Abstract
A potentiometric whole cell biosensor based on immobilized marine bacterium, Pseudomonas carrageenovora producing κ-carrageenase and glycosulfatase enzymes for specific and direct determination of κ-carrageenan, is described. The bacterial cells were immobilized on the self-plasticized hydrogen ion (H+)-selective acrylic membrane electrode surface to form a catalytic layer. Hydrogen ionophore I was incorporated in the poly(n-butyl acrylate) [poly(nBA)] as a pH ionophore. Catalytic decomposition of κ-carrageenan by the bienzymatic cascade reaction produced neoagarobiose, an inorganic sulfate ion and a proton. The latter was detectable by H+ ion transducer for indirect potentiometric quantification of κ-carrageenan concentration. The use of a disposable screen-printed Ag/AgCl electrode (SPE) provided no cleaning requirement and enabled κ-carrageenan detection to be carried out conveniently without cross contamination in a complex food sample. The SPE-based microbial biosensor response was found to be reproducible with high reproducibility and relative standard deviation (RSD) at 2.6% (n = 3). The whole cell biosensor demonstrated a broad dynamic linear response range to κ-carrageenan from 0.2–100 ppm in 20 mM phosphate buffer saline (PBS) at pH 7.5 with a detection limit at 0.05 ppm and a Nernstian sensitivity of 58.78±0.87 mV/decade (R2 = 0.995). The biosensor showed excellent selectivity towards κ-carrageenan compared to other types of carrageenans tested e.g. ι-carrageenan and λ-carrageenan. No pretreatment to the food sample was necessary when the developed whole cell biosensor was employed for direct assay of κ-carrageenan in dairy product.
Highlights
Carrageenans are high molecular weight sulfated polysaccharides made up of repeating Dgalactose residues and 3,6-anhydrogalactose, which are linked via alternating α-(1,3) and β(1,4) linkages [1,2]
We present a simple potentiometric biosensor for direct detection of κ-carrageenan, being the most widely use carrageenan in the food industry as κ-carrageenan gel appears to be more firm than those obtained with ι- and λ-carrageenan
Further dilution of the carrageenan solutions were made with 20 mM phosphate buffer saline (PBS) at pH 7.0, which was prepared from the mixture of 20 mM Na2HPO4 (Hamburg Chemical) and NaH2PO4 (Fluka) solutions
Summary
Carrageenans are high molecular weight sulfated polysaccharides made up of repeating Dgalactose residues and 3,6-anhydrogalactose, which are linked via alternating α-(1,3) and β(1,4) linkages [1,2]. These high molecular weight polysaccharides are the major cell wall. There are three types of commercially available carrageenans such as kappa (κ)-, iota (ι)- and lambda (λ)-carrageenan, which are differentiated according to the number and position of sulfate ester group attached to the disaccharide units, and are used to manufacture a wide variety of commercial products e.g. milk products, processed meats, infant formula, toothpaste, cosmetics and pesticides [6]. Typical sulfate levels in κ-, ι- and λ-carrageenan are 22.0% (w/w), 32.0% (w/w) and 38.0% (w/w), respectively [9,10], whilst the contents of carrageenans in different food products are ranging from 0.03% to 3.0% according to the purpose of their addition [11,12]
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