Elsevier

Food Chemistry

Volume 151, 15 May 2014, Pages 141-147
Food Chemistry

Lupine protein hydrolysates inhibit enzymes involved in the inflammatory pathway

https://doi.org/10.1016/j.foodchem.2013.11.053Get rights and content

Highlights

  • Lupine protein isolate was hydrolysed using Alcalase 2.4 L and/or Izyme AL.

  • Protein hydrolysates inhibited some enzymes involved in the inflammatory pathway.

  • Lupine protein hydrolysates may act as potential anti-inflammatory agents.

Abstract

Lupine protein hydrolysates (LPHs) were obtained from a lupine protein isolate (LPI) by enzymatic hydrolysis using two proteases, Izyme AL and Alcalase 2.4 L, and their potential anti-inflammatory capacities were studied by determining their in vitro inhibition of the following enzymes that are involved in the inflammatory process: phospholipase A2 (PLA2), cyclooxygenase 2 (COX-2), thrombin, and transglutaminase (TG). The strongest inhibitory activities toward PLA2 and TG were found in the hydrolysates obtained by hydrolysis with Izyme and subsequently with Alcalase, with more than 70% inhibition obtained in some cases. All of the hydrolysates tested inhibited more than 60% of the COX-2 activity. In no case did the percentage of thrombin activity inhibition exceed 40%. The best inhibitory activities were found in the LPH obtained after 15 min of hydrolysis with Alcalase and in the LPH obtained after 60 min of hydrolysis with Izyme followed by 15 min of hydrolysis with Alcalase. Enzyme kinetic analyses were conducted to determine the Km and Vmax parameters of these two hydrolysates using the Lineweaver–Burk equation. Both hydrolysates competitively inhibited the thrombin and PLA2 activities. In the case of COX-2 and TG, the inhibition appeared to be the mixed type.

Introduction

Chronic inflammation is involved in the onset and development of numerous chronic diseases, such as cardiovascular diseases, cancer, diabetes, obesity, arthritis, neurological diseases, pulmonary diseases, psychological diseases and autoimmune diseases (Sung, Prasad, Gupta, Patchva, & Aggarwal, 2012). Inflammation is one of the complex biological responses of the immune system of an organism. In the initial steps, the inflammatory response is mediated by phospholipase A2 (PLA2), which is elevated in many inflammatory disorders. PLA2 hydrolyses the sn-2 ester bond of glycerophospholipids to produce free fatty acids, particularly arachidonic acid (AA), and lysophospholipids (Yedgar, Cohen, & Shoseyov, 2006). AA is metabolised mainly via the cyclo-oxygenase (COX) and lipo-oxygenase enzymatic pathways to produce diverse families of eicosanoids, including prostaglandins (PGs), thromboxanes, and leukotrienes. These eicosanoids are involved in the development of many pathological conditions, particularly inflammation-related processes (Yedgar et al., 2006).

There is a close relationship between inflammation and coagulation. Inflammation-induced coagulation is due to tissue factor, which produces thrombin generation (Levi & van der Poll, 2005). Thrombin is a trypsin-like serine protease that has a central role in haemostasis and thrombosis. Once formed, thrombin cleaves fibrinogen to produce the fibrin mesh of the blood clot (Cirino et al., 1996). Thrombin also activates transglutaminase or factor XIII, which cross-links fibrin molecules via the formation of interchain ε-(γ-glutamyl)-lysine isopeptide bonds and stabilises the clot by preventing its hydrolysis by proteases (Moreno, 2006).

Diet is thought to be one of the major risk factors for the development of chronic diseases. Thus, modifying the diet could prevent or delay these diseases (Prasad, Sung, & Aggarwal, 2012). Some dietary agents have shown the potential to inactivate inflammatory molecules by direct binding. Recent studies have provided evidence that peptides can modify crucial regulatory functions of inflammatory processes (Fernandes, Schmidhuber, & Brain, 2009). There are many reports about synthetically derived anti-inflammatory peptides (Selvatici, Siniscalchi, & Spisani, 2013). However, few studies have examined the effect of plant protein hydrolysates on inflammatory markers. It has been reported that soybean protein hydrolysates reduce the level of some of these markers (Vernaza, Dia, González de Mejía, & Chang, 2012).

Lupine (Lupinus angustifolius L.) is an herbaceous plant typical of the Mediterranean region. Lupine seeds can be incorporated as a protein source in both animal feed and in a variety of human foods. Lupine flour is rich in protein (20–40%), and this content can be increased to more than 90% in protein isolates (Lqari, Vioque, Pedroche, & Millán, 2002). Lupine protein hydrolysates (LPHs) have shown bile-acid binding activity, so these may have potential applications for reducing cholesterol in hypercholesterolemic patients (Yoshie-Stark & Wäsche, 2004). More recently, lupine proteins have been shown to exert hypotensive and hypotriglyceridemic effects in vivo (Cam & Gonzalez de Mejia, 2012), but there are no reports of their anti-inflammatory activity.

The objective of this work was to determine the effect of LPHs on the activity of four enzymes implicated in the inflammatory process: PLA2, COX-2, thrombin, and TG. LPHs were obtained by hydrolysis of lupine protein isolate (LPI) with Izyme and Alcalase, two food-grade proteases produced by Novozymes. Izyme has trypsin-like activity whereas Alcalase is a non-specific endoprotease. Both trypsin and Alcalase have been previously used for the generation of bioactive peptides (Korhonen & Pihlanto, 2006). Samples were collected after different periods of incubation, and the activity of the above mentioned enzymes was tested in presence of LPHs with different degrees of hydrolysis. The LPHs that exhibited a higher inhibitory activity were selected, and their enzyme kinetic parameters (Km, Vmax) were determined.

Section snippets

Materials

The L. angustifolius seeds were provided by Koipesol Semillas, S.A. (Seville, Spain). Izyme AL (564 EU/ml) and Alcalase 2.4 L (2.4 AU/g) were provided by Novozymes (Bagsvaerd, Denmark). PLA2 from bovine pancreas, albumin (human, essentially fatty acid-free), thrombin from bovine plasma, antithrombin III (AT III), tosyl-gly-pro-arg-p-nitroanilide acetate salt (Chromozym), heparin sodium salt, protamine sulphate salt from salmon, TG from guinea pig liver, monodansylcadaverine (dansyl-CAD),

Study of the degree of hydrolysis

LPI was hydrolysed using two food-grade commercial enzymes, Izyme and Alcalase. Hydrolysis with Izyme resulted in a DH of approximately 9% (Fig. 1A); the DH was not increased by increasing the time of hydrolysis or the enzyme/substrate ratio, which may be due, partially, to the lysine-plus-argentine content of the LPI (15%, data not shown). Analysis of the results, showed that Izyme performance was approximately 60%. Because most bioactive peptides known at present contain 2–20 amino acids and

Conclusion

Hydrolysates of a lupine protein isolate obtained using commercial proteases inhibited the activity of some of the enzymes involved in the inflammatory pathway. Chronic inflammation is associated with the development of several chronic diseases. Therefore, the results of this study indicate the potential use of lupine protein hydrolysates in the production of functional foods. Additionally, the addition of lupine protein hydrolysates, as ingredients, may improve the nutritional value of a

Acknowledgements

This work was supported by the Grant AGL2012-40247-C02-01 from the Spanish Ministry of Economy and Competitiveness.

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