Mode of inhibition of finger millet malt amylases by the millet phenolics S. Chethan, Y.N. Sreerama, N.G. Malleshi * Department of Grain Science and Technology, Central Food Technological Research Institute, Mysore 570020, India article info Article history: Received 17 December 2007 Received in revised form 14 March 2008 Accepted 18 March 2008 Keywords: Finger millet Eleusine coracana Polyphenols Amylase activity Mode of inhibition abstract The effect of millet polyphenols on starch hydrolysis catalyzed by amylases developed during malting were investigated. The enzyme kinetic studies using Michaelis–Menten and Lineweaver–Burk equations showed the K m remained constant (0.625%) but the maximum velocity (V max ) decreased in the presence of a crude extract of millet polyphenols, indicating mixed non-competitive inhibition. On the other hand, gallic acid, vanillic acid, quercetin and trans-cinnamic acid isolated from the polyphenol extract of the millet showed uncompetitive inhibition. Kinetic studies of amylase inhibition by phenolic compounds indicated the presence of secondary binding sites in malted finger millet amylases similar to other cereal amylases. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Polyphenols are known to inhibit the activity of digestive en- zymes such as amylase, glucosidase, pepsin, trypsin and lipases and the subject has been studied extensively (Rohn, Rawel, & Kroll, 2002). Synergy between phenolics may play a role in mediating amylase inhibition and therefore have the potential to contribute to the management of type 2 Diabetes mellitus, which is character- ized by high blood glucose levels (Saito, Sakai, Sekihara, & Yajima, 1998; Toeller, 1994). Polyphenols may act as inhibitors of amylase and glucosidase (similar to acarbose, miglitol and voglibose) lead- ing to a decrease in post-prandial hyperglycemia (Bailey, 2001). Finger millet or ragi, is one of the minor cereals and is a staple food in south India and parts of Africa. Germination has been suggested as an inexpensive and effective method for improving the overall nutritional quality of cereals and legumes, by enhancing their digestibility (Chavan & Kadam, 1989) and reducing the contents of anti-nutritional factors (Ghorpade & Kadam, 1989). Malting of cereals and germination of legumes is documented but very little has been reported on the effect of polyphenols on the activity of enzymes developed during germination. Millet malt is also a good source of a- and b-amylases (Chandrasekhara & Swaminathan, 1953) and malted millet is extensively used in weaning food, infant food and supplementary food formulations (Malleshi, 2005). The hydrolytic enzymes in the malt are largely confined to the endo- sperm fraction of the malted millet. The seed coat of the millet con- tains nearly 90% of the polyphenols and remaining 10% are distributed in the endosperm as constituents of the cell walls (Che- than & Malleshi, 2007). Millet is being used as a source of amylases in improving the nutrient density and texture of weaning food for- mulations. Although there are reports on the inhibitory activity of the polyphenols on the cereal amylases (Rohn et al., 2002), there are no reports on the inhibition of finger millet malt amylases by its polyphenols. In the present study, the inhibitory effects as well as the mode of action of the millet polyphenols on its malt amy- lases have been investigated through kinetic studies, using Michaelis–Menten and the derived Lineweaver–Burk (LB) equations. 2. Materials and methods 2.1. Materials Finger millet (variety GPU 28) seeds were procured from the University of Agricultural Sciences, Bangalore. Phenolic standards: gallic, vanillic, ferulic, p-coumaric, p-hydroxy benzoic, syringic, trans-cinnamic, p-catechuic, quercetin and trifluoroacetic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The other reagents were of analytical and HPLC grade. 2.2. Malting of finger millet The millet seeds (1 kg) were cleaned, steeped for 24 h and ger- minated on moist cloth at 25 °C in a BOD incubator (Alpha Scien- tific Co., Bangalore, India) up to 120 h. The seeds were withdrawn from the germination bed at intervals of about 24 h, dried at 50 °C in an air oven for 6–8 h and the vegetative growth 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.03.063 * Corresponding author. Tel.: +91 0821 2510843; fax: +91 0821 2517233. E-mail address: malleshi@yahoo.com (N.G. Malleshi). Food Chemistry 111 (2008) 187–191 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem