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

Abstract

Enzymatic time–temperature indicators (TTIs) usually suffer from instability and inefficiency in practical use as food quality indicator during storage. The aim of this study was to address the aforementioned problem by immobilizing laccase on electrospun chitosan fibers to increase the stability and minimize the usage of laccase. The addition of NaN3, as and enzyme inhibitor, was intended to extend this laccase TTI coloration rate and activation energy (Ea) range, so as to expand the application range of TTIs for evaluating changes in the quality of foods during storage. A two-component time–temperature indicator was prepared by immobilizing laccase on electrospun chitosan fibers as a TTI film, and by using guaiacol solution as a coloration substrate. The color difference of the innovative laccase TTI was discovered to be <3, and visually indistinguishable when OD500 reached 3.2; the response reaction time was regarded as the TTI’s coloration endpoint. Enzyme immobilization and the addition of NaN3 increased coloration Km and reduced coloration Vmax. The coloration Vmax decreased to 64% when 0.1 mM NaN3 was added to the TTI, which exhibited noncompetitive inhibition and a slower coloration rate. Coloration hysteresis appeared in the TTI with NaN3, particularly at low temperatures. For TTI coloration, the Ea increased to 29.92–66.39 kJ/mol when 15–25 μg/cm2 of laccase was immobilized, and the endpoint increased to 11.0–199.5 h when 0–0.10 mM NaN3 was added. These modifications expanded the applicability of laccase TTIs in intelligent food packaging.

Ameliorating the prediction ability of laccase time temperature indicator for dynamic monitoring quality of fresh‐cut papaya

“…Following the method of Lin et al . (2021), fungal laccase was immobilised on electrospun fibres and was used to prepare the laccase TTI prototypes. The electrospun film was made by electrospinning on one side of polypropylene film with a chitosan composite gel solution and was then soaked in 3% GA.…”

“…Meanwhile, the normalised absorbance (Abs norm ) was calculated as the OD 500 of the sample divided by OD 500 obtained at the coloration endpoint. The coloration endpoint (Abs norm = 1) was defined as the minimum time taken for Δ E to decrease to <3 (Lin et al ., 2021).…”

“…Each temperature value of k 1 and k 2 was determined using nonlinear regression analysis (SigmaPlot 10.0, Systat Software, Chicago, IL, USA). The 1/4 k 2 is the maximum reaction rate of the sigmoid patter at the inflection point calculated by quadratic differentiation from eqn 6, which was regarded as the reaction rate k and was then substituted into the calculation of Ea (Lin et al ., 2021).…”

Principle, development and application of time–temperature indicators for packaging

“…The first developed enzymatic TTI was based on oxidoreductase and a phenolic derivative with an irreversible colourless‐to‐brown colouration 21 . Afterwards, diverse enzymes, such as phospholipase, 27 urease, 28 lipase, 29,30 laccase, 31,32 glucoamylase 33 and tyrosinase, 34 have been applied for enzymatic TTIs.…”

“…The ceCPL immobilized laccase was transparent right after soaking in guaiacol solution but presented a transparent‐to‐deep purple‐brown colour change as time elapsed when stored at 4°C (Figure 2D). In 2021, Lin et al 32 also prepared a laccase‐based TTI by immobilizing laccase on electrospun chitosan fibres. Variation of the laccase amount and addition of an enzyme inhibitor (sodium azide, NaN 3 ) could tune the discolouration rate and the activation energy range (29.92–66.39 kJ/mol).…”