Enzymatic synthesis of fructooligosaccharides with high 1-kestose concentrations using response surface methodology

doi:10.1016/j.biortech.2011.09.025

Bioresource Technology

Volume 102, Issue 22, November 2011, Pages 10180-10186

https://doi.org/10.1016/j.biortech.2011.09.025 Get rights and content

Abstract

Response surface methodology was used as an optimization tool for the production of short chain fructooligosaccharides (sc-FOS) using the commercial cellulolytic enzyme preparation, Rohapect CM. Three independent variables, temperature, concentrations of sucrose and enzyme were tested in the reaction medium. The responses of the design were, yield (g sc-FOS/100 g initial sucrose), 1-kestose (g/100 g sc-FOS) and volumetric productivity (g sc-FOS/L h). Significant effects on the three responses included a quadratic effect (temperature), a linear effect (sucrose and enzyme concentrations) and an interaction between temperature and sucrose concentration. The cost-effective conditions to support the process in a high competitive market were 50 °C, 6.6 TU/mL enzyme, 2.103 M sucrose in 50 mM acetate buffer at pH 5.5, and the synthesis for a 5 h reaction time. Under these conditions, a high Y_P/S (63.8%), Q_P (91.9 g/L h) and sGF2 (68.2%) was achieved.

Highlights

► An industrial commercial enzyme preparation was used as fructosyltransferase. ► Factors affecting the process can be optimized according to product specifications. ► 1-Kestose was the predominant component in the obtained fructooligosaccharide syrup. ► The reported process is inexpensive, simple and efficient.

Introduction

Short chain fructooligosaccharides (sc-FOS) of the inulin type constitute one of the most recognized groups of prebiotic oligosaccharides (Ballesteros et al., 2007, Nemukula et al., 2009). Their physiological functions are directly related to the indigestibility of sc-FOS in the upper gastrointestinal tract, which promotes the selective growth of bifidobacteria in the large intestine (Hirayama, 2002). This recognition has increased their demand in the food industry; however, the supply of sc-FOS is limited due to the fact that enzymes such as fructosyltransferases (β-fructofuranosidase, EC 3.2.1.26 or β-d-fructosyltransferase, EC 2.4.1.9) are not commercially available. Pectinex Ultra SP-L, a pectinolytic and cellulolytic preparation designed for fruit juice processing, has been suggested as a source of food-grade fructosyltransferase because this enzyme has been found in the commercial preparation (Antošová et al., 2008, Ghazi et al., 2006).

Reaction conditions to obtain high yields of sc-FOS have been determined using fructosyltransferases of Aspergillus japonicus, Pectinex Ultra SP-L and Aureobasidium pullulans (Cruz et al., 1998, Hang and Woodams, 1996, Madlová et al., 1999). Transfructosylation is favored over hydrolysis at high concentrations of sucrose and by the reaction conditions such as, pH (4.5–6.5), temperature (50–60 °C), reaction time (3–5 h) and high ratios of transferase and hydrolase activities of the enzyme (Ghazi et al., 2006, Nemukula et al., 2009). However, there are no studies on the interactions between the reaction conditions. The optimization of the production of syrups consisting largely of a fructooligosaccharide with a specific degree of polymerization at high yield and volumetric productivity has also not been studied.

Transfructosylation is a complex reaction with efficient kinetic controls because sc-FOSs are potential substrates of the reaction (Monsan and Paul, 1995). A higher yield of sc-FOS can be obtained as the duration of the reaction progresses (approximately 55–60%); however, a large amount of 1-kestose is transformed to nystose.

1-Kestose has more sweetening power than other sc-FOS, and 1-kestose-rich sc-FOS syrups can be used as sugar for diabetics (Yun, 1996). The chain length is an important factor influencing the physiological effect of the oligomer in the host (Biedrzycka and Bielecka, 2004, Yoshida et al., 2006) and fermentation by bifidobacteria and lactobacilli species (Kaplan and Hutkins, 2000, Sannohe et al., 2008). Working with mice and in vitro experiments, Suzuki et al. (2006) have observed the superiority of 1-kestose over syrups consisting largely of nystose in the selective growth of bifidobacteria, but the relevance of these studies to the human gut microflora remains unknown. Yoshida et al. (2006) reported that 1-kestose and nystose can modulate the intestinal microflora and immune system in mice with different degrees of effectiveness. The authors suggested that the ratios of 1-kestose and nystose in the sc-FOS mixture can be changed to improve their biological activity in the host. In a study with infants, Shibata et al. (2009) administered 1-kestose for the treatment of atopic dermatitis (AD) and found a significant improvement in the SCORAD (Clinical evaluations of AD using Severity Scoring of Atopic Dermatitis) score in kestose-treated subjects.

It is well-known that high temperatures and high enzyme concentrations in the reaction medium accelerate the transfructosylation rate, which improves volumetric productivity; however, 1-kestose is converted more quickly to nystose under these conditions. Therefore, the factors affecting the process can be optimized according to the required final product specifications. Response surface methodology can be applied to this process as an optimization tool (Montgomery, 2004).

Screening of commercial food-grade enzyme preparations for fructosyltransferases suitable for the production of sc-FOS (Vega and Zuniga-Hansen, 2010), revealed high levels of transfructosylation activity in Rohapect CM. This enzyme preparation, obtained from Trichoderma reesei, is employed in cleaning of UF-membranes used in the fruit juice and wine industries. In the current study, conditions were determined for producing sc-FOS from sucrose while obtaining a high percentage of 1-kestose using Rohapect CM.

Section snippets

Materials

Rohapect CM was obtained from AB Enzymes GmbH (Dimerco Comercial Ltda., Chile). Wako Chemicals (Richmond, VA, USA) provided 1-kestose, nystose and 1^F-fructofuranosylnystose standards. A glucose–oxidase–peroxidase enzymatic kit was obtained from Spinreact (San Esteve de Bas, Spain). Other reagents were purchased from Sigma Chemical (St. Louis, MO, USA) or Merck (Darmstadt, Germany).

Enzyme assay

Rohapect CM had 12,000 TU/mL of transfructosylation activity. One unit of TU was defined as the amount of enzyme

Fitting model, adequacy and model adequacy checking

The results of the experimental design after 3 or 5 h reaction time are shown in Table 3, Table 4, respectively. The second to fourth column are the results obtained for Y_P/S, sGF2 and Q_P, which were the responses of the experimental design. sGF2 and Q_P in all of the experiments presented in Table 3 were greater than those in Table 4, in contrast, the Y_P/S was lower. This low yield represented a large amount of remaining sucrose, which is not sustainable in a highly competitive market. A higher Y

Conclusions

Rohapect CM catalyzed the sc-FOS synthesis under all of the experimental conditions, which is remarkable for its potential use as biocatalyst for the industrial processing of sucrose into sc-FOS syrup. Using response surface methodology, it was possible to determine the cost-effective conditions of temperature (50 °C), sucrose concentration (2.103 M) and enzyme concentration (6.6 TU/mL) to support the process in a high competitive market. Under these conditions, a high Y_P/S (63.8%), Q_P (91.9 g/L h)

Acknowledgements

This research was financially supported by the Project FONDEF DO7I1045 of Chile and the CREAS. In addition, we acknowledge the financial support (scholarship) of CONICYT for our PhD student, R.Vega. We are grateful to Matias Berndt (Dimerco Comercial Ltda., Chile) for the enzyme sample.

References (31)

M. Antošová et al.
Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans
J. Biotechnol.
(2008)
D. Bas et al.
Modeling and optimization I: usability of response surface methodology
J. Food Eng.
(2007)
E. Biedrzycka et al.
Prebiotic effectiveness of fructans of different degrees of polymerization
Trends Food Sci. Technol.
(2004)
W.C. Chen et al.
Production of β-fructofuranosidase by Aspergillus japonicus
Enzyme Microbial. Technol.
(1996)
R. Cruz et al.
Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate
Bioresour. Technol.
(1998)
A. Gosling et al.
Recent advances refining galactooligosaccharide production from lactose
Food Chem.
(2010)
Y.D. Hang et al.
Optimization of enzymatic production of fructo–oligosaccharides from sucrose
Lebensm. Wiss. u. Technol.
(1996)
J.C. Lee et al.
The stabilization of proteins by sucrose
J. Biol. Chem.
(1981)
P. Monsan et al.
Enzymatic synthesis of oligosaccharides
FEMS Microbiol. Rev.
(1995)
S.I. Mussatto et al.
Fructooligosaccharides and β-fructofuranosidase production by Aspergillus japonicus immobilized on lignocellulosic materials
J. Mol. Catal. B Enzym.
(2009)

S.I. Mussatto et al.

Increase in the fructooligosaccharides yield and productivity by solid-state fermentation with Aspergillus japonicus using agro-industrial residues as support and nutrient source

Biochem. Eng. J.

(2010)

A. Nemukula et al.

Response surface methodology: synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

Bioresour. Technol.

(2009)

P.T. Sangeetha et al.

Maximization of fructooligosaccharide production by two stage continuous process and its scale up

J. Food Eng.

(2005)

J.W. Yun

Fructooligosaccharides – occurrence, preparation, and application

Enzyme Microbial. Technol.

(1996)

A. Ballesteros et al.

Enzymatic synthesis of sugar esters and oligosaccharides from renewable resources

Cited by (31)

Enzymatic synthesis of fructooligosaccharides: From carrot discards to prebiotic juice
2023, Food Research International
A great volume of carrots is discarded daily worldwide because they do not meet the required shape and size standards. However, they have the same nutritional characteristics as those commercialized, and can be used in different food products. Carrot juice is an excellent matrix for the development of functional foods with prebiotic compounds, such as fructooligosaccharides (FOS). In this work, the production of FOS in situ in carrot juice was evaluated using a fructosyltransferase from Aspergillus niger, produced by solid-state fermentation on carrot bagasse. The enzyme was partially purified 12.5-fold with a total yield of 93 %, and specific activity of 59 U/mg of protein by Sephadex G-105 molecular exclusion chromatography. It was identified by nano LC-MS/MS as a β-fructofuranosidase with a 63.6 kDa MW and it allowed obtaining a FOS yield of 31.6 % in carrot juice. The result was a prebiotic juice with a final concentration of 32.4 mg/mL of FOS. Using the commercial enzyme Viscozyme L a higher yield of FOS (39.8 %) was obtained in carrot juice, corresponding to a total amount of FOS of 54.6 mg/mL. This circular economy scheme allowed the obtention of a functional juice, that may contribute to improve health of consumers.
Development of a functional prebiotic strawberry preparation by in situ enzymatic conversion of sucrose into fructo-oligosaccharides
2023, Food Research International
Food industry has been pressed to develop products with reduced sugar and low caloric value, while maintaining unchanged their rheological and physicochemical properties. The development of a strawberry preparation for the dairy industry, with prebiotic functionality, was herein investigated by in situ conversion of its intrinsic sucrose content into prebiotic fructo-oligosaccharides (FOS). Two commercial enzymatic complexes, Viscozyme® L and Pectinex® Ultra SP-L, were evaluated for the synthesis of FOS. Operational parameters such as temperature, pH, and enzyme:substrate ratio (E:S) were optimized to maximize FOS yield. The rheological and physicochemical properties of the obtained strawberry preparation were evaluated. For functional analysis, the resistance of FOS to the harsh conditions of the gastro-intestinal digestion was evaluated by applying the standardized INFOGEST static protocol. At optimal conditions (60 ℃, pH 5.0), Pectinex® produced 265 ± 3 g·L⁻¹ FOS, yielding 0.57 ± 0.01 g_FOS·g_{initial sucrose}⁻¹ after 7 h reaction (E:S: 1:40); and Viscozyme® produced 295 ± 1 g·L⁻¹ FOS, yielding 0.66 ± 0.00 g_FOS·g_{initial sucrose}⁻¹ after 5 h (E:S: 1:30). The obtained strawberry preparations contained more than 50 % (w/w) prebiotic FOS incorporated (DP 3–5), with 80 % reduction of its sucrose content. The caloric value was therefore reduced by 26–31 %. FOS showed resistance to gastrointestinal digestion being only slightly hydrolysed (<10 %). 1^F-Fructofuranosylnystose was not digested at any phase of the digestion. Although the physicochemical properties of the prebiotic preparations were different from the original one, parameters such as the lower °Brix, water activity, consistency and viscosity, and its different color, may be easily adjusted. Results indicate that in situ synthesis strategies are efficient alternatives in the manufacture of reduced sugar and low-caloric food products with prebiotic potential.
Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose
2022, Enzyme and Microbial Technology
Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.
Biotransformation of sucrose rich Maple syrups into fructooligosaccharides, oligolevans and levans using levansucrase biocatalyst: Bioprocess optimization and prebiotic activity assessment
2022, Food Chemistry
Maple syrup was investigated as a source to produce FOSs and β-(2–6)-linked-oligolevans/levans. The modulation of this biotransformation was achieved through the control of Maple syrup °Bx and reaction conditions. Reaction time was identified as the most influential factor for the oligolevans/FOSs production in Maple syrup 30°Bx reaction system as well as for the oligolevans/levans synthesis in the 66°Bx one. In the predictive model of oligolevans/levans production in Maple syrup 60°Bx, the interactive effect between levansucrase unit and reaction time was significant (p-value of 0.0008). The optimal conditions for oligolevans/FOSs production (109.20 g/L) in Maple syrup 30°Bx were 3.73 U/mL, pH 6.60 and 23.12 h; while 5 U/mL, pH 6.04 and 29.92 h were identified as the optimal conditions for oligolevans/levans production (147.09 g/L) in Maple syrup 66°Bx. As compared to inulin-type commercial FOSs, the fermentation of oligolevans/FOSs from Maple syrup led to a higher count of Lactobacillus acidophilus and Bifidobacterium lactis and resulted in a higher production of lactic acid. This study lays the foundation for the biotransformation of Maple syrups into functional prebiotic ingredients.
Enzymes in fructooligosaccharides production
2021, Value-Addition in Food Products and Processing Through Enzyme Technology
In recent years, the demand for food products that generate health benefits has been a popular trend. Functional foods are beneficial to health, transcend nutritional characteristics, and involve physiological effects.
Fructooligosaccharides (FOS) are ingredients of functional foods whose intake promoting a positive impact on human health. The consumption of FOS also provides various benefits such as reduces cancer risk, decreases cardiovascular diseases, and improves the immune system.
Microbial enzymes have been widely used to produce traditional foods, such as beer, cheese, bread, etc. Nowadays, enzymes have an important role in other synthesis process like FOS production. The enzymes used for FOS production are fructosyltransferase (FTase; 2.4.1.9) and β-fructofuranosidase (FFase; 3.2.1.26). Recent research focuses on the development of enzymes for FOS production with high activity and stability.
Novel fructooligosaccharide conversion from sugarcane syrup using a specialised enzymatic pH-stat bioreactor
2020, Process Biochemistry
Citation Excerpt :
Several reports to date have focused on modulating the substrate concentration on the enzymatic synthesis of FOS [9–11]. However, few studies have addressed the effects of the interaction of more than one parameter at a time on native sugars [12,13]. Prior research on the enzymatic synthesis of FOS from pure sucrose, beet syrup, and molasses has been successfully carried out using conventional shake flasks [14,15], but there is little research on the use of a stirred tank reactor (STR) in FOS production.
Fructooligosaccharides (FOS) have gained significant attention for their prebiotic properties. Given that sugarcane syrup (SS) is sucrose-rich but with other nutritional benefits, its direct transformation into FOS may add value to this product. Therefore, the aims of this study were to develop FOS conversion from SS and to define the kinetic behaviour of FOS synthesized in a 1-L specialized pH-stat bioreactor (SPSB). The SS was composed of sucrose (58.93%) with considerable antioxidant capacities and Ƴ-aminobutyric acid. The developed SPSB process consisted of three stages: evaporation of sugarcane juice into syrup (68–75  °Brix) (stage 1), optimization of the Viscozyme L and SS mixture at different reaction temperatures (47–55 °C) (stage 2), and upscaling of the optimized reaction system under defined conditions in a 1 L-SPSB system (stage 3). In the 1 L-SPSB system, the enzymatic reaction yielded 32.22% of FOS from SS after a 6 h reaction, which is comparable with a pure system containing an equivalent concentration of 10% of sucrose as initial substrate with 39.55% yield. This result demonstrated the efficient conversion of SS into FOS, supporting the utilization of sugarcane juice for its health benefits.

View all citing articles on Scopus

View full text

Enzymatic synthesis of fructooligosaccharides with high 1-kestose concentrations using response surface methodology

Abstract

Highlights

Introduction

Section snippets

Materials

Enzyme assay

Fitting model, adequacy and model adequacy checking

Conclusions

Acknowledgements

J. Biotechnol.

J. Food Eng.

Trends Food Sci. Technol.

Enzyme Microbial. Technol.

Bioresour. Technol.

Food Chem.

Lebensm. Wiss. u. Technol.

J. Biol. Chem.

FEMS Microbiol. Rev.

J. Mol. Catal. B Enzym.

Biochem. Eng. J.

Bioresour. Technol.

J. Food Eng.

Enzyme Microbial. Technol.

Enzymatic synthesis of sugar esters and oligosaccharides from renewable resources