Interaction of cellulose nanocrystals and amylase: Its influence on enzyme activity and resistant starch content

Highlights

• The cellulose nanocrystals quenched amylase intrinsic fluorescence.

• The cellulose nanocrystals inhibited the activity of α-amylase and glucoamylase.

• The addition of cellulose nanocrystals increased the resistant starch contents.

Abstract

The aim of this work was to evaluate the effects of cellulose nanocrystals (CNCs) on amylolytic enzyme activity and starch digestibility. For the first time, we investigated the interaction between α-amylase or glucoamylase and CNCs using ultraviolet visible (UV–Vis) absorption spectroscopy, fluorescence quenching method, Fourier transform infrared (FTIR) spectroscopy, and circular dichroism (CD). The results of UV and fluorescence spectra suggested that CNCs interacted with α-amylase and glucoamylase. Increasing the concentration of CNCs caused a reduction of α-amylase and glucoamylase activities. The FTIR and CD results indicated that CNCs induced structural changes in the secondary structure of α-amylase and glucoamylase. By incorporating CNCs into maize, potato and pea starches, the contents of rapid digestible starch and slowly digestible starch of the cooked starches decreased while resistant starch content increased.

Introduction

Starch is the most important carbohydrate in the human diet and serves as a major energy source (Hung, Vien, & Phi, 2016). Resistant starch (RS) is a form of dietary fiber and is naturally present in many starchy foods (Raigond, Ezekiel, & Raigond, 2015). The products of RS fermentation help to prevent colorectal cancer, lower the risk of heart disease, and influence metabolic and inflammatory bowel diseases such as diabetes and diverticulitis (Craig, Troup, Auerbach, & Frier, 1998; Topping & Clifton, 2001). Due to the health benefits of RS in the human diet, there has been a great interest in increasing its content in foods through various techniques.

Human α-amylase, as one of the key digestive enzymes in the digestive system, has been shown to function by breaking down starch into maltose and glucose (Sandip et al., 2008). Glucoamylase, also known as amyloglucosidase, is a biocatalyst capable of hydrolyzing α-1, 4 glycosidic linkages in raw or soluble starches and related oligosaccharides with the inversion of the anomeric configuration to produce β-glucose (Norouzian, Akbarzadeh, Scharer, & Moo, 2006). These two enzymes, which are the chief digestive enzymes, catalyze the hydrolysis of α-1, 6- and α-1, 4-glycosidic bonds in starch and other gluco-oligosaccharides, resulting in the formation of glucose (Shigechi et al., 2004). Great effort has been devoted to inhibit the activities of digestive enzymes in order to control the level of glucose. For instance, phenolic compounds can directly bind to digestive enzymes (amylases, sucrase, trypsin, and lipase), decreasing enzyme functionality (Le Bourvellec and Renard, 2005, Asquith and Butler, 1986; Vonk et al., 2000), and further slowing the rate of starch and protein digestion. Amylase inhibition by polyphenol binding could lead to lower blood glucose levels for diabetic patients. Interactions of polyphenols with enzymatic proteins subsequently change their molecular configuration, and this is known to reduce the catalytic activity of various enzymes (Bandyopadhyay, Ghosh, & Ghosh, 2012). In an in vitro study, He, Lv, and Yao (2007) discovered that tea polyphenols inhibit the activity of pepsin (31%) and other digestive enzymes like α-amylase (61%), trypsin (38%) and lipase (54%). This result suggests the possibility of antinutritional effects of tea polyphenols, in terms of reduction in activity of digestive enzymes. It was found that the dietary fiber materials as citrus pectin (Tsujita et al., 2003), pectin of high methylic esterification and guar gum (Isaksson, Lundquist, & Ihse, 1982) could contribute to inhibiting the activity of gastrointestinal tract enzymes. Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, and Narváez-Cuenca (2014) suggested that the inhibition of activity of enzymes by ingested pectic polysaccharides might play an important role to decrease the caloric intake.

Recent developments in bio-nanotechnology have had a large socioeconomic impact in bio-medical industrial sectors because the use of nanomaterials is constantly increasing in industrial activities such as biosensing, diagnostics, biomedicine, and therapeutics (Michalet et al., 2005, Zhang et al., 2008). The interaction of protein/enzyme molecules with nanomaterials is at the core of such applications. On interacting with nanoparticles, the enzyme molecules may alter their conformation, expose new epitopes on the protein surface, or even deviate from their normal function (Lynch, Dawson, & Linse, 2006). In a recent study, Ernest, Shiny, Mukherjee, and Chandrasekaran (2012) reported that silver nanoparticles showed an increased enzyme activity in the endohydrolysis of starch. Saha, Saikia, and Das (2015) investigated the in-depth interaction features of protein molecules with copper sulfide nanoparticles and they were able to extrapolate suitable parameters for developing functional bionanocomposites with the desired activity.

Cellulose nanocrystals (CNCs) is a material obtained from the acid hydrolysis (Incani, Danumah, & Boluk, 2013) or ultrasonic-assisted enzymatic hydrolysis (Cui, Zhang, Ge, Xiong, & Sun, 2016) of native cellulose. As a result of their high content of –OH groups, high aspect ratio and surface area, these nanocrystals could be used for polymer reinforcement, nanocomposite formulation and reinforcement, drug delivery and biomedical uses (de Lima et al., 2012; Male, Leung, Montes, Kamen, & Luong, 2012; Gaspar et al., 2014). To the best of our knowledge, however, there have been neither reports about the interaction of CNCs with α-amylase and glucoamylase nor the effect of CNCs on these enzyme activities. Therefore, in this work, we investigated this interaction using ultraviolet visible (UV–Vis), fluorescence, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopic techniques. We also explored the effect of CNCs on the activities of α-amylase and glucoamylase. Further, we determined the impact of CNCs on starch digestibility, so as to ascertain whether our results could reasonably be extrapolated to CNCs during digestion.