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Screen printing method was employed to produce microbial time temperature indicators (TTI). Bio‐pastes containing lactic acid bacteria loaded Ca‐alginate microparticles (LCAMs) and suitable for printing on polymer films have been produced. Through a spray‐solidification method, polysaccharide gel microparticles allowed for the efficient encapsulation of the lactic acid bacteria, which chromatically induced a colour change in the pH indicator. As the alginate concentration of LCAMs increased, the size of the microparticles decreased. The average diameter of LCAMs ranged from (1.67 ± 0.15) × 103 to (2.93 ± 0.31) × 103 nm. For the evaluation of bio‐pastes, the contact angles and lactic acid production properties were determined. Lower contact angles were obtained with decreasing pullulan concentration, indicating the increase in wettability for printing. The curve of lactic acid production by alginate immobilized cells was determined to take place as a zero‐order reaction favourable to TTI colour change. Visibility of TTIs was greatly improved at microencapsulation sites. As the size of the LCAMs was decreased, the visibility was found to be improved. The Arrhenius activation energy (Ea) of CIFP009‐based TTI was 117 kJ/mol. The results show that the developed manufacturing method would be used for an industrialized, simple and low‐cost manufacturing method for microbial TTIs. Copyright © 2013 John Wiley & Sons, Ltd.

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Two isoenzyme mixture system was developed to freely adjust the Arrhenius activation energy (Ea), which is one of the most important parameters to design a reliable time temperature integrators (TTIs). We hypothesized that the apparent Ea of a mixture of two isozymes would be expressed in a simple linear relationship with the ratio of the mixture, although it could be expressed with a rather complicate equation. To prove our hypothesis, laccase from Pleurotus ostreatus (Ea =27.06 kJ/mol) and PEGylated laccase from Trametes versicolor (Ea =50.35 kJ/ mol) were used to prepare enzyme mixtures with ratios of 0, 0.25, 0.5, 0.75, and 1.0. The enzyme activity was determined by the increase of absorbance of 2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) incubated at 5−30 o C, pH 5.0, and Ea for each enzyme mixture was determined to be in the range of 27.06−50.35 kJ/mol. Relationship between Ea and a ratio of the enzyme mixture not only turned out to be linear, but also was well fitted to the linear mixture rule. This newly found linear dependency is much simpler than kinetically derived one, presumably because microscopic reaction paths and thermodynamic parameters were combined and cancelled out, resulting in linearity. This finding is important in that a mixture of two enzymes with a proper ratio determined from the simple linear mixture rule can customize Ea of an enzymatic TTI. This easier and convenient method can offer a practical and reliable way to adjust Ea of an enzymatic TTI on demand.