Fractionation and antioxidant properties of rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion
Introduction
Recently, findings of natural antioxidants have gained growing interest because the use of synthetic antioxidants is being strictly regulated due to their potential health hazards. Plant-based antioxidant peptides are increasingly being studied and considered as alternative antioxidants for food preservation and health protection. Rice bran is an underutilized component obtained from rice milling processes. According to world rice production, >800 thousand metric tons of rice bran is generated as by-product annually (USDA, 2016). Rice bran contains high quality protein with high amounts of essential amino acids, especially aromatic amino acids (9.46–11.41%) that act as strong antioxidants (Wang, Chen, Fu, Li, & Wei, 2017). There have been numerous reports about rice proteins and their bioactivities, such as their angiotensin converting enzyme (ACE)-inhibitory activity (Chen et al., 2013), their cholesterol-lowering effect (Yang, Chen, Xu, Nie, & Yang, 2013), and their antioxidant activities (Zhou, Sun, & Canning, 2012 and Phongthai, Lim, & Rawdkuen, 2016). However, macroproteins are generally inactive within the sequence of the original structure. Enzymatic hydrolysis is an effective method to expose and release bioactive peptides without affecting nutritional value. Various enzymes such as alkaline and neutral proteases (Zhou, Canning, & Sun, 2013), papain (Zhang et al., 2010), alcalase (Phongthai et al., 2016), flavourzyme, neutrase (Thamnarathip, Jangchud, Nitisinprasert, & Vardhanabhuti, 2016), and trypsin (Wattanasiritham, Theerakulkait, Wickramasekara, Maier, & Stevens, 2016) have been used to produce rice protein hydrolysates that possess antioxidant activities, including the ability to scavenge free radicals, inhibit lipid peroxidation, and chelate metal ions. These activities depend on many variables such as amino acid composition, degree of hydrolysis, molecular weight (MW), and peptides sequences due to the types of enzymes used (Saidi, Deratani, Belleville, & Amar, 2014). Therefore, there is interest in utilizing the high quality protein in rice bran, and there is great potential in the enzymatic hydrolysis method to add value to the protein with highly desired biological, functional, and nutritional properties.
Stimulating bioactivities in proteins through in vitro gastrointestinal (GI) digestion is being used extensively since it is rapid, inexpensive, safe, and is not bound by the same ethical restrictions (Xiao et al., 2014). The peptides are encrypted in the proteins and can be released by the GI tract in the human stomach and small intestine. The proteases in these areas have been reported to have abilities to induce peptide bioactivities from several protein sources such as from loach (Misgurnus anguillicaudatus) (You, Zhao, Regenstein, & Ren, 2010), salmon by-product (Ahn, Kim, & Je, 2014), radix isatidis (Xiao et al., 2014), coconut meat (Jin, Zhou, Li, Lai, & Li, 2015), amaranth (Orsini Delgado et al., 2016), pig meat (Simonetti, Gambacorta, & Perna, 2016), and fish skin gelatin (Ketnawa, Benjakul, Martínez-Alvarez, & Rawdkuen, 2017). However, different protein sources must be individually investigated since the differences in amino acid sequences/compositions influences their activities. In addition, many studies reported that the bioactivities of peptides are also related to their MW (Foh et al., 2010, Girgih et al., 2015, Onuh et al., 2014, Saidi et al., 2014 and Wiriyaphan, Xiao, Decker, & Yongsawatdigul, 2015). Generally, peptides with low MW exhibit high bioactivities since they have a higher chance to cross the intestinal barrier and perform biological functions (Chi et al., 2015). In order to obtain those low MW active peptides, membrane fractionation is widely used over other methods for its productivity, lower capital investment, it’s maintenance of product purity under ambient conditions, and for the ease of keeping the equipment clean (Saidi et al., 2014). Even if the enhancement of peptide bioactivities by digestive enzymes as well as the effect of MW has been vastly reported, there is no data available with regards to the use of membrane technology to fractionate rice bran protein hydrolysate into smaller fractions.
Based on the above rationale, the authors aimed to stimulate antioxidant activities of rice bran protein prepared by α-amylase-assisted extraction through in vitro GI digestion. This study also investigates protein patterns of the hydrolysates by SDS-PAGE, and the effects of fractionation by membrane ultrafiltration (UF) on the antioxidant activities of their fractions. Moreover, the UF fractions were analyzed for aromatic amino acid contents in order to investigate the correlation of their activities. Further, the fraction that possessed the highest antioxidant activity was further investigated for its MW distribution using MALDI-TOF mass spectrometry.
Section snippets
Materials
Organic rice bran was supplied by Urmatt Ltd. (Chiang Rai, Thailand). α-Amylase (E.C.3.2.1.1, ≥500 units/mg protein) from Bacillus licheniformis; pepsin (E.C.3.4.23.1, ≥250 units/mg solid) from porcine gastric mucosa; trypsin (E.C.3.4.21.4, ∼10,000 BAEE units/mg protein) from bovine pancreas; 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid; ABTS); 3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (ferrozine);
Effect of solid-liquid ratio
The result showed that solid-liquid ratio influenced the protein recovery and protein yield (Fig. 1a). The use of solid-liquid ratio at 0.5:10 and 1.0:10 for rice bran protein extraction gave comparable protein recovery and protein yield (P > 0.05), reaching maximum values of 12.06 ± 0.24% and 2.78 ± 0.08%, respectively (P < 0.05) (extraction time and enzyme concentration were kept constant at 3 h and 2500U). By contrast, the extraction with high solid-liquid ratios of 1.5:10 and 2.0:10 apparently
Conclusion
In vitro gastrointestinal digestion (pepsin-trypsin system) influenced the antioxidant properties of rice bran protein. Only digestion by pepsin enhanced the DPPH radical scavenging activity, while further digestion with trypsin improved ferric reducing antioxidant power by 13.9%. In addition, the membrane ultrafiltration of protein hydrolysates had a positive effect on metal chelating and ABTS radical scavenging activities. Tyrosine and phenylalanine exhibited positive correlations with their
Acknowledgements
This study was financially supported by Research and Researcher for Industry (RRI) under the Thailand Research Fund (TRF) to Mr. Suphat Phongthai [Grant numbers PHD57I0018]; Mae Fah Luang University; and the Technology Grants 2014 under Thai-Austrian Cooperation in Science, Technology and Arts. The authors thank Urmatt Ltd (Chiang Rai, Thailand) for providing the Thai Jasmine organic rice bran. Thank you to Matthew Robert Ferguson of Mahidol University International College for language editing
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