Enzymatic Time-Temperature Indicator Prototype Developed by Immobilizing Laccase on Electrospun Fibers to Predict Lactic Acid Bacterial Growth in Milk during Storage

Abstract

Laccase was immobilized on a chitosan/polyvinyl alcohol/tetraethylorthosilicate electrospun film (ceCPTL) and colored with guaiacol to obtain a laccase time–temperature indicator (TTI) prototype. The activation energy (Ea) of coloration of the prototype was 50.89–33.62 kJ/mol when 8–25 μg/cm2 laccase was immobilized on ceCPTL, and that of lactic acid bacteria (LAB) growth in milk was 73.32 kJ/mol. The Ea of coloration of the TTI prototype onto which 8–10 μg/cm2 laccase was immobilized was in the required range for predicting LAB growth in milk. The coloration endpoint of the TTI prototype onto which 10 μg/cm2 (0.01 U) laccase was immobilized could respond to the LAB count reaching 106 colony-forming units (CFU)/mL in milk during a static temperature response test, and the prediction error was discovered to be low. In dynamic temperature response experiments with intermittent temperature changes between 4 and 25 °C, the coloration rate of the laccase TTI prototype was consistent with LAB growth. The results of this study indicate that the laccase TTI prototype can be applied as a visual monitoring indicator to assist in evaluating milk quality in cold chains.

Expanding the Applicability of an Innovative Laccase TTI in Intelligent Packaging by Adding an Enzyme Inhibitor to Change Its Coloration Kinetics

“…Following the method of Tsai et al [12], a chitosan (CS, DD: 76%, 50-190 kDa, Sigma-Aldrich, St. Louis, MO, USA) composite gel solution with polyvinyl alcohol (PVA, 118-124 kDa, First Chemical Manufacture, Taipei, Taiwan) and tetraethoxysilane (Si(OEt) 4 , Sigma-Aldrich) was electrospun on a polypropylene (PP) film using an electrospinning apparatus (NE-300, Falco Enterprise, New Taipei City, Taiwan). The electrospun fibers were collected on a polypropylene (PP) film, which was attached to the drum collector, 12 cm from the tip of the needle.…”

“…Following the method of Tsai et al [12], temperature fluctuations between 4 • C (refrigeration temperature) and 25 • C (room temperature) were applied to simulate dynamic storage conditions. Incubation of the laccase TTI prototype at 4 • C for 8 h, followed by storage at 25 • C for 8 h, was performed three times.…”

“…The coloration of laccase-catalyzed oxidation of guaiacol (Figure S1B, see Supplementary Materials) can be expressed as follows: However, the disadvantages of enzymatic TTI sinclude enzymatic instability, high cost, and a small monitoring range, all of which must be addressed [11]. To reduce the influence of enzymatic instability and the high cost of enzymes, Tsai et al [12] immobilized laccase on electrospun chitosan fibers to develop an innovative TTI. Electrospinning is a progressive and desirable method that produces fibers with diameters ranging from submicrons to several nanometers in order to obtain materials with a large specific surface area.…”

“…In addition, the electrospun fibrous network can alleviate the three-dimensional obstacles faced by reaction products [16]. The amount of enzyme required is diminished, thereby minimizing TTI production costs [12]. The immobilization of laccase with covalent crosslinking on the electrospun chitosan fibers also results in increased enzyme stability [17].…”

“…Nevertheless, the enzymatic TTI that immobilizes laccase on electrospun fibers still has some disadvantages, including the pigmentation appearing after the long coloration duration and a narrow range of coloration Ea, affecting its applicability and monitoring range, respectively [12,17]. The former disadvantage might be resolved by adding dispersing substances [18], and the latter might be resolved by adding an enzyme inhibitor [6].…”