Food Chemistry 172 (2015) 407–415
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of hydrolysis time on the physicochemical and functional properties of corn glutelin by Protamex hydrolysis Xi-qun Zheng, Jun-tong Wang, Xiao-lan Liu ⇑, Ying Sun, Yong-jie Zheng, Xiao-jie Wang, Yue Liu Heilongjiang Provincial University Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, PR China
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Article history: Received 5 March 2014 Received in revised form 25 July 2014 Accepted 15 September 2014 Available online 1 October 2014 Keywords: Corn glutelin Protamex Hydrolysates Physicochemical properties Functionalities Antioxidant activity
a b s t r a c t The physicochemical and functional properties, such as surface hydrophobicity, disulphide bond content, thermal properties, molecular weight distribution, antioxidant properties, of corn glutelin hydrolysates catalysed by Protamex at different hydrolysis times were evaluated. The hydrolysis influenced the properties of corn glutelin significantly, and not only decreased its molecular weight and disulphide bond content, but also eventually transformed its insoluble native aggregates to soluble aggregates during the hydrolysis process. Corn glutelin hydrolysates were found to have a higher solubility, which was associated with their relatively higher foaming and emulsifying properties compared to the original glutelin. Corn glutelin and its hydrolysates maintained a high thermal stability. In addition, the hydrolysates exhibited excellent antioxidant properties measured through in vitro assays, namely DPPH and OH radical scavenging activity, Fe2+-chelating capacity and reducing power; the values were 58.86%, 82.64%, 29.92% and 0.236% at 2.0 mg/mL, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Proteins are important nutrients in human and animal diet, whereas the lack of protein rich diet causes malnutrition. There are many people who do not get adequate amounts of protein (Chambal, Bergenståhl, & Dejmek, 2012). Cereals and legumes, which are the cheapest and most abundant protein food, have become a major source of dietary protein in many developing countries (Du et al., 2012). Corn, which is the third most widely cultivated cereal in the world after wheat and rice, supplies approximately 42 million tons of protein per annum (Malumba, Vanderghem, Deroanne, & Béra, 2008). China is the second largest country for production and consumption of corn (http://www.igc. int/en/grainsupdate/sd.aspx?crop=Maize). Corn gluten meal (CGM), a major co-product of corn wet-milling process, contains 62–71% (w/w) protein (Hardwick & Glatz, 1989). Apart from its rich protein content, it has been reported that CGM proteins also have special functionalities, such as antioxidative activity (Zheng, Liu, Wang, Lin, & Li, 2006; Zhou, Sun, & Canning, 2012), angiotensin I converting enzyme-inhibitory activity (Suh, Whang, Kim, Bae, & Noh, 2003) and alcohol metabolism activity (Ma, Zhang, Yu, He, ⇑ Corresponding author at: College of Food and Bioengineering, 42 Wenhua Street, Qiqihar 161006, Heilongjiang Province, PR China. Tel.: +86 04522738341; fax: +86 04522725454. E-mail address: [email protected] (X.-l. Liu). http://dx.doi.org/10.1016/j.foodchem.2014.09.080 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
& Zhang, 2012). The major protein fractions of CGM consist of approximately 65% (w/w) of zein and 30% (w/w) of glutelin (Hardwick & Glatz, 1989). Zein contains a high proportion of hydrophobic amino acids, such as glutamine (21.4%), leucine (19.3–21.1%), alanine (8.3–10.5%), proline (9.0–10.5%), and isoleucine (5.7–6.2%) (Pomes, 1971), so it is soluble in alcohol-aqueous solution. The glutelin molecule has excessive intra and intermolecular disulphide bonds and hydrophobic interactions (Paraman, Hettiarachchy, Schaefer, & Beck, 2006), and it is soluble only in dilute acid or alkali solutions. Due to these characteristics, glutelins have limited solubility in aqueous systems under the conditions of pH occurring in most food products, and hence CGM has rarely been applied in food industry. Studies have been performed for improving CGM protein’s solubility and increasing its physiological functional properties. Limited hydrolysis of corn gluten by using commercial proteases not only improved its water solubility, but also the prepared hydrolysates showed powerful angiotensin converting enzyme inhibitory (ACEI) activity (Kim, Whang, Kim, Koh, & Suh, 2004; Suh et al., 2003). Several CGM hydrolysates with improved processing functionality and antioxidative activities also have been reported (Zhang, Luo, & Wang, 2011; Zheng, Liu, & Liu, 2012; Zheng et al., 2006; Zhou et al., 2012). Zheng et al. reported that the aqueous solubility of two kinds of extruded corn gluten meal hydrolysates was increased by using a commercial protease Alcalase and fermentation with Bacillus natto, respectively; the hydrolysates also
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exhibited antioxidative activity (Zheng et al., 2006, 2012). The effect of acid and base treatments on the structural, rheological, and antioxidant properties of a-zein was investigated, and the antioxidant activity of treated a-zein was improved (Zhang et al., 2011). Corn protein hydrolysates, prepared by three types of microbial proteases, were separated by sequential ultra-filtration into several fractions, and the fraction of 1–3 kDa showed the highest activity in scavenging peroxyl radicals. Additionally, this fraction inhibited lipid oxidation in ground beef both at 250 and 500 lg/g levels (Zhou et al., 2012). There are several reports on modifying the functionality of isolated glutelins from barley. Zhao et al. found that the structure and functional properties of a deamidated barley glutelin could be improved (Zhao, Tian, & Chen, 2011). In addition, barley glutelin hydrolysates with antioxidant capacity were obtained by enzymatic hydrolysis (Xia, Bamdad, Gänzle, & Chen, 2012). However, in the available literature, no reports could be found about the use of enzymatic proteolysis for modifying the functional properties of corn glutelin. The aim of the present work was to evaluate the feasibility of improving the functional properties of corn glutelin in an aqueous solution by Protamex hydrolysis, investigate the effects of hydrolysis time on the physicochemical and functional properties (solubility, emulsification, foaming properties and antioxidative activity) of corn glutelin, thus providing useful information regarding its potential commercial applications.
2. Materials and methods 2.1. Materials and chemicals Corn gluten meal (CGM) was obtained from Longfeng Corn Co. Ltd. (Heilongjiang, China), with a total protein content of 56.42% (w/w). Protamex from Bacillus subtilis was purchased from Novo Nordisk (Bagsvaerd, Denmark), and a-amylase was purchased from Aoboxing Biotechnology Co. Ltd. (Beijing, China). 1-Anilinonaphthalene-8-sulphonic acid (ANS) and 5,50 -dithiobis-2-nitrobenzoic acid (DNTB) were purchased from Sigma Chemical Co. Ltd. (California, America). Superdex peptide (10/300) column and gel filtration calibration kit were purchased from GE Life sciences (USA). All other chemicals were of reagent grade unless otherwise stated. 2.2. Extrusion of CGM
2.4. Pigment and zein removal of pretreated CGM CGM pretreated by extrusion and starch removal was extracted with acetone for 30 min (acetone:CGM = 10:1, w/w) to remove pigment, and the extract was centrifuged at 4000g for 15 min at room temperature. The residues were collected and dispersed with 70% ethanol water solution (ethanol:CGM = 10:1, w/w) at 60 °C for 2 h, followed by centrifugation at 4000g for 15 min. The precipitate was collected. 2.5. Extraction of glutelin The residues were dispersed with 0.1 M NaOH (residues:NaOH = 1:10, w/v) at 60 °C for 2 h, then centrifuged at 4000g for 15 min at room temperature. The process was repeated twice. The supernatants were adjusted to pH 4.8 using 4 M HCl and centrifuged at 4000g for 15 min. The residues were washed three times with 70% ethanol–water solution and distilled water, respectively. Finally, the residues were freeze-dried and then ground and sieved to collect the fraction of 0.05). The high Tp value usually implies high thermal stability. Thus, the high Tp value of corn glutelin and its hydrolysates indicated that both of them exhibited good thermal stability which was not reduced by Protamex hydrolysis. As shown in Table 2, the thermal enthalpy (DH) of the corn glutelin was 192.00 °C, which was significantly higher than that of glutelin hydrolysates (p < 0.05). This indicated that hydrolysis could markedly decrease the DH of corn glutelin, and that the ordered structures and aggregation tendency of the protein were decreased during the hydrolysis process. Many factors can effectively influence the thermal properties of proteins, such as hydrogen bonds, hydrophobic interactions, –SH/–SS– and hydration (Bouaouina, Desrumaux, Loisel, & Legrand, 2006; Shimada & Cheftel, 1988). The synergism among these factors resulted in the complex changes observed in Tp and DH.
3.6. Solubility
Fig. 2. (A) Molecular weight distribution profiles of glutelin hydrolysate at hydrolysis time of 15 min (a), 30 min (b), 60 min (c), 90 min (d), 120 min (e), and 150 min (f). (B) Relative area (%) of the peptide peaks in glutelin hydrolysate fractions. (C) Molecular weight standard on HPLC with a gel filtration column Superdex peptide PE 10/300, 1 Blue dextran 2000 (2,000 kDa), 2 aprotinine (6.5 kDa), 3 bacitracin (1.4 kDa), 4 glutathione disulfide (0.6 kDa), 5 glutathione reduced (0.3 kDa).
peptide fragments (Mw < 1 kDa). Fig. 2C summarises the quantitative changes of these three fractions during hydrolysis, represented by the area of each fraction relative to the total area of profiles. The results show that the hydrolysis by Protamex resulted in large-
Solubility is an important prerequisite for the functional properties of food proteins and directly affects their applications in the food industry (Zhao et al., 2012). The solubilities of corn glutelin and glutelin hydrolysates are shown in Table 2. The corn glutelin showed limited solubility (1.99%) in distilled water (pH 7.0). The poor solubility was due to the rigid macromolecule structure of corn glutelin which contained exorbitant intermolecular and intramolecular disulphide bonds and hydrophobic interactions (Paraman et al., 2006). However, even limited hydrolysis by Protamex was able to significantly improve the corn glutelin solubility. The maximum solubility of glutelin hydrolysates was found at 150 min (94.65%). Protein solubility over 90% has been reported for other protein hydrolysates (Chen et al., 2012). Compared with the original glutelin, the improved solubility of glutelin hydrolysates may be attributed to the unfolding of the peptide chains, a size reduction caused by protease hydrolysis, and the exposure of more charged residues and polar groups into the surrounding aqueous phase. These structural changes resulted in an improvement of protein–water interaction, thereby increasing the solubility (Chen et al., 2012).
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3.7. Foaming properties The foam capacity (FC) and foam stability (FS) of corn glutelin and its hydrolysates at pH 7.0 are shown in Fig. 3A. The FC of corn glutelin was 78.17%, which was lower (p < 0.05) than that of rice protein (118.8%) (Zhao et al., 2012). This might be due to the poor solubility of corn glutelin in water at pH 7.0. The FC and FS of glutelin hydrolysates were higher (p < 0.05) than those of corn glutelin. Lee and Chen (2002) also reported that the FC and FS of protein increased after enzymatic hydrolysis. Limited hydrolysis by Protamex significantly improved protein solubility, reduced the molecular weight and the SS content, which facilitated the formation of an interfacial membrane and foam production. The FC and FS of glutelin hydrolysates were also affected by hydrolysis time, and their maximum values were obtained at 15 min. The structure of the corn glutelin was excessively destroyed with longer hydrolysis, which adversely affected FC and FS. Thus, prolonged hydrolysis should be avoided as it leads to undesirable property changes. 3.8. Emulsifying activity and emulsifying stability The emulsifying activity index (EAI) and emulsifying stability index (ESI) of the corn glutelin and its hydrolysates at pH 7.0 are shown in Fig. 3B. The EAI of glutelin hydrolysates was improved and was significant higher (p < 0.05) than that of corn glutelin.
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The EAI was increased from 3.20% to 68.23% when the hydrolysis time was increased from 0 to 15 min, and by further increasing the hydrolysis time from 15 to 150 min, the EAI of glutelin hydrolysates were maintained at a rather high value (more than 60%). The same trend was also observed for the ESI of the glutelin hydrolysates. The increase of EAI reflected the enhanced solubility of corn glutelin after hydrolysis, enabling protein diffusion to the oil/water surface easily. However, this phenomenon was different from that observed for other proteins, such as rice and barley, in which extensive hydrolysis greatly decreased the protein emulsifying activity (Paraman, Hettiarachchy, & Schaefer, 2008). This was due to the fact that extensive hydrolysis prevents the formation of a continuous protein film at the oil/water interface to stabilise the emulsions (Chen, Chi, & Xu, 2011; Xia et al., 2012). In this work, the reason for the stable emulsion capacity of the hydrolysates with different DHs is attributed to the soluble aggregates formed during hydrolysis, which could form a rigid film at the oil/water interface to prevent the close contact of oil droplets and avoid flocculation and coalescence. Similar phenomenon was also found by (Agyare, Addo, & Xiong, 2009). 3.9. Antioxidant properties of corn glutelin hydrolysates The antioxidant properties of samples are assessed using different assays, because there different mechanism of antioxidant action exist (Pan, Jiang, & Pan, 2011). In this study, the antioxidant properties of corn glutelin hydrolysates prepared at different hydrolysis times were evaluated based on their radical scavenging capacity against DPPH and OH, Fe2+-chelating effect and reducing power. Peptide concentrations of 2 mg/mL were used in each assay. Ascorbic acid at concentrations of 0.01 mg/mL was used as positive control. The antioxidant activity of corn glutelin was not tested because it barely dissolved in distilled water. 3.9.1. DPPH radical scavenging ability DPPH is a stable free radical, which can accept protons from an antioxidant to form a stable diamagnetic molecule. The absorbance of DPPH that has accepted proton is lower than that of DPPH free radical (at 517 nm), and there is a quantitative relation between the fading degree of DPPH and the accepted proton-donating number. Therefore, this assay has been widely used to evaluate the antioxidant activity of natural compounds (Lu et al., 2010). The DPPH radical scavenging capacities of corn glutelin hydrolysates are presented in Fig. 4A. All of the samples tested exhibited good scavenging ability against DPPH (41–59%). In particular, the activity of hydrolysates (58.86 ± 1.40%) at hydrolysis time of 120 min was prominently higher than that of others (p < 0.05). The DPPH radical scavenging effects of barley glutelin hydrolysates by Flavourzyme and rapeseed protein hydrolysates were 56–61% at a concentration of 1.0 mg/mL and 50% at the 0.71 mg/mL, respectively (Pan et al., 2011; Xia et al., 2012). Thus, the activity of corn glutelin hydrolysates was comparable to the hydrolysates reported in these studies. The result suggested that corn glutelin hydrolysates contained substances that were electron donors and could react with free radicals to convert them to stable products and terminate the radical chain reaction.
Fig. 3. (A) The foam capacity and foam stability of corn glutelin and its hydrolysate. (B) Emulsifying activity index (EAI) and emulsifying stability index (ESI) of corn glutelin and its hydrolysate.
3.9.2. Hydroxyl radical scavenging ability Hydroxyl radical is the strongest oxidant among the oxygen free radicals. It can activate lipid peroxidation and react with almost any adjacent biomolecules such as DNA, proteins and amino acids (Xia et al., 2012). Since this damage can cause food-spoilage or several diseases in living beings, its removal is probably one of the most effective way to prevent putrefaction of food and occurrence of various diseases (Pan et al., 2011). The hydroxyl radical scavenging activities of corn glutelin hydrolysates are shown in Fig. 4B. The
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Fig. 4. (A) DPPH radical scavenging activity (2.0 mg/mL). (B) hydroxyl radical scavenging activity (2.0 mg/mL). (C) Fe2+-chelating activity (2.0 mg/mL) and (E) reducing power (2.0 mg/mL) of corn glutelin hydrolysates treated by protamex.
hydrolysates exhibited strong inhibitory effect against hydroxyl radicals (71–83%). The activities of the hydrolysates were 82.89 ± 1.53%, 82.45 ± 1.32% and 82.64 ± 0.75% at an hydrolysis time of 15, 90 and 120 min, respectively. The results suggest that corn glutelin hydrolysates with excellent hydroxyl scavenging activity could have the potential to protect food or living system against hydroxyl radical-induced damages.
3.9.3. Fe2+-chelating ability Since certain transition metal ions can catalyse the Haber– Weiss reaction and induce superoxide to become harmful hydroxyl radical, measuring the metal ions chelating ability of compounds is important for evaluating their antioxidant activity (Zhang et al., 2011). The Fe2+-chelating ability of corn glutelin hydrolysates was assayed, and the results are shown in Fig. 4C. The Fe2+-chelating capacity of the hydrolysates increased considerably (10–30%) after an hydrolysis time of 120 min, then reduced slightly until 150 min, while, ascorbic acid at concentrations of 0.01 mg/mL had almost no chelating ability on Fe2+. Xia et al. (2012) reported a similar Fe2+-chelating ability for Flavourzyme hydrolysates (about 23–30%) derived from barley glutelin at 2.0 mg/mL. Compared with the native protein, the enhancement of the metal ion binding power of the hydrolysates might be due to an increased
concentration of carboxylic groups in the side chain of the acidic and basic amino acids (Zhang et al., 2010). 3.9.4. Reducing power There is a direct correlation between antioxidant activity and reducing power of a compound (Pan et al., 2011; Zhang et al., 2011). Compounds with a higher reducing capacity have better abilities to donate electron or hydrogen, leading to the interruption of the free radical chain reactions. As a result, the reducing power assay is often used to evaluate the potential antioxidant activity of natural products (Chen et al., 2012). Fig. 4D describes the reducing power of corn glutelin hydrolysates. This was increased significantly (0.191–0.236) by prolonging the hydrolysis time up to 120 min, and then decreased slightly afterwards. The corn glutelin hydrolysates also exhibited more effective reducing capacity than those of chickpea protein hydrolysates (0.2 at 2 mg/mL) and barley glutelin hydrolysates (0.121 at 2 mg/mL) (Li, Jiang, Zhang, Mu, & Liu, 2008; Xia et al., 2012). These results revealed that corn glutelin hydrolysates had good reducing power. The above results indicated that corn glutelin hydrolysates prepared by Protamex possessed excellent antioxidant activity. The hydroxyl radical scavenging capacity and reducing power were related to hydrolysis time, while the DPPH radical scavenging activity and Fe2+-chelating capacity were slightly affected by
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hydrolysis time. The corn glutelin hydrolysates that were obtained at hydrolysis time of 120 min exhibiting best antioxidant activity using anyone of the assay methods, and the percentage of their small-sized peptide was higher than that of others (Fig. 2C). The results suggested that the antioxidant activity might be influenced by the peptide size. However, Xia et al. (2012) reported that the large-sized peptide fraction (Mw > 10 kDa) in barley glutelin hydrolysates was more effective in DPPH scavenging activity and reducing power, whereas the small-sized peptide fraction (Mw < 1 kDa) possessed stronger Fe2+-chelating capacity and radical scavenging activity against OH. Therefore, investigation of the effect of molecular weight on the functional properties and bioactivities of corn glutelin hydrolysates requires further study. 4. Conclusions Corn glutelin can be effectively hydrolysed by Protamex to obtain glutelin hydrolysates with strong antioxidant activities. The hydrolysis of glutelin increased its hydrophobic surface, but also decreased the molecular weight and disulphide bond of the proteins, and eventually transformed its insoluble native aggregates to soluble aggregates during the hydrolysis process. Both corn glutelin and its hydrolysates maintained a high thermal stability and had similar amino acid composition. On the other hand, the corn glutelin hydrolysates were found to have a higher solubility which most likely resulted in improved foaming and emulsifying properties compared to the original glutelin. Overall, this research indicated that corn glutelin hydrolysates can be potentially used as functional ingredients with an increased antioxidant effect in both food and non-food applications. Acknowledgement This work was supported by National Natural Science Foundation of China (No. 31071629). References Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins, Vols. 9–56. Elsevier Applied Science Publishers, pp. 122–144. Agyare, K. K., Addo, K., & Xiong, Y. L. (2009). Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt. Food Hydrocolloids, 23, 72–81. Beveridge, T., Toma, S., & Nakai, S. (1974). Determination of SH and SS groups in some food proteins using Ellman’s reagent. Journal of Food Science, 39, 49–51. Bouaouina, H., Desrumaux, A., Loisel, C., & Legrand, J. (2006). Functional properties of whey proteins as affected by dynamic high-pressure treatment. International Dairy Journal, 16, 275–284. Cabra, V., Vázquez-Contreras, E., Moreno, A., & Arreguin-Espinosa, R. (2008). The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 a-zein. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1784, 1028–1036. Chambal, B., Bergenståhl, B., & Dejmek, P. (2012). Edible proteins from coconut milk press cake; one step alkaline extraction and characterization by electrophoresis and mass spectrometry. Food Research International, 47, 146–151. Chen, C., Chi, Y. J., & Xu, W. (2011). Comparisons on the functional properties and antioxidant activity of spray-dried and freeze-dried egg white protein hydrolysate. Food and Bioprocess Technology, 5, 2342–2352. Chen, C., Chi, Y.-J., Zhao, M.-Y., & Xu, W. (2012). Influence of degree of hydrolysis on functional properties, antioxidant and ACE inhibitory activities of egg white protein hydrolysate. Food Science and Biotechnology, 21, 27–34. Cromwell, M. E., Hilario, E., & Jacobson, F. (2006). Protein aggregation and bioprocessing. The AAPS Journal, 8, E572–E579. Du, Y., Jiang, Y., Zhu, X., Xiong, H., Shi, S., Hu, J., et al. (2012). Physicochemical and functional properties of the protein isolate and major fractions prepared from Akebia trifoliata var. australis seed. Food Chemistry, 133, 923–929. Halliwell, B. (1978). Superoxide-dependent formation of hydroxyl radicals in the presence of iron chelates: Is it a mechanism for hydroxyl radical production in biochemical systems? FEBS Letters, 92, 321–326.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Effect of hydrolysis time on the physicochemical and functional properties of corn glutelin by Protamex hydrolysis Xi-qun Zheng, Jun-tong Wang, Xiao-lan Liu ⇑, Ying Sun, Yong-jie Zheng, Xiao-jie Wang, Yue Liu Heilongjiang Provincial University Key Laboratory of Processing Agricultural Products, College of Food and Bioengineering, Qiqihar University, Qiqihar 161006, PR China
a r t i c l e
i n f o
Article history: Received 5 March 2014 Received in revised form 25 July 2014 Accepted 15 September 2014 Available online 1 October 2014 Keywords: Corn glutelin Protamex Hydrolysates Physicochemical properties Functionalities Antioxidant activity
a b s t r a c t The physicochemical and functional properties, such as surface hydrophobicity, disulphide bond content, thermal properties, molecular weight distribution, antioxidant properties, of corn glutelin hydrolysates catalysed by Protamex at different hydrolysis times were evaluated. The hydrolysis influenced the properties of corn glutelin significantly, and not only decreased its molecular weight and disulphide bond content, but also eventually transformed its insoluble native aggregates to soluble aggregates during the hydrolysis process. Corn glutelin hydrolysates were found to have a higher solubility, which was associated with their relatively higher foaming and emulsifying properties compared to the original glutelin. Corn glutelin and its hydrolysates maintained a high thermal stability. In addition, the hydrolysates exhibited excellent antioxidant properties measured through in vitro assays, namely DPPH and OH radical scavenging activity, Fe2+-chelating capacity and reducing power; the values were 58.86%, 82.64%, 29.92% and 0.236% at 2.0 mg/mL, respectively. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Proteins are important nutrients in human and animal diet, whereas the lack of protein rich diet causes malnutrition. There are many people who do not get adequate amounts of protein (Chambal, Bergenståhl, & Dejmek, 2012). Cereals and legumes, which are the cheapest and most abundant protein food, have become a major source of dietary protein in many developing countries (Du et al., 2012). Corn, which is the third most widely cultivated cereal in the world after wheat and rice, supplies approximately 42 million tons of protein per annum (Malumba, Vanderghem, Deroanne, & Béra, 2008). China is the second largest country for production and consumption of corn (http://www.igc. int/en/grainsupdate/sd.aspx?crop=Maize). Corn gluten meal (CGM), a major co-product of corn wet-milling process, contains 62–71% (w/w) protein (Hardwick & Glatz, 1989). Apart from its rich protein content, it has been reported that CGM proteins also have special functionalities, such as antioxidative activity (Zheng, Liu, Wang, Lin, & Li, 2006; Zhou, Sun, & Canning, 2012), angiotensin I converting enzyme-inhibitory activity (Suh, Whang, Kim, Bae, & Noh, 2003) and alcohol metabolism activity (Ma, Zhang, Yu, He, ⇑ Corresponding author at: College of Food and Bioengineering, 42 Wenhua Street, Qiqihar 161006, Heilongjiang Province, PR China. Tel.: +86 04522738341; fax: +86 04522725454. E-mail address: [email protected] (X.-l. Liu). http://dx.doi.org/10.1016/j.foodchem.2014.09.080 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
& Zhang, 2012). The major protein fractions of CGM consist of approximately 65% (w/w) of zein and 30% (w/w) of glutelin (Hardwick & Glatz, 1989). Zein contains a high proportion of hydrophobic amino acids, such as glutamine (21.4%), leucine (19.3–21.1%), alanine (8.3–10.5%), proline (9.0–10.5%), and isoleucine (5.7–6.2%) (Pomes, 1971), so it is soluble in alcohol-aqueous solution. The glutelin molecule has excessive intra and intermolecular disulphide bonds and hydrophobic interactions (Paraman, Hettiarachchy, Schaefer, & Beck, 2006), and it is soluble only in dilute acid or alkali solutions. Due to these characteristics, glutelins have limited solubility in aqueous systems under the conditions of pH occurring in most food products, and hence CGM has rarely been applied in food industry. Studies have been performed for improving CGM protein’s solubility and increasing its physiological functional properties. Limited hydrolysis of corn gluten by using commercial proteases not only improved its water solubility, but also the prepared hydrolysates showed powerful angiotensin converting enzyme inhibitory (ACEI) activity (Kim, Whang, Kim, Koh, & Suh, 2004; Suh et al., 2003). Several CGM hydrolysates with improved processing functionality and antioxidative activities also have been reported (Zhang, Luo, & Wang, 2011; Zheng, Liu, & Liu, 2012; Zheng et al., 2006; Zhou et al., 2012). Zheng et al. reported that the aqueous solubility of two kinds of extruded corn gluten meal hydrolysates was increased by using a commercial protease Alcalase and fermentation with Bacillus natto, respectively; the hydrolysates also
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exhibited antioxidative activity (Zheng et al., 2006, 2012). The effect of acid and base treatments on the structural, rheological, and antioxidant properties of a-zein was investigated, and the antioxidant activity of treated a-zein was improved (Zhang et al., 2011). Corn protein hydrolysates, prepared by three types of microbial proteases, were separated by sequential ultra-filtration into several fractions, and the fraction of 1–3 kDa showed the highest activity in scavenging peroxyl radicals. Additionally, this fraction inhibited lipid oxidation in ground beef both at 250 and 500 lg/g levels (Zhou et al., 2012). There are several reports on modifying the functionality of isolated glutelins from barley. Zhao et al. found that the structure and functional properties of a deamidated barley glutelin could be improved (Zhao, Tian, & Chen, 2011). In addition, barley glutelin hydrolysates with antioxidant capacity were obtained by enzymatic hydrolysis (Xia, Bamdad, Gänzle, & Chen, 2012). However, in the available literature, no reports could be found about the use of enzymatic proteolysis for modifying the functional properties of corn glutelin. The aim of the present work was to evaluate the feasibility of improving the functional properties of corn glutelin in an aqueous solution by Protamex hydrolysis, investigate the effects of hydrolysis time on the physicochemical and functional properties (solubility, emulsification, foaming properties and antioxidative activity) of corn glutelin, thus providing useful information regarding its potential commercial applications.
2. Materials and methods 2.1. Materials and chemicals Corn gluten meal (CGM) was obtained from Longfeng Corn Co. Ltd. (Heilongjiang, China), with a total protein content of 56.42% (w/w). Protamex from Bacillus subtilis was purchased from Novo Nordisk (Bagsvaerd, Denmark), and a-amylase was purchased from Aoboxing Biotechnology Co. Ltd. (Beijing, China). 1-Anilinonaphthalene-8-sulphonic acid (ANS) and 5,50 -dithiobis-2-nitrobenzoic acid (DNTB) were purchased from Sigma Chemical Co. Ltd. (California, America). Superdex peptide (10/300) column and gel filtration calibration kit were purchased from GE Life sciences (USA). All other chemicals were of reagent grade unless otherwise stated. 2.2. Extrusion of CGM
2.4. Pigment and zein removal of pretreated CGM CGM pretreated by extrusion and starch removal was extracted with acetone for 30 min (acetone:CGM = 10:1, w/w) to remove pigment, and the extract was centrifuged at 4000g for 15 min at room temperature. The residues were collected and dispersed with 70% ethanol water solution (ethanol:CGM = 10:1, w/w) at 60 °C for 2 h, followed by centrifugation at 4000g for 15 min. The precipitate was collected. 2.5. Extraction of glutelin The residues were dispersed with 0.1 M NaOH (residues:NaOH = 1:10, w/v) at 60 °C for 2 h, then centrifuged at 4000g for 15 min at room temperature. The process was repeated twice. The supernatants were adjusted to pH 4.8 using 4 M HCl and centrifuged at 4000g for 15 min. The residues were washed three times with 70% ethanol–water solution and distilled water, respectively. Finally, the residues were freeze-dried and then ground and sieved to collect the fraction of 0.05). The high Tp value usually implies high thermal stability. Thus, the high Tp value of corn glutelin and its hydrolysates indicated that both of them exhibited good thermal stability which was not reduced by Protamex hydrolysis. As shown in Table 2, the thermal enthalpy (DH) of the corn glutelin was 192.00 °C, which was significantly higher than that of glutelin hydrolysates (p < 0.05). This indicated that hydrolysis could markedly decrease the DH of corn glutelin, and that the ordered structures and aggregation tendency of the protein were decreased during the hydrolysis process. Many factors can effectively influence the thermal properties of proteins, such as hydrogen bonds, hydrophobic interactions, –SH/–SS– and hydration (Bouaouina, Desrumaux, Loisel, & Legrand, 2006; Shimada & Cheftel, 1988). The synergism among these factors resulted in the complex changes observed in Tp and DH.
3.6. Solubility
Fig. 2. (A) Molecular weight distribution profiles of glutelin hydrolysate at hydrolysis time of 15 min (a), 30 min (b), 60 min (c), 90 min (d), 120 min (e), and 150 min (f). (B) Relative area (%) of the peptide peaks in glutelin hydrolysate fractions. (C) Molecular weight standard on HPLC with a gel filtration column Superdex peptide PE 10/300, 1 Blue dextran 2000 (2,000 kDa), 2 aprotinine (6.5 kDa), 3 bacitracin (1.4 kDa), 4 glutathione disulfide (0.6 kDa), 5 glutathione reduced (0.3 kDa).
peptide fragments (Mw < 1 kDa). Fig. 2C summarises the quantitative changes of these three fractions during hydrolysis, represented by the area of each fraction relative to the total area of profiles. The results show that the hydrolysis by Protamex resulted in large-
Solubility is an important prerequisite for the functional properties of food proteins and directly affects their applications in the food industry (Zhao et al., 2012). The solubilities of corn glutelin and glutelin hydrolysates are shown in Table 2. The corn glutelin showed limited solubility (1.99%) in distilled water (pH 7.0). The poor solubility was due to the rigid macromolecule structure of corn glutelin which contained exorbitant intermolecular and intramolecular disulphide bonds and hydrophobic interactions (Paraman et al., 2006). However, even limited hydrolysis by Protamex was able to significantly improve the corn glutelin solubility. The maximum solubility of glutelin hydrolysates was found at 150 min (94.65%). Protein solubility over 90% has been reported for other protein hydrolysates (Chen et al., 2012). Compared with the original glutelin, the improved solubility of glutelin hydrolysates may be attributed to the unfolding of the peptide chains, a size reduction caused by protease hydrolysis, and the exposure of more charged residues and polar groups into the surrounding aqueous phase. These structural changes resulted in an improvement of protein–water interaction, thereby increasing the solubility (Chen et al., 2012).
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3.7. Foaming properties The foam capacity (FC) and foam stability (FS) of corn glutelin and its hydrolysates at pH 7.0 are shown in Fig. 3A. The FC of corn glutelin was 78.17%, which was lower (p < 0.05) than that of rice protein (118.8%) (Zhao et al., 2012). This might be due to the poor solubility of corn glutelin in water at pH 7.0. The FC and FS of glutelin hydrolysates were higher (p < 0.05) than those of corn glutelin. Lee and Chen (2002) also reported that the FC and FS of protein increased after enzymatic hydrolysis. Limited hydrolysis by Protamex significantly improved protein solubility, reduced the molecular weight and the SS content, which facilitated the formation of an interfacial membrane and foam production. The FC and FS of glutelin hydrolysates were also affected by hydrolysis time, and their maximum values were obtained at 15 min. The structure of the corn glutelin was excessively destroyed with longer hydrolysis, which adversely affected FC and FS. Thus, prolonged hydrolysis should be avoided as it leads to undesirable property changes. 3.8. Emulsifying activity and emulsifying stability The emulsifying activity index (EAI) and emulsifying stability index (ESI) of the corn glutelin and its hydrolysates at pH 7.0 are shown in Fig. 3B. The EAI of glutelin hydrolysates was improved and was significant higher (p < 0.05) than that of corn glutelin.
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The EAI was increased from 3.20% to 68.23% when the hydrolysis time was increased from 0 to 15 min, and by further increasing the hydrolysis time from 15 to 150 min, the EAI of glutelin hydrolysates were maintained at a rather high value (more than 60%). The same trend was also observed for the ESI of the glutelin hydrolysates. The increase of EAI reflected the enhanced solubility of corn glutelin after hydrolysis, enabling protein diffusion to the oil/water surface easily. However, this phenomenon was different from that observed for other proteins, such as rice and barley, in which extensive hydrolysis greatly decreased the protein emulsifying activity (Paraman, Hettiarachchy, & Schaefer, 2008). This was due to the fact that extensive hydrolysis prevents the formation of a continuous protein film at the oil/water interface to stabilise the emulsions (Chen, Chi, & Xu, 2011; Xia et al., 2012). In this work, the reason for the stable emulsion capacity of the hydrolysates with different DHs is attributed to the soluble aggregates formed during hydrolysis, which could form a rigid film at the oil/water interface to prevent the close contact of oil droplets and avoid flocculation and coalescence. Similar phenomenon was also found by (Agyare, Addo, & Xiong, 2009). 3.9. Antioxidant properties of corn glutelin hydrolysates The antioxidant properties of samples are assessed using different assays, because there different mechanism of antioxidant action exist (Pan, Jiang, & Pan, 2011). In this study, the antioxidant properties of corn glutelin hydrolysates prepared at different hydrolysis times were evaluated based on their radical scavenging capacity against DPPH and OH, Fe2+-chelating effect and reducing power. Peptide concentrations of 2 mg/mL were used in each assay. Ascorbic acid at concentrations of 0.01 mg/mL was used as positive control. The antioxidant activity of corn glutelin was not tested because it barely dissolved in distilled water. 3.9.1. DPPH radical scavenging ability DPPH is a stable free radical, which can accept protons from an antioxidant to form a stable diamagnetic molecule. The absorbance of DPPH that has accepted proton is lower than that of DPPH free radical (at 517 nm), and there is a quantitative relation between the fading degree of DPPH and the accepted proton-donating number. Therefore, this assay has been widely used to evaluate the antioxidant activity of natural compounds (Lu et al., 2010). The DPPH radical scavenging capacities of corn glutelin hydrolysates are presented in Fig. 4A. All of the samples tested exhibited good scavenging ability against DPPH (41–59%). In particular, the activity of hydrolysates (58.86 ± 1.40%) at hydrolysis time of 120 min was prominently higher than that of others (p < 0.05). The DPPH radical scavenging effects of barley glutelin hydrolysates by Flavourzyme and rapeseed protein hydrolysates were 56–61% at a concentration of 1.0 mg/mL and 50% at the 0.71 mg/mL, respectively (Pan et al., 2011; Xia et al., 2012). Thus, the activity of corn glutelin hydrolysates was comparable to the hydrolysates reported in these studies. The result suggested that corn glutelin hydrolysates contained substances that were electron donors and could react with free radicals to convert them to stable products and terminate the radical chain reaction.
Fig. 3. (A) The foam capacity and foam stability of corn glutelin and its hydrolysate. (B) Emulsifying activity index (EAI) and emulsifying stability index (ESI) of corn glutelin and its hydrolysate.
3.9.2. Hydroxyl radical scavenging ability Hydroxyl radical is the strongest oxidant among the oxygen free radicals. It can activate lipid peroxidation and react with almost any adjacent biomolecules such as DNA, proteins and amino acids (Xia et al., 2012). Since this damage can cause food-spoilage or several diseases in living beings, its removal is probably one of the most effective way to prevent putrefaction of food and occurrence of various diseases (Pan et al., 2011). The hydroxyl radical scavenging activities of corn glutelin hydrolysates are shown in Fig. 4B. The
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Fig. 4. (A) DPPH radical scavenging activity (2.0 mg/mL). (B) hydroxyl radical scavenging activity (2.0 mg/mL). (C) Fe2+-chelating activity (2.0 mg/mL) and (E) reducing power (2.0 mg/mL) of corn glutelin hydrolysates treated by protamex.
hydrolysates exhibited strong inhibitory effect against hydroxyl radicals (71–83%). The activities of the hydrolysates were 82.89 ± 1.53%, 82.45 ± 1.32% and 82.64 ± 0.75% at an hydrolysis time of 15, 90 and 120 min, respectively. The results suggest that corn glutelin hydrolysates with excellent hydroxyl scavenging activity could have the potential to protect food or living system against hydroxyl radical-induced damages.
3.9.3. Fe2+-chelating ability Since certain transition metal ions can catalyse the Haber– Weiss reaction and induce superoxide to become harmful hydroxyl radical, measuring the metal ions chelating ability of compounds is important for evaluating their antioxidant activity (Zhang et al., 2011). The Fe2+-chelating ability of corn glutelin hydrolysates was assayed, and the results are shown in Fig. 4C. The Fe2+-chelating capacity of the hydrolysates increased considerably (10–30%) after an hydrolysis time of 120 min, then reduced slightly until 150 min, while, ascorbic acid at concentrations of 0.01 mg/mL had almost no chelating ability on Fe2+. Xia et al. (2012) reported a similar Fe2+-chelating ability for Flavourzyme hydrolysates (about 23–30%) derived from barley glutelin at 2.0 mg/mL. Compared with the native protein, the enhancement of the metal ion binding power of the hydrolysates might be due to an increased
concentration of carboxylic groups in the side chain of the acidic and basic amino acids (Zhang et al., 2010). 3.9.4. Reducing power There is a direct correlation between antioxidant activity and reducing power of a compound (Pan et al., 2011; Zhang et al., 2011). Compounds with a higher reducing capacity have better abilities to donate electron or hydrogen, leading to the interruption of the free radical chain reactions. As a result, the reducing power assay is often used to evaluate the potential antioxidant activity of natural products (Chen et al., 2012). Fig. 4D describes the reducing power of corn glutelin hydrolysates. This was increased significantly (0.191–0.236) by prolonging the hydrolysis time up to 120 min, and then decreased slightly afterwards. The corn glutelin hydrolysates also exhibited more effective reducing capacity than those of chickpea protein hydrolysates (0.2 at 2 mg/mL) and barley glutelin hydrolysates (0.121 at 2 mg/mL) (Li, Jiang, Zhang, Mu, & Liu, 2008; Xia et al., 2012). These results revealed that corn glutelin hydrolysates had good reducing power. The above results indicated that corn glutelin hydrolysates prepared by Protamex possessed excellent antioxidant activity. The hydroxyl radical scavenging capacity and reducing power were related to hydrolysis time, while the DPPH radical scavenging activity and Fe2+-chelating capacity were slightly affected by
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hydrolysis time. The corn glutelin hydrolysates that were obtained at hydrolysis time of 120 min exhibiting best antioxidant activity using anyone of the assay methods, and the percentage of their small-sized peptide was higher than that of others (Fig. 2C). The results suggested that the antioxidant activity might be influenced by the peptide size. However, Xia et al. (2012) reported that the large-sized peptide fraction (Mw > 10 kDa) in barley glutelin hydrolysates was more effective in DPPH scavenging activity and reducing power, whereas the small-sized peptide fraction (Mw < 1 kDa) possessed stronger Fe2+-chelating capacity and radical scavenging activity against OH. Therefore, investigation of the effect of molecular weight on the functional properties and bioactivities of corn glutelin hydrolysates requires further study. 4. Conclusions Corn glutelin can be effectively hydrolysed by Protamex to obtain glutelin hydrolysates with strong antioxidant activities. The hydrolysis of glutelin increased its hydrophobic surface, but also decreased the molecular weight and disulphide bond of the proteins, and eventually transformed its insoluble native aggregates to soluble aggregates during the hydrolysis process. Both corn glutelin and its hydrolysates maintained a high thermal stability and had similar amino acid composition. On the other hand, the corn glutelin hydrolysates were found to have a higher solubility which most likely resulted in improved foaming and emulsifying properties compared to the original glutelin. Overall, this research indicated that corn glutelin hydrolysates can be potentially used as functional ingredients with an increased antioxidant effect in both food and non-food applications. Acknowledgement This work was supported by National Natural Science Foundation of China (No. 31071629). References Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins, Vols. 9–56. Elsevier Applied Science Publishers, pp. 122–144. Agyare, K. K., Addo, K., & Xiong, Y. L. (2009). Emulsifying and foaming properties of transglutaminase-treated wheat gluten hydrolysate as influenced by pH, temperature and salt. Food Hydrocolloids, 23, 72–81. Beveridge, T., Toma, S., & Nakai, S. (1974). Determination of SH and SS groups in some food proteins using Ellman’s reagent. Journal of Food Science, 39, 49–51. Bouaouina, H., Desrumaux, A., Loisel, C., & Legrand, J. (2006). Functional properties of whey proteins as affected by dynamic high-pressure treatment. International Dairy Journal, 16, 275–284. Cabra, V., Vázquez-Contreras, E., Moreno, A., & Arreguin-Espinosa, R. (2008). The effect of sulfhydryl groups and disulphide linkage in the thermal aggregation of Z19 a-zein. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1784, 1028–1036. Chambal, B., Bergenståhl, B., & Dejmek, P. (2012). Edible proteins from coconut milk press cake; one step alkaline extraction and characterization by electrophoresis and mass spectrometry. Food Research International, 47, 146–151. Chen, C., Chi, Y. J., & Xu, W. (2011). Comparisons on the functional properties and antioxidant activity of spray-dried and freeze-dried egg white protein hydrolysate. Food and Bioprocess Technology, 5, 2342–2352. Chen, C., Chi, Y.-J., Zhao, M.-Y., & Xu, W. (2012). Influence of degree of hydrolysis on functional properties, antioxidant and ACE inhibitory activities of egg white protein hydrolysate. Food Science and Biotechnology, 21, 27–34. Cromwell, M. E., Hilario, E., & Jacobson, F. (2006). Protein aggregation and bioprocessing. The AAPS Journal, 8, E572–E579. Du, Y., Jiang, Y., Zhu, X., Xiong, H., Shi, S., Hu, J., et al. (2012). Physicochemical and functional properties of the protein isolate and major fractions prepared from Akebia trifoliata var. australis seed. Food Chemistry, 133, 923–929. Halliwell, B. (1978). Superoxide-dependent formation of hydroxyl radicals in the presence of iron chelates: Is it a mechanism for hydroxyl radical production in biochemical systems? FEBS Letters, 92, 321–326.
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