Potential application of commercial enzyme preparations for industrial production of short-chain fructooligosaccharides

doi:10.1016/j.molcatb.2011.12.007

Journal of Molecular Catalysis B: Enzymatic

Volume 76, April 2012, Pages 44-51

https://doi.org/10.1016/j.molcatb.2011.12.007 Get rights and content

Abstract

Twenty-five commercial enzyme preparations for use in the food industry were assayed for transfructosylation activity. Three preparations showed high transfructosylation activity from sucrose as well as the ratio of transferase and hydrolase activities. Short-chain fructooligosaccharides (sc-FOS) were not hydrolyzed by the three enzyme preparations after a 12 h reaction time. At a 6 h reaction time, yield and volumetric productivity were in the range from 58.8 to 62.6% (g sc-FOS/100 g initial sucrose) and 52.5 to 55.9 g sc-FOS/L h, respectively. One enzyme preparation was then evaluated for sc-FOS synthesis. Thus, environmental factors influencing the reaction were studied on products. Total sc-FOS concentration was not affected by temperature, pH and enzyme concentration at the studied levels, but high concentrations of sucrose affected the sc-FOS formation. The results suggest that these enzyme preparations can be exploited as a source of food-grade fructosyltransferase, in addition to Pectinex Ultra SP-L.

Graphical abstract

Highlights

► Twenty-five enzyme preparations were assayed for transfructosylation activity. ► Twenty-four of them have not been tested for this purpose. ► Three preparations exhibited a high ratio of transferase and hydrolase activities. ► Kestose was the predominant oligosaccharide under the reaction conditions tested. ► The process described here is inexpensive, simple and efficient.

Introduction

Short-chain fructooligosaccharides (sc-FOS) are a mixture of 1-kestose (GF₂), nystose (GF₃) and 1^F-fructofuranosylnystose (GF₄), which have been regarded as prebiotics since the mid-1990s. They have important physiological functions due to their indigestibility in the upper gastrointestinal tract, which stimulate the selective growth of bifidobacteria in the large intestine [1], [2]. Sc-FOS have received GRAS status (generally recognized as safe), which has promoted their use as ingredients for both food and feed in East Asia, North America and Europe [3].

Sc-FOS are produced either from sucrose by transfructosylation with fructosyltransferases (β-fructofuranosidase, EC 3.2.1.26 or β-d-fructosyltransferase, EC 2.4.1.9) or from inulin by controlled enzymatic hydrolysis. The transfructosylation process has a greater potential because it is possible to synthesize sc-FOS oligomers of either defined chain length [4] or desired composition mixtures by modulating the reaction time. GF₂ has more sweetening power than other sc-FOS and can be used as a sweetener for diabetics [5], so the reaction should be stopped at the maximum production of sc-FOS.

A fructosyltransferase is considered efficient if it possesses the ability to bind the acceptor, fructosyl moiety, and to exclude H₂O via a double-displacement mechanism, as shown in Fig. 1 [6], [7], [8], [9]. This efficiency is confirmed by the synthesis yield because it depends on the relative rates of transfructosylation and hydrolysis [10].

The main disadvantage of the sc-FOS synthesis process is the lack of a reasonably priced and efficient catalyst. Some studies have described screening microorganisms for transfructosylation activity [7], [11], [12], but this approach is complicated and tedious because a large number of positive hits may still fail. Only a few of these enzymes have the level of transfructosylation activity necessary for industrial applications [10]. Moreover, these isolated enzymes are not yet commercially available. Another approach is the development of heterologous recombinant enzymes that efficiently and selectively synthesize GF₂ [13], [14]. These enzymes are not currently used in industrial production of oligosaccharides in Japan, according to a report by Taniguchi [15], because manufacturers are nervous about consumers’ response to their use.

Currently, one of the most common alternatives discussed in the literature is the immobilization of fructosyltransferases [16], [17], [18], but this process is only justified if the enzyme is expensive or inactivated under reaction conditions. An alternative is to use low-cost commercial enzyme preparations designed for use in the food industry. Many are obtained from filamentous fungi that are considered good producers of fructosyltransferases [19]. In addition to the main enzymatic activity, some enzyme preparations have secondary activities, including transfructosylation, which has been found in Pectinex Ultra SP-L [20].

On the other hand, compared with previous approaches, commercial enzyme preparations have economic and technical advantages, such as low price, versatility and stability of enzymatic activity under reaction conditions. Because of advances in biotechnology, it is now possible to have more varieties of food-grade enzymes, which increases their potential application in sc-FOS production. More studies are needed to find other preparations with transfructosylation activity and thermal stability as Pectinex Ultra SP-L. In the current study, three commercial enzyme preparations were selected from twenty-five for the synthesis of sc-FOS from sucrose because they showed high transfructosylation activity as well as the ratio of transferase and hydrolase activities. In addition, the effect of reaction conditions on the synthesis was studied by the action of an enzyme preparation previously selected.

Section snippets

Materials

1-Kestose, nystose and 1^F-fructofuranosylnystose standards were obtained from Wako Chemicals (Richmond, VA, USA). Glucose–oxidase–peroxidase enzymatic kit was purchased from Spinreact (San Esteve de Bas, Spain). Other reagents were purchased from Sigma Chemical (St. Louis, MO, USA) or Merck (Darmstadt, Germany).

Enzymes

Twenty-five enzyme preparations from fungal strains that are designed for use in the food industry were kindly donated by Biocatalysts Ltd. (Parc Nantgarw, Wales, UK); Amano Enzyme Co.,

Screening of enzyme preparations for transfructosylation activity

The twenty-five enzyme preparations have several known activities for their use in the food industry, but the current preparations were assayed using sucrose to measure conversion into transfructosylation products. The transfructosylation and hydrolysis activities of these preparations are shown in Fig. 2a and b.

Enzyme preparations exhibited one or both activities, except for Pectinex Smash XXL, Cellulase 13L and Depol 692L. In order to select enzyme preparations for subsequent experiments were

Conclusions

Three low-cost enzyme preparations were selected from a screening for transfructosylation activity. These preparations and Pectinex Ultra SP-L exhibited a high ratio (U_T/U_H), selectivity for the synthesis of sc-FOS and did not hydrolyze the produced sc-FOS after a 12 h reaction time. Pectinex Ultra SP-L, Rohapect CM, Viscozyme L and Pectinex Smash could serve as a source of food-grade fructosyltransferase for the inexpensive and efficient production of sc-FOS.

Sc-FOS synthesis catalyzed by

Acknowledgements

This research was financially supported by the Project FONDEF DO7I1045 of Chile and the CREAS. Also, we acknowledge financial support (scholarship) of CONICYT and PUCV for the PhD student, R. Vega. We are very grateful by the generous donations of Biocatalysts Ltd. (Wales, UK), Amano Enzyme USA Co., Ltd., DSM Food Specialties Unltd (Santiago, Chile), Dimerco Comercial Ltda (Santiago, Chile) and Blumos SA (Santiago, Chile).

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Potential application of commercial enzyme preparations for industrial production of short-chain fructooligosaccharides

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Materials

Enzymes

Screening of enzyme preparations for transfructosylation activity

Conclusions

Acknowledgements

J. Nutr.

Bioresour. Technol.

Carbohydr. Res.

J. Mol. Catal. B: Enzym.

J. Biol. Chem.

Enzyme Microb. Technol.

Enzyme Microb. Technol.

J. Biotechnol.

Carbohydr. Res.

Bioresour. Technol.

Lebensm. Wiss. Technol.

Enzyme Microb. Technol.

J. Mol. Catal. B: Enzym.

J. Food Eng.

Food Bioprod. Process

J. Mol. Catal. B: Enzym.

Biochem. Eng. J.

J. Biotechnol.

Enzyme Microb. Technol.

Process Biochem.