Elsevier

Journal of Functional Foods

Volume 20, January 2016, Pages 486-495
Journal of Functional Foods

Screening for potential novel probiotic Lactobacillus strains based on high dipeptidyl peptidase IV and α-glucosidase inhibitory activity

https://doi.org/10.1016/j.jff.2015.11.030Get rights and content

Highlights

  • Lactobacillus strains displayed DPP-IV inhibitory activity in vitro.

  • DPP-IV inhibition was concentration-dependent and stable to temperature and pH.

  • Trypsin treatment significantly enhanced DPP-IV inhibitory property.

  • Some strains showed both DPP-IV and α-glucosidase inhibitory property in vitro.

Abstract

Lactobacillus strains were screened for inhibition of dipeptidyl peptidase IV (DPP-IV) and α-glucosidase. Cell-free excretory supernatants (CFS) and cell-free extracts (CFE) of 21 strains were prepared, and most of them showed DPP-IV inhibition in a concentration-dependent manner, which started at the exponential phase and peaked at the stationary phase of the Lactobacillus. The CFS bioactivity was heat-resistant, stable to acid–alkali and glucosidase treatment. Trypsin treatment specifically elevated CFS's DPP-IV inhibition. Lactobacillus plantarum strains CFS displayed different protein patterns and trypsin sensitive bands on SDS-PAGE, demonstrating specific peptide-cleavage contributes to higher inhibition. Seven strains with highest DPP-IV inhibition also displayed α-glucosidase inhibitory activity, of which, three strains of L.plantarum and one of Lactobacillus brevis showed tolerance to simulated gastrointestinal tract conditions and high HT-29 cell adhesion. This initial report of lactobacilli secreting strong DPP-IV and α-glucosidase inhibition components will be beneficial for selection and application of anti-diabetic probiotics.

Introduction

Diabetes has been recognized as a serious metabolic disorder characterized by chronic hyperglycaemia as a result of insufficient insulin production (type 1) or insulin resistance (type 2) (Imamura, Tsuyama, & Hirata, 2011). It has become a major disease, causing enormous damage to human life and health, and a number of anti-diabetic agents have been developed to combat it (Mazziotti, Gazzaruso, & Giustina, 2011).

Several classes of hypoglycaemic agents are available for the treatment of diabetes (Mazziotti et al., 2011). Among them, dipeptidyl peptidase IV (DPP-IV; EC 3.4.14.5) inhibitors are the newest and most promising anti-diabetic drugs. These oral medicines play a beneficial role in glycaemia management mainly by prolonging the incretin effect of insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) (Deacon, 2011). GIP and GLP-1 are incretins secreted by intestinal L cells; they have many physiological functions, such as promoting glucose-dependent insulin secretion, suppressing pancreatic glucagon release and inhibiting gastric emptying (Verspohl, 2009). Both GIP and GLP-1 are endogenous substrates of DPP-IV, which rapidly inactivates them by proteolytic cleavage (Mentlein, 1999). A number of synthesized DPP-IV inhibitors have shown promising results in the treatment of type 2 diabetes (T2D) (Drucker & Nauck, 2006). However, they are all chemically synthesized compounds, and their long-term safety remains unknown. The more recently discovered DPP-IV inhibitors from natural sources, such as dairy proteins, might be more desirable as novel anti-diabetes drugs (Lacroix, Li-Chan, 2012, Nongonierma, FitzGerald, 2013a, Nongonierma, FitzGerald, 2013b, Uenishi et al, 2012). At the same time, probiotics are also being recognized for their remarkable role in diabetes management (Thomas & Versalovic, 2010).

Another strategy for managing type 2 diabetes (T2D) involves the use of α-glucosidase inhibitors. α-Glucosidase is an enzyme located in the brush-border membrane of the intestine that catalyses the transformation of complex carbohydrates into glucose to be absorbed by the gut (Sales, Souza, Simeoni, Magalhaes, & Silveira, 2012). Curbing the activity of intestinal α-glucosidases can effectively reduce polysaccharide and disaccharide hydrolysis, and glucose liberation and absorption, thereby decreasing blood glucose levels (Hansawasdi, Kawabata, & Kasai, 2001). Therefore, α-glucosidase inhibitors have become useful oral hypoglycaemic drugs and the commercially available acarbose is used as a strong α-glucosidase inhibitor for diabetes management. However, the drug commonly has side effects, such as flatulence and diarrhea (Chiasson et al., 2002). In recent years, some lactic acid bacteria (LAB) have been shown to possess α-glucosidase inhibitory activity (Chen et al, 2014a, Muganga et al, 2015, Panwar et al, 2014), and might serve to alleviate the effects of diabetes (Chen et al, 2014b, Panwar et al, 2014). Thus, oral administration of probiotics with α-glucosidase inhibitory properties might be beneficial to T2D patients.

Probiotics have been used for centuries in the field of fermented dairy products. They are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002). Recently, probiotics have emerged as potentially novel and natural therapeutic drugs for controlling diabetes, alleviating hyperglycaemia and increasing insulin sensitivity; thus they have the potential to act as an adjunct to diabetes treatment (Baboota et al, 2013, Cani et al, 2007, Miyoshi et al, 2014, Panwar et al, 2013, Panwar et al, 2014, Zhang et al, 2014). However, most of the data on probiotic control of diabetes have been obtained directly in animal experiments and little is known about the specific mechanisms of glycaemia regulation. Because inhibition of DPP-IV and α-glucosidase constitutes two important approaches to T2D management, potential mechanisms of hyperglycaemia regulation by probiotics might involve this inhibitory activity. Screening for potential probiotics with DPP-IV and α-glucosidase inhibitory activity may produce dual beneficial effects on glycaemia regulation, enabling their use as an adjunct to diabetes treatment.

The aim of this study was to screen lactobacilli with the ability to inhibit DPP-IV. The screened strains with the highest DPP-IV inhibition were further tested for their α-glucosidase inhibitory activity, gastrointestinal tract (GIT) tolerance and cell adhesion – basic features of probiotics.

Section snippets

Bacterial strains and growth conditions

The 21 Lactobacillus strains used in this study are listed in Table 1. Lactobacillus rhamnosus strain GG (ATCC 53103; Valio Ltd., Helsinki, Finland), which is used commercially in functional foods, served as the reference strain. All strains were cultured in de Man, Rogosa and Sharpe (MRS) broth at 37 °C for 12 h. They were subcultured three times prior to their use in the experiments.

Preparation of cell-free excretory supernatant (CFS)

The cells of the 21 Lactobacillus strains were harvested by centrifugation at 12,000 × g for 15 min after

DPP-IV inhibition by Lactobacillus

The DPP-IV inhibitory activity of the Lactobacillus strains is shown in Table 1. All strains used in this study showed DPP-IV inhibitory activity in vitro. Both CFS and CFE showed varying levels of DPP-IV inhibitory activity. The DIR of the CFS samples (from 5 × 1010 cfu mL−1, 6 h) ranged from 7.2 to 20.7%. Lactobacillus plantarum strain ZF06-3, Lactobacillus paracasei IF13 and L.plantarum IF2-14 showed high levels of inhibition, followed by L.plantarum ZF06-1 (the other strains' CFS had

Conclusion

L.plantarum strains ZF06-1, ZF06-3 and IF2-14 and L.brevis strain IF2-17 were found able to inhibit DPP-IV and porcine intestinal α-glucosidase, and displayed good probiotic properties in vitro. Specific peptide-cleavage and fragmental release in CFS contribute higher DPP-IV inhibition and bioactivities. Lactobacilli with DPP-IV and α-glucosidase inhibitory activities may serve as potential candidates for the management of T2D by means of their dual ability to slow both inactivation of the

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31071507), the National High Technology Research and Development Program (“863” Program, No. 2008AA10Z310), and the National Science and Technology Support Program, Ministry of Science and Technology of the People's Republic of China (2011BAD09B03).

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