Effect of sucrose concentration on the composition of enzymatically synthesized short-chain fructo-oligosaccharides as determined by FTIR and multivariate analysis
Introduction
Fructo-oligosaccharides (FOS) are small chain oligosaccharides composed of fructose units linked by (2→1)-β-glycosidic bonds and a single d-glucosyl unit at the non-reducing end. In most cases, FOS are mixtures of short chain oligosaccharides, namely 1-kestose [degree of polymerization (DP) equal to 3], nystose (DP4) and 1F-fructofuranosylnystose (DP5) (Crittenden & Playne, 2009).
FOS are well recognized prebiotics, that is, non-digestible food components that beneficially affect the host health by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Gibson & Roberfroid, 1995). They have a great economic importance as they are used in infant formula and other functional food products (Romano, Tymczyszyn, Mobili, & Gómez-Zavaglia, 2015). FOS are also low-calorie and non-cariogenic sweeteners, and effective protectants of biological structures during dehydration processes (Romano et al., 2014, Schwab et al., 2007). This latter property has been ascribed to their capacity to interact with lipid membranes, which in turn is dependent on their structure and on their DP (Hincha et al., 2006, Hincha et al., 2007).
Short chain FOS (i.e.: DP3, DP4, DP5) are generally produced from sucrose by transfructosylation reactions using fructosyltransferases (β-fructofuranosidase, EC 3.2.1.26 or β-d-fructosyltransferase, EC 2.4.1.9) as biocatalysts (Vega and Zuniga-Hansen, 2011, Vega and Zuñiga-Hansen, 2014, Vega-Paulino and Zuniga-Hansen, 2012). The composition of the FOS obtained can be modulated by adjusting the reaction parameters (i.e., time, temperature, pH, enzyme source and substrate concentrations).
Commercial enzyme preparations have economic and technical advantages, including low price, versatility and stability under reaction conditions of industrial processes. Most of them have both transfructosylase and hydrolase activities, thus to synthesize short chain oligosaccharides, preparations with high transfructosylase activity are those to be selected. Viscozyme L from Aspergillus aculeatus (Novozyme, Denmark) is an adequate enzymatic preparation because it has both high transfructosylation activity and high transferase/hydrolase ratio (Lorenzoni et al., 2014, Lorenzoni et al., 2015, Vega-Paulino and Zuniga-Hansen, 2012).
The economic importance of FOS requires on quick and reliable methods to monitor their synthesis. Even though the usefulness of high performance liquid chromatography (HPLC) in determining the composition of sugar mixtures is unquestionable, Fourier transform infrared spectroscopy (FTIR) is nowadays a trustworthy technique to ascertain structural and physical properties of carbohydrates. As no exogenous chemical reagents are needed, samples require almost no preparation and analytical testing does not generate hazardous waste, FTIR is definitely a useful tool to determine the composition of complex oligo- and polysaccharides in a quick and environmentally friendly way (Anjos et al., 2015, Coimbra et al., 2002, Santos et al., 2015). The use of FTIR in tandem with multivariate analysis has enabled an expeditious determination of the oligo and polysaccharide composition of different products, including commercial sugars (Kačurakóvá & Wilson, 2001), cellulose, pectins (Fellah, Anjukandi, Waterland, & Williams, 2009), starch, hemicelluloses, carrageenans, hyaluronates (Fellah et al., 2009, Kačurakóvá and Wilson, 2001), fruits (Bureau et al., 2009), cereals (Cozzolino, Roumeliotis, & Eglinton, 2014), honey (Anjos et al., 2015) and wine extracts (Coimbra et al., 2002). Moreover, different enzymatic reactions including sugars either as substrates or as products have been monitored using FTIR (Baum et al., 2013, Chiş et al., 2010, Schindler et al., 1998).
With regard to FOS, FTIR and multivariate analysis were used to qualitatively characterize FOS in strawberries (Blanch, Goñi, Sanchez-Ballesta, Escribano, & Merodio, 2012), barley (Cozzolino et al., 2014) and agave (Mellado-Mojica & López, 2015). Trollope, Nieuwoudt, Görgens, and Volschenk (2014) used FTIR to quantify the consumption of sucrose as an indicator of the activity of β-fructofuranosidases from different origins. More recently, they developed partial least square (PLS) calibration models based on FTIR spectra for the screening of β-fructofuranosidases libraries generated by random mutagenesis (Trollope, Volschenk, Görgens, Bro, & Nieuwoudt, 2015).
Considering that the composition of FOS determines both their prebiotic properties and their capacity to interact with lipid membranes, and that this composition is in turn determined by the conditions of synthesis, the goal of this work was twofold: (a) to obtain FOS of different composition by adjusting the initial sucrose concentration; and (b) to define models based on multivariate analysis to determine the composition of FOS throughout the synthesis, directly from the FTIR spectra.
Section snippets
Materials
Viscozyme L was donated by Blumos SA-Chile. 1-kestose (DP3), nystose (DP4) and 1F-fructofuranosylnystose (DP5) standards were purchased from Wako Chemicals (Richmond, VA, USA). Sucrose, glucose, fructose and other reagents were obtained from Sigma Chemical (St. Louis, MO, USA).
Synthesis of FOS
Sucrose solutions within 10% and 60% w/v prepared in distilled water were used as substrate for the enzymatic synthesis. The pH was adjusted to 5.5 with 2 M NaOH and 4% v/v Viscozyme L (56 FU/mL; FU: fructosyltransferase
Results
Sucrose concentrations of 10%, 20%, 40% and 60% (w/v) were tested to modulate the composition of oligosaccharides in the FOS syrup. The higher the sucrose concentration, the lower its percentage of conversion (X) at a given time (Fig. 1A). The exponential fitting of the experimental data enabled the description of X over time as shown in Eq. (3):where X is the sucrose conversion, Xmax is the maximum sucrose conversion, t is the reaction time and k is the sucrose decay constant. X
Discussion
The composition of FOS is directly related to their prebiotic and physico-chemical properties, and this composition can be modulated by adjusting the synthesis conditions. Bearing in mind this background, the results obtained in this work can be analyzed from two different points of view: (a) the effect of the initial sucrose concentration on the composition of FOS, and (b) the definition of quantification models based on FTIR and multivariate analysis to determine the composition of
Conclusions
Even though FOS are mainly known for their prebiotic properties, they have other interesting properties, including their role as low-calorie and non-cariogenic sweeteners, and their capacity to protect lipid membranes during dehydration processes. Each of these properties is mainly ascribed to a particular type of FOS in the mixture of oligosaccharides.
In this context, this work integrates two complementary aspects related to the production of FOS: a rationalization of their enzymatic synthesis
Competing interests
The authors declare that they have no competing interests.
Author’s contributions
N.R. did the experimental work. M.S. did the multivariate analysis and R.V., the enzymatic analysis. A.G.-Z. and P.M. coordinated the work (analysis of results, discussion and writing of the manuscript). All authors have approved the final version of the manuscript.
Acknowledgments
The Center for the Study of Healthy Foods (CREAS) is especially acknowledged for providing the infrastructure for the synthesis of FOS. M.E. Zuniga-Hansen and C. Altamirano are also acknowledged for the helpful discussions. Authors acknowledge C. Reyes for technical assistance in HPLC.
This work was supported by the Argentinean Agency for the Scientific and Technological Promotion (ANPCyT) (Projects PICT/2011/0226 and PICT/2014/0912), the Ministry for Science and Technology (MINCYT, project
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