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Fish gelatin (FG) is a potential alternative to current mammalian (beef and pork) gelatin. However, its physical and thermal properties limit its use in many applications. The treatment of microbial transglutaminase (MTGase) on FG could be a practical way to increase the use of FG films in various applications. Physical properties, barrier properties, and molecular weight change of FG films were measured. The viscosity of the MTGase‐treated FG solution significantly (P < 0.05) increased from 6.81 ± 0.65 cP to 35.04 ± 3.59 cP as the reaction time and concentration of MTGase increased. After employing 2% of MTGase into FG solution, the tensile strength increased from 48.03 ± 5.45 MPa to 68.00 ± 1.9 MPa, while percent elongation decreased from 13.1%± 2.90% to 1.47%± 0.05%. Oxygen barrier property was significantly (P < 0.05) increased from 7.24 ± 1.48 cc·m/m2·d to 17.69 ± 3.08 cc·m/m2·d, while water vapor permeability was not statistically (P < 0.05) different. The melting temperature (Tm) measured by differential scanning calorimetry increased from 124.78 ± 1.98 °C to 158.49 ± 2.68 °C as the enzyme reaction time increased. The color values of L*, a*, and b* were changed from 95.56 ± 0.09 to 95.50 ± 0.06, from −0.17 ± 0.01 to −0.23 ± 0.00, and from 3.17 ± 0.04 to 3.47 ± 0.09, respectively. The opacity significantly (P < 0.05) increased from 1.43%± 0.32% to 2.87%± 0.06%. SDS‐PAGE results showed that the molecular weight of fish gelatin films increased when the MTGase reaction takes place.

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