Food Chemistry 163 (2014) 1–5
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Hydrolysis kinetics and radical-scavenging activity of gelatin under simulated gastrointestinal digestion Lin Wang a,⇑, Qiufang Liang a, Qiuhong Chen a, Junmin Xu b, Zhujun Shi a, Zhenbin Wang a, Yang Liu c, Haile Ma a a b c
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Zhenjiang Kehua Aquaculture Development Company Limited, Zhenjiang 212134, PR China Zhenjiang Entry-Exit Inspection and Quarantine Burea, Zhenjiang 212008, PR China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 11 March 2014 Accepted 23 April 2014 Available online 2 May 2014 Keywords: Gelatin Hydrolysis kinetics Radical-scavenging activity Simulated gastrointestinal digestion
a b s t r a c t The hydrolysis kinetics and radical-scavenging activity of gelatin were investigated under simulated gastrointestinal digestion in this study. In the gastric phase, the degree of gelatin hydrolysis increased from 0.17% to 1.20%, while the DPPH radical-scavenging rate increased from 6.27% to 24.56%. Further digestion in the intestinal phase brought the degree of hydrolysis and radical-scavenging rate to 26.08% and 44.76%, respectively. After digestion, the gelatin hydrolysates were separated into two fractions by ultrafiltration. The fraction with an average molecular weight of 312.98 Da exhibited the higher yield (78.26%) and radical-scavenging activity (IC50 = 2.09 mg/ml), suggesting the high digestibility and bioactivity of gelatin after oral administration. The fraction was further purified with multi-step column chromatography and identified to be Gly-Pro-Met (303.38 Da) by UPLC–ESI-MS. These results may help us to better understand its physiological effects and to use it properly in foods and pharmaceuticals. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin, a denatured form of collagen, has long been used in foods, pharmaceuticals and cosmetics. It has been used to improve blood circulation, arrest bleeding and to improve joint condition in traditional medicine (Ohara, Matsumoto, Ito, Iwai, & Sato, 2007). Recent animal experiments and human trials have also suggested that the oral ingestion of gelatin had beneficial effects on skin ageing (Tanaka, Koyama, & Nomura, 2009), osteoarthritis and osteoporosis (Moskowitz, 2000), rheumatoid arthritis (Trentham et al., 1993), properties of nail, hair and Achilles tendon (Matsuda et al., 2006; Minaguchi et al., 2005). Free radicals are very reactive molecules, which can react with every cellular component and give rise to functional and morphologic disturbances in cells (Martínez-Cayuela, 1995). There is evidence to implicate oxygen free radicals as important pathologic mediators in many human diseases and ageing processes, such as skin ageing (Terra et al., 2012), osteoarthritis (Davies, Guilak, Weinberg, & Fermor, 2008), osteoporosis (Xu et al., 2011), rheumatoid arthritis (Bhowmick, Chakraborti, Gudi, Moideen, & Shetty, 2008) and hair and nail damage (Fernández et al., 2011; Khengar
et al., 2010). Recently, the antioxidant activity of collagen peptides has been widely demonstrated in many different oxidative systems (Gómez-Guillén, Giménez, López-Caballero, & Montero, 2011). Therefore, we hypothesized that the physiological effects of gelatin was related with antioxidant activity. However, it is not clear how gelatin is digested and further exerts antioxidant activity in vivo. In vitro, simulated gastrointestinal digestion is extensively used since it is rapid, safe, inexpensive and does not have the same ethical restrictions as in vivo methods (Liang et al., 2012). In recent years, the method has already been developed for the bioavailability and bioactivity assessment of protein. Thus, it might also be reasonable to investigate the hydrolysis kinetics and radical-scavenging activity of gelatin after oral administration. In this study, the hydrolysis kinetics and radical-scavenging activity of gelatin was investigated under simulated gastrointestinal digestion, which may help us to better understand its physiological effects and to use it properly in foods and pharmaceuticals.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel./fax: +86 511 88780201. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.foodchem.2014.04.083 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
The gelatin was prepared from scales of the Nile tilapia (Oreochromis niloticus) following the method of Wangtueai and
L. Wang et al. / Food Chemistry 163 (2014) 1–5
Noomhorm (2009). Simulated gastric and intestinal fluids were prepared according to the United States Pharmacopeial Convention Council of Experts (2007).
2.3. Assay of the degree of hydrolysis (DH) The DH of gelatin was determined using the o-phthaldialdehyde (OPA) method (Marambe, Shand, & Wanasundara, 2008). The average chain length (ACL) and average molecular weight (AMW) of the ˇ urda, & hydrolysates were calculated by the equation (Mišún, C Jelen, 2008): ACL = 100/DH, AMM = ACL 90. 2.4. Determination of DPPH radical-scavenging activity The DPPH radical-scavenging activity was determined as described by Hu et al. with slight modifications (Hu, Xu, Chen, & Yang, 2004). Two millilitre of sample was added to 2 ml of 0.2 mM ethanol solution of DPPH. After shaking vigorously, the absorbance was monitored immediately at 517 nm and recorded every 5 min interval until 240 min. The scavenging rate was calculated as follows: Scavenging rate (%) = [(A0 A1)/A0] 100, where A0 was the absorbance of the control and A1 was the absorbance of the sample. 2.5. Separation of gelatin hydrolysates by ultrafiltration The ultrafiltration was performed on a Millipore Pellicon system equipped with a membrane with the nominal molecular weight cut-off of 1 kDa. The digests were pumped to the membrane surface and the filtrate was collected, while the retentate was recirculated until the absorbance of the filtrate at 220 nm was close to 0. Then, the filtrate (F1) and retentate (F2) were concentrated and lyophilized separately. The yield was obtained by comparing hydroxyproline content in the fractions with that in gelatin. The hydroxyproline content was determined as recommended by ISO 3496 (Anonymous, 1994). 2.6. Purification of radical-scavenging peptide by chromatography The sample was loaded on a Toyopearl SP-650M column (2.5 30 cm) equilibrated with 0.05 M sodium acetate buffer (pH 4.5), and eluted with a linear gradient of NaCl (0–1.0 M) in the same buffer at 1.0 ml/min. Then, the potent fraction was subjected to a Sephadex G10 column (1.5 75 cm) eluted with distilled water at 0.5 ml/min. Subsequently, the active fraction was injected onto an Agilent ZORBAX Eclipse XDB C18 semi-preparative column (9.4 250 mm, 5 lm) and separated by a gradient of acetonitrile (2–10% in 0–15 min, 10–30% in 15–25 min, 30–70% in 25–30 min) at 2.5 ml/min. The radical-scavenging activity was expressed as the DPPH radical-scavenging rate per absorbance unit. The detection wavelength was 220 nm.
Degree of hydrolysis (%)
The simulated digestion of gelatin was carried out by modification of the method of Chen and Li (2012). Briefly, the gelatin was hydrolyzed with simulated gastric fluid (pH 1.2) at 1:20 (w/v) for 4 h at 37 °C in a shaking incubator at 150 rpm. Then, pancreatin was added at 1:100 (w/v) after adjusting the concentration of the KH2PO4 buffer to 0.05 M and to pH 6.8 with 1.0 M NaOH. The mixture was further incubated for 6 h. To terminate the digestion, the samples were kept in boiling water for 10 min. Finally, the digests were cooled and centrifuged at 10,000g for 15 min. The control was conducted in the same manner, with distilled water used instead of gelatin.
50
Intestinal phase
30
40
30
20
20 10 10
0
0
2
4
6
8
10
0
Digestion time (h) Fig. 1. Kinetics of hydrolysis and radical-scavenging of gelatin under stimulated gastrointestinal digestion. ( ) Degree of hydrolysis; ( ) DPPH radical-scavenging rate.
Table 1 The yield, AMW and IC50 value of gelatin and its hydrolysates.
F1 F2 Gelatin
Yielda (%)
AMW (Da)
IC50 (mg/ml)
78.26 ± 4.28a 14.29 ± 1.75b
312.98 ± 2.04c 1822.46 ± 66.74b 52,714.81 ± 3183.33a
2.09 ± 0.02c 3.17 ± 0.03b 17.22 ± 0.76a
a Mean value ± standard deviation from three separate samples. Values followed by different letters are significantly different at P < 0.05. AMW, average molecular weight; IC50, the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%; , no data.
2.7. Identification of amino acid sequences by UPLC–ESI-MS The purified peptide was identified using a Waters ACQUITY UPLC coupled to SYNAPT Q-TOF MS with electrospray ionization. The sample was dissolved in distilled water, and 5 ll was then injected onto a BEH130 C18 column (2.1 150 mm, 1.7 lm) with a linear gradient of acetonitrile (0–20% in 20 min) at 0.2 ml/min. The mass spectrometer was operated in positive ion mode with a capillary voltage 3.5 kV, ion source temperature 100 °C, desolvation temperature 300 °C, desolvation gas flow 10 l/min, cone voltage 45 V, cone gas flow 1 l/min and scanning range 50–1000 m/z. The amino acid sequence was analyzed using BioLynx software.
1.2 1.0
Absorbance at 517 nm
2.2. Simulated gastrointestinal digestion
Gastric phase
DPPH radical-scavenging rate (%)
2
0.8 0.6 0.4 0.2 0.0
0
60
120
180
240
Time (min) Fig. 2. DPPH radical-scavenging activity of gelatin and its hydrolysates (5 mg/ml), compared to BHT and a-tocopherol (0.2 mg/ml). (j) Control; ( ) F1; ( ) F2; ( ) gelatin; ( ) BHT; ( ) a-tocopherol.
3
L. Wang et al. / Food Chemistry 163 (2014) 1–5
20
0 mV
% 150
a
15 100 10 50 5
0 0
60
0 mV
120
% 800
C
b
30
0 240
180
600 20 400
10
200
B D
A 0
0 0
1500
60
120
180
360
mAu
540
% 1200
cc
1200
900
900 600 600 300
300
0
0
5
10
15
20
25
0 30
Elution time (min) Fig. 3. Chromatographic and antioxidant profiles of F1 using Toyopearl SP-650M (a), Sephadex G10 (b), Eclipse XDB C18 semi-preparative column (c). ( concentration (0–1.0 M); ( ) voltage (mV) or absorbance (mAu); ( ) as well as histogram, DPPH radical-scavenging rate per absorbance unit (%).
2.8. Statistical analysis The data are presented as mean ± standard deviation of three determinations. Statistical analyses were performed using oneway analysis of variance. Multiple comparisons of means were done by LSD test. A P value F2 (3.17 mg/ml) > gelatin (17.22 mg/ml). Combined with the bioavailability, it was indicated that the F1 fraction might be responsible for the various physiological effects of gelatin. Thus, the F1 fraction was used for further purification and identification of the radical-scavenging peptide.
3.2. Separation of gelatin hydrolysates The gelatin hydrolysates after digestion were separated into two fractions by ultrafiltration. Most of the gelatin hydrolysates (78.26%) were recovered in F1, with a lower AMW of 312.98 Da, while the yields of F2 were only 14.29% (Table 1). This suggests that gelatin was digested almost completely in the gastrointestinal tract into oligopeptides or amino acids, which could be absorbed by the small intestine. Therefore, it seemed that the gelatin was digestible and absorbable after oral administration, although the final DH was still lower than amaranth protein (35.8%) and loach protein (46.6%) (Delgado, Tironi, & Añón, 2011; You et al., 2010).
3.4. Purification of radical-scavenging peptide The radical-scavenging peptide was purified by multi-step column chromatography (Fig. 3). The F1 fraction was separated by a Toyopearl SP-650M cation-exchange column, and the fraction with the highest activity (90–120 min) was bound by the column and washed out at a low NaCl concentration (Fig. 3a). It signified that the fraction might be weakly basic, which agrees with Ren’s report that basic peptides were more effective than acidic (Ren et al., 2008). The fraction with the highest activity was then separated into four subfractions (A–D) by a Sephadex G10 molecular exclusion column (Fig. 3b), in which molecules are separated by their size. Both fractions A and B were peptides, but only fraction B with the lower molecular weight had radical-scavenging activity. Fractions C and D might be free amino acids and acetates, respectively. The fraction B was further separated by a C18 semi-preparative column, which was based on the hydrophobic or hydrophilic property of a peptide (Fig. 3c). The fraction displaying the strongest activity was eluted in 5–6 min with an IC50 value of 28.05 lg/ml, which was almost 75-fold greater than the gelatin hydrolysates (2.09 mg/ ml). This indicated that the radical-scavenging peptide was
3.3. Radical-scavenging activity of gelatin hydrolysates Fig. 2 illustrates the kinetic behaviour of gelatin and its hydrolysates as scavengers toward the DPPH radical. All samples tested exhibited remarkable radical-scavenging activity, although this was lower than a-tocopherol and BHT. The a-tocopherol reacted rapidly with DPPH radical and reached a steady state in 5 min, while the BHT was stable after 150 min. However, the F1 fraction reached a plateau at 180 min, and the gelatin and F2 fraction did not reach steady states until 240 min, which suggested that they were typical slow-acting antioxidants. From the IC50 values (Table 1), radical-scavenging activity decreased in the order: F1 (2.09 mg/
10
0.0
a
TOF MS ES+ TIC
%
4.66e4
0.0
0.5
1.0
1.5
2.0
2.5
b
3.0
3.5
264.1
min
TOF MS ES+ 625
%
304.0
144.0 80.9
122.0 82.9 104.0 70.0
50
90.9
100
112.0
134.0138.9 164.9 148.1158.0
150
191.0 196.8198.8 184.0
200
219.0
282.1
229.1 256.8 266.1 240.1 246.0
250
284.1300.1
300
322.1
344.1 337.1
350 m/z
Fig. 4. Identification of amino acid sequences of the purified peptide by UPLC–ESI-MS. (a) UPLC profile with BEH130 C18 column; (b) mass spectrum using ESI-MS.
L. Wang et al. / Food Chemistry 163 (2014) 1–5
hydrophilic, which contradicts most reports which show that the hydrophobic peptides present in hydrolysates had a greater impact on antioxidant activity (Rajapakse, Mendis, Byun, & Kim, 2005). 3.5. Identification of amino acid sequences of radical-scavenging peptide The UPLC chromatogram reveals a major peak (1.09 min) corresponding to the target peptide (Fig. 4a). The target peptide was further determined to be Gly-Pro-Met (303.38 Da) by ESI-MS (Fig. 4b), on which there was no report yet. It is commonly believed that the antioxidant activity of a peptide is associated with molecular size. Most studies have shown that antioxidant peptides usually contained less than 20 amino acid residues and resulted in a better chance to cross the intestinal barrier and exert biological effects (Kou et al., 2013). In recent years, several antioxidant peptides have been isolated from collagen or other collagenous sources, and the molecular weights of these peptides were not more than 1500 Da (Gómez-Guillén et al., 2011). The molecular weight of this peptide was 303.38 Da, which supported the fact that the peptide with high antioxidant activity had lower molecular weight. Furthermore, amino acid compositions and sequences of a peptide may also be important to antioxidant activity. It is known that Met can be readily oxidized to Met sulfoxide by reactive oxygen species and Pro is often included in antioxidative peptides (Elias, Kellerby, & Decker, 2008), which would contribute to the antioxidant activity of this peptide. However, the antioxidant activity of a peptide is higher than free amino acids, which could be attributed to the unique physico-chemical properties of amino acid sequences (Elias et al., 2008). The Gly-Pro sequence is often present in antioxidant peptides and has been proposed to play an important role in antioxidant activity (Sun, Zhang, & Zhuang, 2013). This peptide also contained this sequence, which might result in the high antioxidant activity. 4. Conclusion The hydrolysis kinetics and radical-scavenging activity of gelatin were investigated under simulated gastrointestinal digestion in this study. The gelatin could be digested nearly completely into oligopeptides or amino acids, suggesting high digestibility by oral administration. The gelatin hydrolysates exhibited high radicalscavenging activity, which might be responsible for the various physiological effects of gelatin. Moreover, a radical-scavenging peptide was purified and identified from the gelatin hydrolysates, but further studies are needed to validate its radical-scavenging activity and physiological effects after synthesizing. Acknowledgements This research was supported by Scientific Research Starting Foundation for Talented Scholars of Jiangsu University (10JDG053) and China Postdoctoral Science Foundation (2011M500872). References Anonymous (1994). Meat and meat products – Determination of hydroxyproline content. International standard. ISO 3496. Bhowmick, K., Chakraborti, G., Gudi, N. S., Moideen, A. V. K., & Shetty, H. V. (2008). Free radical and antioxidant status in rheumatoid arthritis. Indian Journal of Rheumatology, 3(1), 8–12. Chen, M., & Li, B. (2012). The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion. Innovative Food Science and Emerging Technologies, 16, 341–348.
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Davies, C. M., Guilak, F., Weinberg, J. B., & Fermor, B. (2008). Reactive nitrogen and oxygen species in interleukin-1-mediated DNA damage associated with osteoarthritis. Osteoarthritis Cartilage, 16(5), 624–630. Delgado, M. C. O., Tironi, V. A., & Añón, M. C. (2011). Antioxidant activity of amaranth protein or their hydrolysates under simulated gastrointestinal digestion. LWT – Food Science and Technology, 44(8), 1752–1760. Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and peptides. Critical Reviews in Food Science and Nutrition, 48(5), 430–441. Fernández, E., Barba, C., Alonso, C., Martí, M., Parra, J. L., & Coderch, L. (2011). Photodamage determination of human hair. Journal of Photochemistry and Photobiology B: Biology, 106, 101–106. Gómez-Guillén, M. C., Giménez, B., López-Caballero, M. E., & Montero, M. P. (2011). Functional and bioactive properties of collagen and gelatin from alternative sources: A review. Food Hydrocolloids, 25(8), 1813–1827. Hu, Q., Xu, J., Chen, S., & Yang, F. (2004). Antioxidant activity of extracts of black sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction. Journal of Agricultural and Food Chemistry, 52(4), 943–947. Khengar, R. H., Brown, M. B., Turner, R. B., Traynor, M. J., Holt, K. B., & Jones, S. A. (2010). Free radical facilitated damage of ungual keratin. Free Radical Biology and Medicine, 49(5), 865–871. Kou, X., Gao, J., Xue, Z., Zhang, Z., Wang, H., & Wang, X. (2013). Purification and identification of antioxidant peptides from chickpea (Cicer arietinum L.) albumin hydrolysates. LWT – Food Science and Technology, 50(2), 591–598. Liang, L., Wu, X., Zhao, T., Zhao, J., Li, F., Zou, Y., et al. (2012). In vitro bioaccessibility and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.) following simulated gastro-intestinal digestion. Food Research International, 46(1), 76–82. Marambe, P. W. M. L. H. K., Shand, P. J., & Wanasundara, J. P. D. (2008). An in-vitro investigation of selected biological activities of hydrolysed flaxseed (Linum usitatissimum L.) proteins. Journal of the American Oil Chemists’ Society, 85, 1155–1164. Martínez-Cayuela, M. (1995). Oxygen free radicals and human disease. Biochimie, 77(3), 147–161. Matsuda, N., Koyama, Y., Hosaka, Y., Ueda, H., Watanabe, T., Araya, T., et al. (2006). Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in the dermis. Journal of Nutritional Science and Vitaminology, 52(3), 211–215. Minaguchi, J., Koyama, Y., Meguri, N., Hosaka, Y., Ueda, H., Kusubata, M., et al. (2005). Effects of ingestion of collagen peptide on collagen fibrils and glycosaminoglycans in Achilles tendon. Journal of Nutritional Science and Vitaminology, 51(3), 169–174. ˇ urda, L., & Jelen, P. (2008). Batch and continuous hydrolysis of ovine Mišún, D., C whey proteins. Small Ruminant Research, 79(1), 51–56. Moskowitz, R. W. (2000). Role of collagen hydrolysate in bone and joint disease. Seminars in Arthritis and Rheumatism, 30(2), 87–99. Ohara, H., Matsumoto, H., Ito, K., Iwai, K., & Sato, K. (2007). Comparison of quantity and structures of hydroxyproline-containing peptides in human blood after oral ingestion of gelatin hydrolysates from different sources. Journal of Agricultural and Food Chemistry, 55(4), 1532–1535. Rajapakse, N., Mendis, E., Byun, H. G., & Kim, S. K. (2005). Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems. The Journal of Nutritional Biochemistry, 16(9), 562–569. Ren, J., Zhao, M., Shi, J., Wang, J., Jiang, Y., Cui, C., et al. (2008). Purification and identification of antioxidant peptides from grass carp muscle hydrolysates by consecutive chromatography and electrospray ionization-mass spectrometry. Food Chemistry, 108(2), 727–736. Sun, L., Zhang, Y., & Zhuang, Y. (2013). Antiphotoaging effect and purification of an antioxidant peptide from tilapia (Oreochromis niloticus) gelatin peptides. Journal of Functional Foods, 5(1), 154–162. Tanaka, M., Koyama, Y., & Nomura, Y. (2009). Effects of collagen peptide ingestion on UV-B-induced skin damage. Bioscience, Biotechnology, and Biochemistry, 73(4), 930–932. Terra, V. A., Souza-Neto, F. P., Pereira, R. C., Silva, T. N. X., Costa, A. C. C., Luiz, R. C., et al. (2012). Time-dependent reactive species formation and oxidative stress damage in the skin after UVB irradiation. Journal of Photochemistry and Photobiology B: Biology, 109, 34–41. Trentham, D. E., Dynesius-Trentham, R. A., Orav, E. J., Combitchi, D., Lorenzo, C., Sewell, K. L., et al. (1993). Effects of oral administration of type II collagen on rheumatoid arthritis. Science, 261(5129), 1727–1730. United States Pharmacopeial Convention Council of Experts (2007). Simulated gastric and intestinal fluids, TS. In Board of Trustees (Ed.), United States pharmacopeial convention and national formulary (pp. 2728). Rockville: United States Pharmacopeial Convention Inc. Wangtueai, S., & Noomhorm, A. (2009). Processing optimization and characterization of gelatin from lizardfish (Saurida spp.) scales. LWT – Food Science and Technology, 42(4), 825–834. Xu, Z. S., Wang, X. Y., Xiao, D. M., Hu, L. F., Lu, M., Wu, Z. Y., et al. (2011). Hydrogen sulfide protects MC3T3-E1 osteoblastic cells against H2O2-induced oxidative damage – Implications for the treatment of osteoporosis. Free Radical Biology and Medicine, 50(10), 1314–1323. You, L., Zhao, M., Regenstein, J. M., & Ren, J. (2010). Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chemistry, 120(3), 810–816.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Hydrolysis kinetics and radical-scavenging activity of gelatin under simulated gastrointestinal digestion Lin Wang a,⇑, Qiufang Liang a, Qiuhong Chen a, Junmin Xu b, Zhujun Shi a, Zhenbin Wang a, Yang Liu c, Haile Ma a a b c
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, PR China Zhenjiang Kehua Aquaculture Development Company Limited, Zhenjiang 212134, PR China Zhenjiang Entry-Exit Inspection and Quarantine Burea, Zhenjiang 212008, PR China
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 11 March 2014 Accepted 23 April 2014 Available online 2 May 2014 Keywords: Gelatin Hydrolysis kinetics Radical-scavenging activity Simulated gastrointestinal digestion
a b s t r a c t The hydrolysis kinetics and radical-scavenging activity of gelatin were investigated under simulated gastrointestinal digestion in this study. In the gastric phase, the degree of gelatin hydrolysis increased from 0.17% to 1.20%, while the DPPH radical-scavenging rate increased from 6.27% to 24.56%. Further digestion in the intestinal phase brought the degree of hydrolysis and radical-scavenging rate to 26.08% and 44.76%, respectively. After digestion, the gelatin hydrolysates were separated into two fractions by ultrafiltration. The fraction with an average molecular weight of 312.98 Da exhibited the higher yield (78.26%) and radical-scavenging activity (IC50 = 2.09 mg/ml), suggesting the high digestibility and bioactivity of gelatin after oral administration. The fraction was further purified with multi-step column chromatography and identified to be Gly-Pro-Met (303.38 Da) by UPLC–ESI-MS. These results may help us to better understand its physiological effects and to use it properly in foods and pharmaceuticals. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Gelatin, a denatured form of collagen, has long been used in foods, pharmaceuticals and cosmetics. It has been used to improve blood circulation, arrest bleeding and to improve joint condition in traditional medicine (Ohara, Matsumoto, Ito, Iwai, & Sato, 2007). Recent animal experiments and human trials have also suggested that the oral ingestion of gelatin had beneficial effects on skin ageing (Tanaka, Koyama, & Nomura, 2009), osteoarthritis and osteoporosis (Moskowitz, 2000), rheumatoid arthritis (Trentham et al., 1993), properties of nail, hair and Achilles tendon (Matsuda et al., 2006; Minaguchi et al., 2005). Free radicals are very reactive molecules, which can react with every cellular component and give rise to functional and morphologic disturbances in cells (Martínez-Cayuela, 1995). There is evidence to implicate oxygen free radicals as important pathologic mediators in many human diseases and ageing processes, such as skin ageing (Terra et al., 2012), osteoarthritis (Davies, Guilak, Weinberg, & Fermor, 2008), osteoporosis (Xu et al., 2011), rheumatoid arthritis (Bhowmick, Chakraborti, Gudi, Moideen, & Shetty, 2008) and hair and nail damage (Fernández et al., 2011; Khengar
et al., 2010). Recently, the antioxidant activity of collagen peptides has been widely demonstrated in many different oxidative systems (Gómez-Guillén, Giménez, López-Caballero, & Montero, 2011). Therefore, we hypothesized that the physiological effects of gelatin was related with antioxidant activity. However, it is not clear how gelatin is digested and further exerts antioxidant activity in vivo. In vitro, simulated gastrointestinal digestion is extensively used since it is rapid, safe, inexpensive and does not have the same ethical restrictions as in vivo methods (Liang et al., 2012). In recent years, the method has already been developed for the bioavailability and bioactivity assessment of protein. Thus, it might also be reasonable to investigate the hydrolysis kinetics and radical-scavenging activity of gelatin after oral administration. In this study, the hydrolysis kinetics and radical-scavenging activity of gelatin was investigated under simulated gastrointestinal digestion, which may help us to better understand its physiological effects and to use it properly in foods and pharmaceuticals.
2. Materials and methods 2.1. Materials
⇑ Corresponding author. Tel./fax: +86 511 88780201. E-mail address: [email protected] (L. Wang). http://dx.doi.org/10.1016/j.foodchem.2014.04.083 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
The gelatin was prepared from scales of the Nile tilapia (Oreochromis niloticus) following the method of Wangtueai and
L. Wang et al. / Food Chemistry 163 (2014) 1–5
Noomhorm (2009). Simulated gastric and intestinal fluids were prepared according to the United States Pharmacopeial Convention Council of Experts (2007).
2.3. Assay of the degree of hydrolysis (DH) The DH of gelatin was determined using the o-phthaldialdehyde (OPA) method (Marambe, Shand, & Wanasundara, 2008). The average chain length (ACL) and average molecular weight (AMW) of the ˇ urda, & hydrolysates were calculated by the equation (Mišún, C Jelen, 2008): ACL = 100/DH, AMM = ACL 90. 2.4. Determination of DPPH radical-scavenging activity The DPPH radical-scavenging activity was determined as described by Hu et al. with slight modifications (Hu, Xu, Chen, & Yang, 2004). Two millilitre of sample was added to 2 ml of 0.2 mM ethanol solution of DPPH. After shaking vigorously, the absorbance was monitored immediately at 517 nm and recorded every 5 min interval until 240 min. The scavenging rate was calculated as follows: Scavenging rate (%) = [(A0 A1)/A0] 100, where A0 was the absorbance of the control and A1 was the absorbance of the sample. 2.5. Separation of gelatin hydrolysates by ultrafiltration The ultrafiltration was performed on a Millipore Pellicon system equipped with a membrane with the nominal molecular weight cut-off of 1 kDa. The digests were pumped to the membrane surface and the filtrate was collected, while the retentate was recirculated until the absorbance of the filtrate at 220 nm was close to 0. Then, the filtrate (F1) and retentate (F2) were concentrated and lyophilized separately. The yield was obtained by comparing hydroxyproline content in the fractions with that in gelatin. The hydroxyproline content was determined as recommended by ISO 3496 (Anonymous, 1994). 2.6. Purification of radical-scavenging peptide by chromatography The sample was loaded on a Toyopearl SP-650M column (2.5 30 cm) equilibrated with 0.05 M sodium acetate buffer (pH 4.5), and eluted with a linear gradient of NaCl (0–1.0 M) in the same buffer at 1.0 ml/min. Then, the potent fraction was subjected to a Sephadex G10 column (1.5 75 cm) eluted with distilled water at 0.5 ml/min. Subsequently, the active fraction was injected onto an Agilent ZORBAX Eclipse XDB C18 semi-preparative column (9.4 250 mm, 5 lm) and separated by a gradient of acetonitrile (2–10% in 0–15 min, 10–30% in 15–25 min, 30–70% in 25–30 min) at 2.5 ml/min. The radical-scavenging activity was expressed as the DPPH radical-scavenging rate per absorbance unit. The detection wavelength was 220 nm.
Degree of hydrolysis (%)
The simulated digestion of gelatin was carried out by modification of the method of Chen and Li (2012). Briefly, the gelatin was hydrolyzed with simulated gastric fluid (pH 1.2) at 1:20 (w/v) for 4 h at 37 °C in a shaking incubator at 150 rpm. Then, pancreatin was added at 1:100 (w/v) after adjusting the concentration of the KH2PO4 buffer to 0.05 M and to pH 6.8 with 1.0 M NaOH. The mixture was further incubated for 6 h. To terminate the digestion, the samples were kept in boiling water for 10 min. Finally, the digests were cooled and centrifuged at 10,000g for 15 min. The control was conducted in the same manner, with distilled water used instead of gelatin.
50
Intestinal phase
30
40
30
20
20 10 10
0
0
2
4
6
8
10
0
Digestion time (h) Fig. 1. Kinetics of hydrolysis and radical-scavenging of gelatin under stimulated gastrointestinal digestion. ( ) Degree of hydrolysis; ( ) DPPH radical-scavenging rate.
Table 1 The yield, AMW and IC50 value of gelatin and its hydrolysates.
F1 F2 Gelatin
Yielda (%)
AMW (Da)
IC50 (mg/ml)
78.26 ± 4.28a 14.29 ± 1.75b
312.98 ± 2.04c 1822.46 ± 66.74b 52,714.81 ± 3183.33a
2.09 ± 0.02c 3.17 ± 0.03b 17.22 ± 0.76a
a Mean value ± standard deviation from three separate samples. Values followed by different letters are significantly different at P < 0.05. AMW, average molecular weight; IC50, the amount of antioxidant necessary to decrease the initial DPPH concentration by 50%; , no data.
2.7. Identification of amino acid sequences by UPLC–ESI-MS The purified peptide was identified using a Waters ACQUITY UPLC coupled to SYNAPT Q-TOF MS with electrospray ionization. The sample was dissolved in distilled water, and 5 ll was then injected onto a BEH130 C18 column (2.1 150 mm, 1.7 lm) with a linear gradient of acetonitrile (0–20% in 20 min) at 0.2 ml/min. The mass spectrometer was operated in positive ion mode with a capillary voltage 3.5 kV, ion source temperature 100 °C, desolvation temperature 300 °C, desolvation gas flow 10 l/min, cone voltage 45 V, cone gas flow 1 l/min and scanning range 50–1000 m/z. The amino acid sequence was analyzed using BioLynx software.
1.2 1.0
Absorbance at 517 nm
2.2. Simulated gastrointestinal digestion
Gastric phase
DPPH radical-scavenging rate (%)
2
0.8 0.6 0.4 0.2 0.0
0
60
120
180
240
Time (min) Fig. 2. DPPH radical-scavenging activity of gelatin and its hydrolysates (5 mg/ml), compared to BHT and a-tocopherol (0.2 mg/ml). (j) Control; ( ) F1; ( ) F2; ( ) gelatin; ( ) BHT; ( ) a-tocopherol.
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L. Wang et al. / Food Chemistry 163 (2014) 1–5
20
0 mV
% 150
a
15 100 10 50 5
0 0
60
0 mV
120
% 800
C
b
30
0 240
180
600 20 400
10
200
B D
A 0
0 0
1500
60
120
180
360
mAu
540
% 1200
cc
1200
900
900 600 600 300
300
0
0
5
10
15
20
25
0 30
Elution time (min) Fig. 3. Chromatographic and antioxidant profiles of F1 using Toyopearl SP-650M (a), Sephadex G10 (b), Eclipse XDB C18 semi-preparative column (c). ( concentration (0–1.0 M); ( ) voltage (mV) or absorbance (mAu); ( ) as well as histogram, DPPH radical-scavenging rate per absorbance unit (%).
2.8. Statistical analysis The data are presented as mean ± standard deviation of three determinations. Statistical analyses were performed using oneway analysis of variance. Multiple comparisons of means were done by LSD test. A P value F2 (3.17 mg/ml) > gelatin (17.22 mg/ml). Combined with the bioavailability, it was indicated that the F1 fraction might be responsible for the various physiological effects of gelatin. Thus, the F1 fraction was used for further purification and identification of the radical-scavenging peptide.
3.2. Separation of gelatin hydrolysates The gelatin hydrolysates after digestion were separated into two fractions by ultrafiltration. Most of the gelatin hydrolysates (78.26%) were recovered in F1, with a lower AMW of 312.98 Da, while the yields of F2 were only 14.29% (Table 1). This suggests that gelatin was digested almost completely in the gastrointestinal tract into oligopeptides or amino acids, which could be absorbed by the small intestine. Therefore, it seemed that the gelatin was digestible and absorbable after oral administration, although the final DH was still lower than amaranth protein (35.8%) and loach protein (46.6%) (Delgado, Tironi, & Añón, 2011; You et al., 2010).
3.4. Purification of radical-scavenging peptide The radical-scavenging peptide was purified by multi-step column chromatography (Fig. 3). The F1 fraction was separated by a Toyopearl SP-650M cation-exchange column, and the fraction with the highest activity (90–120 min) was bound by the column and washed out at a low NaCl concentration (Fig. 3a). It signified that the fraction might be weakly basic, which agrees with Ren’s report that basic peptides were more effective than acidic (Ren et al., 2008). The fraction with the highest activity was then separated into four subfractions (A–D) by a Sephadex G10 molecular exclusion column (Fig. 3b), in which molecules are separated by their size. Both fractions A and B were peptides, but only fraction B with the lower molecular weight had radical-scavenging activity. Fractions C and D might be free amino acids and acetates, respectively. The fraction B was further separated by a C18 semi-preparative column, which was based on the hydrophobic or hydrophilic property of a peptide (Fig. 3c). The fraction displaying the strongest activity was eluted in 5–6 min with an IC50 value of 28.05 lg/ml, which was almost 75-fold greater than the gelatin hydrolysates (2.09 mg/ ml). This indicated that the radical-scavenging peptide was
3.3. Radical-scavenging activity of gelatin hydrolysates Fig. 2 illustrates the kinetic behaviour of gelatin and its hydrolysates as scavengers toward the DPPH radical. All samples tested exhibited remarkable radical-scavenging activity, although this was lower than a-tocopherol and BHT. The a-tocopherol reacted rapidly with DPPH radical and reached a steady state in 5 min, while the BHT was stable after 150 min. However, the F1 fraction reached a plateau at 180 min, and the gelatin and F2 fraction did not reach steady states until 240 min, which suggested that they were typical slow-acting antioxidants. From the IC50 values (Table 1), radical-scavenging activity decreased in the order: F1 (2.09 mg/
10
0.0
a
TOF MS ES+ TIC
%
4.66e4
0.0
0.5
1.0
1.5
2.0
2.5
b
3.0
3.5
264.1
min
TOF MS ES+ 625
%
304.0
144.0 80.9
122.0 82.9 104.0 70.0
50
90.9
100
112.0
134.0138.9 164.9 148.1158.0
150
191.0 196.8198.8 184.0
200
219.0
282.1
229.1 256.8 266.1 240.1 246.0
250
284.1300.1
300
322.1
344.1 337.1
350 m/z
Fig. 4. Identification of amino acid sequences of the purified peptide by UPLC–ESI-MS. (a) UPLC profile with BEH130 C18 column; (b) mass spectrum using ESI-MS.
L. Wang et al. / Food Chemistry 163 (2014) 1–5
hydrophilic, which contradicts most reports which show that the hydrophobic peptides present in hydrolysates had a greater impact on antioxidant activity (Rajapakse, Mendis, Byun, & Kim, 2005). 3.5. Identification of amino acid sequences of radical-scavenging peptide The UPLC chromatogram reveals a major peak (1.09 min) corresponding to the target peptide (Fig. 4a). The target peptide was further determined to be Gly-Pro-Met (303.38 Da) by ESI-MS (Fig. 4b), on which there was no report yet. It is commonly believed that the antioxidant activity of a peptide is associated with molecular size. Most studies have shown that antioxidant peptides usually contained less than 20 amino acid residues and resulted in a better chance to cross the intestinal barrier and exert biological effects (Kou et al., 2013). In recent years, several antioxidant peptides have been isolated from collagen or other collagenous sources, and the molecular weights of these peptides were not more than 1500 Da (Gómez-Guillén et al., 2011). The molecular weight of this peptide was 303.38 Da, which supported the fact that the peptide with high antioxidant activity had lower molecular weight. Furthermore, amino acid compositions and sequences of a peptide may also be important to antioxidant activity. It is known that Met can be readily oxidized to Met sulfoxide by reactive oxygen species and Pro is often included in antioxidative peptides (Elias, Kellerby, & Decker, 2008), which would contribute to the antioxidant activity of this peptide. However, the antioxidant activity of a peptide is higher than free amino acids, which could be attributed to the unique physico-chemical properties of amino acid sequences (Elias et al., 2008). The Gly-Pro sequence is often present in antioxidant peptides and has been proposed to play an important role in antioxidant activity (Sun, Zhang, & Zhuang, 2013). This peptide also contained this sequence, which might result in the high antioxidant activity. 4. Conclusion The hydrolysis kinetics and radical-scavenging activity of gelatin were investigated under simulated gastrointestinal digestion in this study. The gelatin could be digested nearly completely into oligopeptides or amino acids, suggesting high digestibility by oral administration. The gelatin hydrolysates exhibited high radicalscavenging activity, which might be responsible for the various physiological effects of gelatin. Moreover, a radical-scavenging peptide was purified and identified from the gelatin hydrolysates, but further studies are needed to validate its radical-scavenging activity and physiological effects after synthesizing. Acknowledgements This research was supported by Scientific Research Starting Foundation for Talented Scholars of Jiangsu University (10JDG053) and China Postdoctoral Science Foundation (2011M500872). References Anonymous (1994). Meat and meat products – Determination of hydroxyproline content. International standard. ISO 3496. Bhowmick, K., Chakraborti, G., Gudi, N. S., Moideen, A. V. K., & Shetty, H. V. (2008). Free radical and antioxidant status in rheumatoid arthritis. Indian Journal of Rheumatology, 3(1), 8–12. Chen, M., & Li, B. (2012). The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion. Innovative Food Science and Emerging Technologies, 16, 341–348.
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