Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

Gerrit J Gerwig,Johannis P Kamerling,Evelien M Te Poele,Davy Van De Walle,Anna K H Hirsch,Koen Dewettinck,Wim Soetaert,Tim Devlamynck,Lubbert Dijkhuizen,Manuel Jäger

doi:10.1038/s41598-018-19622-5

Gerrit J Gerwig, Johannis P Kamerling + Show 8 more

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https://doi.org/10.1038/s41598-018-19622-5

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Abstract

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties.

Highlights

The world-wide increasing incidence of obesity, diabetes type II, cardio vascular diseases, and dental caries leads to an increased consumer demand for food products and beverages without high-calorie sugars[1]
(α1→4)-glucosylation of the Glc(β1→ residue at the steviol C-19 site resulted in an increased bitter aftertaste and a lower sweetness intensity12,17–19. α-Glucosylation of stevioside using Biozyme L (β-amylase preparation, probably contaminated with an α-glucosidase) and maltose as donor substrate, resulted in a product with a decreased sweetness, but a remarkable improvement in the quality of taste [Glc(α1→6) residue attached at the Glc(β1→C-19 residue], a product with a much lower sweetness [Glc(α1→6) residue attached to the terminal Glc(β1→2) residue of the β-sophorosyl-C-13 unit] and a product with a bitter taste [Glc(α1→3) residue attached to the terminal Glc(β1→2) residue of the β-sophorosyl-C-13 unit]20
Aiming to obtain steviol glycoside derivatives with improved organoleptic properties, we studied the α-glucosylation potential of mutant glucansucrase enzymes of the generally recognized as safe (GRAS) bacterium Lactobacillus reuteri 180 on rebaudioside A (RebA)

Summary

Introduction

The world-wide increasing incidence of obesity, diabetes type II, cardio vascular diseases, and dental caries leads to an increased consumer demand for food products and beverages without high-calorie sugars[1]. To improve the taste of steviol glycosides, especially for food applications, various (enzymatic) modifications of the carbohydrate moieties of steviol glycosides have been reported, mainly using cyclodextrin glycosyltransferase (CGTase), α- and β-glucosidase, α- and β-galactosidase and β-fructosidase transglycosylation and β-glycosyltransferase glycosylation systems as biocatalysts [see review15]. Several early studies have shown that both mono- and di-(α1→4)-glucosylation of the carbohydrate moiety at the steviol C-13 site of stevioside and rubusoside gave products with a remarkable improvement in both intensity and quality of sweetness. (α1→4)-glucosylation of the Glc(β1→ residue at the steviol C-19 site resulted in an increased bitter aftertaste and a lower sweetness intensity. Glucansucrases (EC 2.1.4.5; glucosyltransferases, Gtfs) are extracellular enzymes catalyzing the synthesis of α-D-glucan polymers from the donor substrate sucrose, thereby introducing different ratios of glycosidic linkages [(α1→2), (α1→3), (α1→4), (α1→6)] in their glucan products, depending on the enzyme specificities[21,22]

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