Elsevier

Journal of Functional Foods

Volume 34, July 2017, Pages 287-296
Journal of Functional Foods

Identification and characterization of multifunctional cationic peptides derived from peptic hydrolysates of rice bran protein

https://doi.org/10.1016/j.jff.2017.04.046Get rights and content

Highlights

  • Peptides were fractionated from hydrolysates of rice bran protein by autofocusing.

  • Cationic peptides were purified by RP-HPLC and then identified by MALDI-TOF MS.

  • Of identified peptides, three cationic peptides exhibited multiple functions.

  • Three peptides exhibited angiogenic, LPS-neutralizing, and antimicrobial activities.

  • Multifunctional cationic peptides are expected to be used as ingredients in foods.

Abstract

In this study, to prepare the fraction containing multifunctional cationic peptides, we first hydrolyzed rice bran protein (RBP) with pepsin. We separated the enzymatic hydrolysate of RBP into 20 fractions containing peptides with different isoelectric point (pI) values by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we examined the antimicrobial activity of each fraction against four pathogens. In addition, we purified the cationic peptides from fractions exhibiting antimicrobial activity by reversed-phase high-performance liquid chromatography and identified them by matrix-assisted laser/desorption ionization–time-of-flight mass spectroscopy. Of five cationic peptides identified, we chemically synthesized three peptides with high pI values and evaluated their multiple functions, including antimicrobial, lipopolysaccharide-neutralizing and angiogenic activities. Our results demonstrated that the three identified cationic peptides exhibited multiple functions with little or no haemolytic activity. Fractions containing cationic peptides obtained from RBP hydrolysate have the potential to be used as dietary supplements and functional ingredients in food products.

Introduction

Food proteins provide organisms with the amino acids necessary for the maintenance of life. Recently, food proteins have also been identified as a source of bioactive peptides, including antimicrobial peptides (AMPs), immunomodulatory peptides, antioxidative peptides, hypocholesterol peptides and inhibitors of various enzymes including angiotensin I-converting enzyme (ACE), dipeptidyl peptidase IV (DPP-IV), α-glucosidase and arginine-specific gingipain (de Castro and Sato, 2015, Li-Chan, 2015). These peptides are inactive prior to cleavage from the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Bioactive peptides can be generated from proteins of different food origins, including milk (Nongonierma & FitzGerald, 2016), eggs (Ibrahim, Inazaki, Abdou, Aoki, & Kim, 2005), fish (Sila & Bougatef, 2016), wheat (Egorov, Odintsova, Pukhalsky, & Grishin, 2005) and soybean (Lammi, Zanori, & Arnoldi, 2015). In addition, increasing evidence indicates that peptides derived from food proteins exhibit multifunctional effects, including antimicrobial, antioxidant, anti-inflammatory, enzyme-inhibitory and/or growth-stimulating activities. Girgih et al. (2014) reported that peptides obtained from enzymatic hydrolysates of hemp seed proteins showed antioxidant and antihypertensive activity. Mandal et al. (2014) isolated and identified multifunctional peptides from human breast milk that displayed antimicrobial, antioxidant and growth-stimulating activities. Zambrowicz et al. (2015) found that four peptides, purified and identified from an enzymatic hydrolysate of egg yolk protein by-product following ethanol extraction of phospholipids, possessed antioxidant, ACE-inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. These multifunctional peptides are superior to other peptides that possess specific activity in the promotion of health and/or the treatment of diseases because they are potent host-defence peptides that can protect against infection induced by pathogens while also expressing bioactivity such as immunomodulatory activity (Hilchie et al., 2013, Lai and Gallo, 2009). However, few studies have investigated multifunctional peptides derived from food proteins (Girgih et al., 2014, Mandal et al., 2014, Zambrowicz et al., 2015).

In our previous studies (Takei et al., 2013, Taniguchi, Matsuhashi et al., 2014, Taniguchi, Takahashi et al., 2014), we reported that CL(14-25), a novel cationic α-helical dodecapeptide derived of cyanate lyase from rice (Oryza sativa L. japonica), exhibited both arginine-specific cysteine proteinase (Arg-gingipain)-inhibitory activity and antimicrobial activity against Porphyromonas gingivalis, a major etiological agent of chronic periodontitis. We also investigated the inhibitory activity of CL(14-25) against the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli (Takayama et al., 2016) and found that CL(14-25) could inhibit the production of both LPS-induced interleukin-6 in human aortic endothelial cells and LPS-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264). Moreover, CL(14-25) exhibited a protective effect against lethal toxicity induced by LPS in mice. Recently, we found that AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice, is a novel cationic α-helical peptide that exhibits antimicrobial activity against several human pathogens (Taniguchi et al., 2015). We previously examined its inhibitory ability against the endotoxic activities of LPS and lipid A from E. coli and found that AmyI-1-18 inhibits the production of endotoxin-induced NO in RAW264 in a concentration-dependent manner (Taniguchi et al., 2016a). Thus, these results illustrated that cationic peptides, including CL(14-25) and AmyI-1-18, found as partial amino acid sequences in rice proteins, directly kill pathogens and exhibit endotoxin-neutralizing activity, which is similar to human multifunctional peptides, including human LL-37 (Nan, Bang, Jacob, Park, & Shin, 2012) and β-defensins (Semple & Dorin, 2012). However, the two cationic peptides, CL(14-25) and AmyI-1-18, were not obtained from enzymatic hydrolysates of rice proteins but represent chemically synthesized peptides based on their partial sequences rich with cationic amino acids (lysine and arginine). Therefore, they cannot be widely applied to dietary supplements and functional ingredients in food products because of safety concerns and high production cost.

To widely use multifunctional peptides as dietary supplements and functional ingredients in food products, it is necessary to obtain them from enzymatic hydrolysate of an inexpensive protein source suitable for the production of natural bioactive components (de Castro and Sato, 2015, Li-Chan, 2015). In addition, all proteinases have a degree of specificity for proteins as substrates, based on the sequence of amino acids. This specificity affects the size and amino acid sequences in peptide chains produced from proteins, and in turn, their bioactive characteristics. In this study, we selected rice bran protein (RBP) as the protein source and pepsin as a hydrolytic enzyme for RBP based on preliminary experiments in which RBP was hydrolysed with pepsin, papain, trypsin, or a mixture of trypsin and chymotrypsin for various reaction times. After hydrolysis of RBP with pepsin, the fractions containing peptides with different isoelectric point (pI) values were prepared using ampholyte-free isoelectric focusing (autofocusing). The antimicrobial activity of each fraction against various human pathogens was examined and peptides in fractions exhibiting antimicrobial activity were purified and identified using matrix-assisted laser/desorption ionization–time-of-flight mass spectroscopy (MALDI-TOF MS). Subsequently, we chemically synthesized three identified peptides with high pI values and evaluated their multifunctional potential, testing antimicrobial, LPS-neutralizing and angiogenic activities. Finally, we compared the properties of peptides to multiple functions obtained in this study.

Section snippets

Materials

RBP 55 (protein content, 55%) was kindly provided by Tsuno Food Industrial Co., Ltd. (Wakayama, Japan). Pepsin was purchased from Sigma-Aldrich Co. (St. Louis, MO). We used smooth-type LPS from E. coli O55:B5 (List Biological Laboratories, CA) as an endotoxin. All other regents were of analytical grade and were purchased from Wako Pure Chemicals Ltd. (Osaka, Japan).

Peptides used in this study

Amino acid sequences and properties of five peptides identified in this study are summarized in Table 1. Of five peptides, three

Fractionation of RBP hydrolysate by autofocusing

In our preliminary experiments, RBP was hydrolyzed with pepsin for different reaction times (3, 4, 5 and 6 h). After each hydrolyzed RBP was fractionated into 20 fractions by autofocusing, we determined the antimicrobial activity of each fraction against P. gingivalis, P. acnes, S mutans and C. albicans. Maximum antimicrobial activity was obtained when the hydrolysis time was 5 h. Antimicrobial activity against P. gingivalis and P. acnes was observed in fractions 18, 19 and 20, while

Discussion

Among the various types of bioactive peptides, increasing attention has been paid to cationic bioactive peptides as promising multifunctional agents. Numerous cationic bioactive peptides, including LL-37 and defensins in humans, cecropins and melittin in insects, magainin and buforin II in amphibians and fowlicidins in chickens, have been identified and extensively characterized (Hilchie et al., 2013, Lai and Gallo, 2009, Nan et al., 2012, Semple and Dorin, 2012). Cationic bioactive peptides

Conclusion

In this study, we selected RBP as a starting material for producing multifunctional cationic peptides. We successfully prepared 20 fractions containing peptides with different pI values from peptic hydrolysate of RBP by autofocusing using a Rotofor. Subsequently, we examined the antimicrobial activity of each fraction against human pathogens. In addition, we purified cationic peptides in fractions exhibiting antimicrobial activity and identified them by MALDI-TOF MS. Of identified cationic

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant number 16K06869).

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