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Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered β-fructofuranosidases

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Abstract

Fructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving β-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening β-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of β-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of β-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up.

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References

  1. Waterhouse AL, Chatterton NJ (1993) In: Suzuki M, Chatterton N (eds) Sci. Technol. Fruct. CRC Press, Boca Raton

  2. Gibson GR, Roberfroid MB (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 125:1401–1412

    CAS  Google Scholar 

  3. Buddington RK, Williams CH, Chen SC, Witherly SA (1996) Dietary supplement of neosugar alters the fecal flora and decreases activities of some reductive enzymes in human subjects. Am J Clin Nutr 63:709–716

    CAS  Google Scholar 

  4. Singh R, Singh R (2010) Production of fructooligosaccharides from inulin by endoinulinases and their prebiotic potential. Food Technol Biotechnol 48:435–450

    CAS  Google Scholar 

  5. Hidaka H, Hirayama M, Sumi N (1988) A fructooligosaccharide-producing enzyme from Aspergillus niger ATCC 20611. Agric Biol Chem 52:1181–1187

    Article  CAS  Google Scholar 

  6. Sangeetha PT, Ramesh MN, Prapulla SG (2005) Recent trends in the microbial production, analysis and application of fructooligosaccharides. Trends Food Sci Technol 16:442–457

    Article  CAS  Google Scholar 

  7. Nemukula A, Mutanda T, Wilhelmi BS, Whiteley CG (2009) Response surface methodology: synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus. Bioresour Technol 100:2040–2045

    Article  CAS  Google Scholar 

  8. Vega R, Zúniga-Hansen ME (2011) Enzymatic synthesis of fructooligosaccharides with high 1-kestose concentrations using response surface methodology. Bioresour Technol 102:10180–10186

    Article  CAS  Google Scholar 

  9. Sangeetha PT, Ramesh MN, Prapulla SG (2005) Maximization of fructooligosaccharide production by two stage continuous process and its scale up. J Food Eng 68:57–64

    Article  Google Scholar 

  10. Dominguez A, Nobre C, Rodrigues LR et al (2012) New improved method for fructooligosaccharides production by Aureobasidium pullulans. Carbohydr Polym 89:1174–1179

    Article  CAS  Google Scholar 

  11. Kuchner O, Arnold FH (1997) Directed evolution of enzyme catalysts. Trends Biotechnol 15:523–530

    Article  CAS  Google Scholar 

  12. De Abreu MA, Alvaro-Benito M, Plou FJ et al (2011) Screening β-fructofuranosidases mutant libraries to enhance the transglycosylation rates of β-(2 → 6) fructooligosaccharides. Comb Chem High Throughput Screen 14:730–738

    Article  Google Scholar 

  13. Maugeri F, Hernalsteens S (2007) Screening of yeast strains for transfructosylating activity. J Mol Catal B Enzym 49:43–49

    Article  CAS  Google Scholar 

  14. L’homme C, Peschet JL, Puigserver A, Biagini A (2001) Evaluation of fructans in various fresh and stewed fruits by high-performance anion-exchange chromatography with pulsed amperometric detection. J Chromatogr A 920:291–297

    Article  Google Scholar 

  15. Corradini C, Lantano C, Cavazza A (2013) Innovative analytical tools to characterize prebiotic carbohydrates of functional food interest. Anal Bioanal Chem 405:4591–4605

    Article  CAS  Google Scholar 

  16. Beine R, Moraru R, Nimtz M et al (2008) Synthesis of novel fructooligosaccharides by substrate and enzyme engineering. J Biotechnol 138:33–41

    Article  CAS  Google Scholar 

  17. Bureau S, Ruiz D, Reich M et al (2009) Application of ATR-FTIR for a rapid and simultaneous determination of sugars and organic acids in apricot fruit. Food Chem 115:1133–1140

    Article  CAS  Google Scholar 

  18. Duarte IF, Barros A, Delgadillo I et al (2002) Application of FTIR spectroscopy for the quantification of sugars in mango juice as a function of ripening. J Agric Food Chem 50:3104–111

    Article  CAS  Google Scholar 

  19. Cozzolino D, Curtin C (2012) The use of attenuated total reflectance as tool to monitor the time course of fermentation in wild ferments. Food Control 26:241–246

    Article  CAS  Google Scholar 

  20. Wang J, Kliks MM, Jun S et al (2010) Rapid analysis of glucose, fructose, sucrose, and maltose in honeys from different geographic regions using fourier transform infrared spectroscopy and multivariate analysis. J Food Sci 75:C208–214

    Article  CAS  Google Scholar 

  21. Blanch M, Goñi O, Sanchez-Ballesta MT et al (2012) Characterisation and functionality of fructo-oligosaccharides affecting water status of strawberry fruit (Fragraria vesca cv. Mara de Bois) during postharvest storage. Food Chem 134:912–919

    Article  CAS  Google Scholar 

  22. Cadet F, Pin FW, Rouch C et al (1995) Enzyme kinetics by mid-infrared spectroscopy: beta-fructosidase study by a one-step assay. Biochim Biophys Acta 1246:142–150

    Article  Google Scholar 

  23. Schindler R, Le Thanh H, Lendl B, Kellner R (1998) Determination of enzyme kinetics and chemometric evaluation of reaction products by FTIR spectroscopy on the example of β-fructofuranosidase. Vib Spectrosc 16:127–135

    Article  CAS  Google Scholar 

  24. Riley MR, Arnold MA, Murhammer DW et al (1998) Adaptive calibration scheme for quantification of nutrients and byproducts in insect cell bioreactors by near-infrared spectroscopy. Biotechnol Prog 14:527–533

    Article  CAS  Google Scholar 

  25. Geladi P, Kowalski BR (1986) Partial least-squares regression: a tutorial. Anal Chim Acta 185:1–17

    Article  CAS  Google Scholar 

  26. Trollope KM, Nieuwoudt HH, Görgens JF, Volschenk H (2014) Screening a random mutagenesis library of a fungal β-fructofuranosidase using FT-MIR ATR spectroscopy and multivariate analysis. Appl Microbiol Biotechnol 98:4063–4073

    Article  CAS  Google Scholar 

  27. Norgaard L, Saudland A, Wagner J et al (2000) Interval partial least-squares regression (iPLS): a comparative chemometric study with an example from near-infrared spectroscopy. Appl Spectrosc 54:413–419

    Article  CAS  Google Scholar 

  28. Jolliffe IT (2002) Principal component analysis, 2nd edn. Springer-Verlag, New York

    Google Scholar 

  29. Max J-J, Chapados C (2007) Glucose and fructose hydrates in aqueous solution by IR spectroscopy. J Phys Chem A 111:2679–2689

    Article  CAS  Google Scholar 

  30. Thongnopkun P, Ekgasit S (2005) FTIR spectra of faceted diamonds and diamond simulants. Diam Relat Mater 14:1592–1599

    Article  CAS  Google Scholar 

  31. Li-Chan E, Chalmers JM, Griffiths P (2010) Applications of vibrational spectroscopy in food science: volumes I and II: analysis of food, drink and related materials. John Wiley & Sons, Chichester

    Google Scholar 

  32. Max J-J, Chapados C (2001) Sucrose hydrates in aqueous solution by IR spectroscopy. J Phys Chem A 105:10681–10688

    Article  CAS  Google Scholar 

  33. Xie L, Ye X, Liu D, Ying Y (2009) Quantification of glucose, fructose and sucrose in bayberry juice by NIR and PLS. Food Chem 114:1135–1140

    Article  CAS  Google Scholar 

  34. Bolton E, Wang Y, Thiessen P, Bryant S (2008) In: Wheeler R, Spellmeyer D (eds) Annu. Rep. Comput. Chem. Elsevier, Oxford

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Acknowledgments

The authors would like to acknowledge Jamie Gallant for technical assistance with scanning FT-MIR ATR samples. Financial support for this work was provided by the Technology Innovation Agency (TIA), project PB110/08. The funders had no involvement in conducting the research or publication thereof. Kim Trollope was supported by grants from TIA, the National Research Foundation and Stellenbosch University. She also holds a L’Oreal-UNESCO For Women in Science Regional Fellowship in Sub-Saharan Africa.

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Authors and Affiliations

  1. Department of Microbiology, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa

    Kim M. Trollope & Heinrich Volschenk

  2. Department of Process Engineering, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa

    Johann F. Görgens

  3. Department of Food Science, University of Copenhagen, Rolighedsvej 30, 1958, Frederiksberg C, Denmark

    Rasmus Bro

  4. Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, Private Bag X1, Stellenbosch, 7602, South Africa

    Rasmus Bro & Hélène H. Nieuwoudt

Authors
  1. Kim M. Trollope
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  2. Heinrich Volschenk
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  3. Johann F. Görgens
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  4. Rasmus Bro
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  5. Hélène H. Nieuwoudt
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Correspondence to Hélène H. Nieuwoudt.

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Trollope, K.M., Volschenk, H., Görgens, J.F. et al. Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered β-fructofuranosidases. Anal Bioanal Chem 407, 1661–1671 (2015). https://doi.org/10.1007/s00216-014-8417-0

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  • DOI: https://doi.org/10.1007/s00216-014-8417-0

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