumen, and if absorbed through the intestinal barrier they could reach the bloodstream and inhibit LDL oxidation (Burkitt, 2001), as well as the progression of certain diseases that have been linked to oxidative processes such as Alzheimer´s disease and Parkinson´s disease (Lin, Xue, Wang, Zhao, & Chen, 2006). It is interesting to note that direct incubation in SIF caused a much lower decrease in ACE-inhibitory activity and copper chelating activity than incubation in SIF following incubation in SGF. Thus, protecting the bioactive peptides by encapsulation in microgels in conditions that facilitate their release in the intestine could greatly increase bioavailability of these peptides.
Slope (CP)
1498
Fig. 3. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min. After incubation in SGF for 1 h, pH was adjusted and pancreatin added in order to carry out incubation in SIF for 3 h. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
3.2. Stability of sunflower bioactive peptides to Caco-2 cells extracts Confluent monolayers of Caco-2 cells, derived originally from a colorectal carcinoma, constitute a model of the normal, healthy intestinal epithelium when they are allowed to grow for a certain time (Engle, Goetz, & Alpers, 1998). Thus, this type of confluent cultures is a very popular in vitro model of the intestinal barrier for studies of absorption of nutrients, bioactive diet components, and drugs (Artursson, Palm, & Lothman, 2001). Caco-2 cells extracts have now been used as an in vitro model to test the stability of the ACE inhibitory activity and the copper chelating activity in sunflower bioactive hydrolysates previously described by us. These studies complement those that were carried out using SGF and SIF. A high ACE inhibitory activity in a sunflower hydrolysate that was produced by incubation of a protein isolate with pepsin for 3 h and pancreatin for another 3 h was previously described. The peptide of sequence FVNPQAGS, corresponding to a fragment of
0,000064
0,0004 100
0,00006
90
Slope (CP)
IC 50
70 60 50
0,000056 0,0002
Slope (PH)
80
0,0003
0,000052
40 30 0,0001
20 10 0
SIF 0 h
SIF 1 h
SIF 2 h
SIF 3 h
Fig. 2. Incubation of an ACE inhibitory sunflower hydrolysate with simulated intestinal fluid (SIF). The hydrolysate, produced by treatment of a sunflower protein isolate with alcalase for 5 min was incubated with SIF for 3 h. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
SIF 0 h
SIF 1.5 h
SIF 3 h
0,000048
Fig. 4. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with simulated intestinal fluid (SIF). The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min, and a peptididc fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
C. Megı´as et al. / LWT - Food Science and Technology 42 (2009) 1496–1500
0,0004
100
0,00006
0,0003
0,00004
0,0002
0,00002
0,0001
Slope (PH)
0,00008
60
Slope (CP)
120
80
IC 50
1499
40 0
0h
1h
2h
0
20
0
0h
1h
2h
Fig. 5. Incubation of two ACE inhibitory hydrolysates obtained by hydrolysis with alcalase and the ACE inhibitory peptide FVNPQAGS with Caco-2 extracts. The hydrolysates were produced by treatment with alcalase for 5 min (–C– A5), or pepsin for 180 min plus pancreatin for another 180 min (–,– PP360). The FVNPQAGS (–6–) was previously purified from the PP360 hydrolysate. ACE inhibitory activity, shown as IC50 (mg/ml), was determined in aliquots taken at different time points.
0,0001
0,0005
0,00008
0,0004
0,00006
0,0003
0,00004
0,0002
0,00002
0,0001
incubation with Caco-2 extracts (Fig. 5), indicating that the bioavailability of this peptide is not limited at all by the peptidases produced by the Caco-2 cells. Incubation of the copper chelating hydrolysate produced by treatment with alcalase for 30 min and the corresponding affinity purified fraction (Megı´as et al., 2007) with Caco-2 extracts lead to a reduction of chelating activity of about 50% after 1 h (Fig. 6). The chelating activity was not further reduced, or even was increased, during the second hour of incubation. Similarly, incubation of the hydrolysate produced by treatment with pepsin plus pancreatin and the fraction purified by affinity chromatography from this hydrolysate (Megı´as et al., 2008), showed that the copper chelating activity did not change during the first hour of incubation, but decreased by about 50% by the end of the 2 h incubation (Fig. 7). In conclusion, the bioactive properties of sunflower protein hydrolysates and related fractions that are described in this article have been found to be partially resistant to hydrolysis by SGF, SIF, and Caco-2 extracts, which represent in vitro models of physiological digestion. It is particularly interesting the case of the FVNPQAGS peptide, which is produced by treatment with pepsin and pancreatin, and is resistant to hydrolysis by Caco-2 extracts. This peptide has been shown to be a true inhibitor and not a substrate of ACE (Megı´as et al., 2004).
Acknowledgements
Slope (CP)
Slope (PH)
helianthin, the main storage protein in sunflower seeds, was found in this hydrolysate (Megı´as et al., 2004). Incubation of this hydrolysate and the FVNPQAGS peptide with Caco-2 extracts was carried out in order to determine stability to the peptidases that are produced by these cells. The sunflower hydrolysate produced by incubation with alcalse for 5 min as described in the previous section was incubated with Caco-2 extracts as well. As shown in Fig. 5, the ACE inhibitory activity in both hydrolysates decreased by about 60% after incubation for 2 h. Similar results were obtained by Vermeirssen et al. (2005) when pea and whey peptides were incubated with Caco-2 extracts. Thus, the ACE inhibitory activity in the sunflower hydrolysates is reduced not only by incubation in SGF and SIF, but also by the peptidases produced by Caco-2 cells. Nevertheless, a significant portion of the inhibitory activity survives exposure to all the proteases and peptidases contained in these in vitro systems that mimic in vivo digestion. In contrast, the activity of the FVNPQAGS peptide was not affected by
Fig. 7. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with Caco-2 extracts. The hydrolysate was produced by treatment of a sunflower protein isolate with pepsin and pancreatin, and a peptidic fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented).
This work was supported by research grants AGL 2004-03930 (F.M.) and AGL 2005-01120 (J.G.-C.) from the Spanish Ministry of Education and Science partially supported by FEDER funds from the EU.
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0
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0
Fig. 6. Incubation of a copper chelating sunflower hydrolysate (–C– PH) and the corresponding peptidic fraction purified by affinity chromatography (–,– CP) with Caco-2 extracts. The hydrolysate was produced by treatment of a sunflower protein isolate with alcalase for 30 min, and a peptidic fraction was purified using immobilized copper. Antioxidant activity (inhibition of copper-mediated oxidative degradation of b-carotene) was determined as an indirect measure of copper chelating activity (the slope of b-carotene degradation is presented) (Pedroche et al., 2006).
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